1. health and fitness
2. disease

Review of Literature
RE VIEW OF LITERATURE
-
2. DISCOVERY OF ENTEROCOCCI A ROADMAP
The history of enterococci dates back to a century when Thiercelin in 1899 used the term
"enterocoque" in a French publication to describe bacteria seen in pairs and short chains
in human feces. Later MacCallum and Hastings in the same year described a fatal case of
endocarditis from the John Hopkins hospital caused by a bacterium that was "very hard
and tenacious of life" which they termed as "Micrococcus zymogenes". They confumed
the pathogenicity of the new organism by satisfying Koch's postulates to reproduce
endocarditis using the organism in a canine model [13, 261. However. Andrews and
Horder first coined the name Srrep[ococcus ,faccalrs in 1906, for an isolate recovered
from the blood of a patient with endocarditis, and considered that this streptowccus was
"so characteristic of the human intestine that the term Srreptococcus,faetalis may justly
be applied to it". Later in 1918. Orla-Jensen described a second organism of this group
Streprococcus faecium that difTered from the fermentation patterns of Streprococcus
faecalis but was not formally recognized as a separate species for several decades A
third species Streptococcus durans was proposed In 1935 by Sherman and Wing. which
was similar to Strcptocomus,fac~c~urn
but had less fermentation activity. In 1937 Sherman
had been used to mean different things ranging
emphasized that the term E~ll~~rococclts
from the broad definition of any fecal streptococcus to a restricted definition of
organisms that appeared to be identical to S, faecalis. He proposed a classification
scheme, which separated streptococci into four divisions: pyogenic, viridans, lactic, and
Enterococcus. The term Enterococcus was used for organisms that grew at 10 and 4 5 ' ~ ,
in 6.5% NaCI, and at pH 9.6 and which survived 6 0 ' ~ for 30 min. Many of these
characteristics
became
widely
used
to
distinguish
between
enterococci
and
nonenterococcal streptococci, such as S. I w ~ i s and
,
some are still used today to help
identify enterococci. Sherman's classification scheme also correlated with the serological
scheme originated by Lancefield in the early 1930s. In that system, the enterococci
reacted with group D antisera, while the pyogenic streptococci reacted with group A, B,
C, E, F, or G and the viridans streptococci were nongroupable; S. bovis, classified by
REVIEW OF LITERATURE
Sherman as a viridans streptococcus, was later shown to react with p u p D antiserum. In
1967, Nowlan and Deibel added a new species S. avium to the enterococcal group, which
was found to react not only with Lancefield's group D antiserum but often with group Q
as well [12, 131. Through the following years a number of other types (species) of
enterococci were recognized and added to the enterococcal group. To date, a proposed 28
species of enterococci belong to the growing list of this genus (141.
3. MOLECULAR TAXONOMY AND PHYLOGENY OF ENTEROCOCCUS
Microbial differentiation and identification
IS
a developing area, which is based on
'class~cal' and 'advanced' methods Phenotypic differences among enterococci may
misrepresent the phylogenetic relationships, complicating the classification of
enterococcal species. With the advenr of novel DNA-based identification techniques,
several methods focus on the unlque nucleic acid composit~onof the microorganism
rather than on phenotypic expression of products that
IS
encoded by the respective gene.
Polyphasic taxonomy comb~nesphenotypic and genotypic information and forms the
bas~sfor actual systematic bacteriology [27, 281 Over most of the last century barring the
last three decades, enterococci were classified as group D streptococci on the basis of
their colony morphology and reaction with group specific antiserum. In 1970, Kalina
proposed a separate genus "Enterococcus" for the enterococcal streptococci based on
cellular arrangement and phenotypic characteristics. But. the use of genus name
Streptococcus continued since no action on this proposal was taken [12]. Later Schleifer
and Kilpper-Balz in 1984 provided genetic evidence using DNA-DNA and DNA-rRNA
hybridization to prove that Streptococcus faccalis and Streprococcus faecium were
sufficiently different from other members of the genus Streptococci including S. bovis,
and suggested to merit a separate genus. The DNA G + C content ranged from 37 to 45
mol% as revealed by them. It was similarly proposed that the group N lactic streptococci
should be transfemed to a new genus Lactococcus [29]. Shortly after the proposal of
Schleifer and Kilpper-Balz, Collins and his colleagues used similar methodology to show
that strains priorly denominated as S. avium. S. carseliflovus. S. durans. S.fiecalis
subspecies malodoratus, and S. g a l l i n a ~ mwere sufficiently closely related to other
REVIEW OF LITERArURE
members of the genus Enierococcus to be transferred to this genus but sufficiently
distinct to be considered separate species. They proposed the names Enierococcus avium.
E. casseliflavus.
E.
durans,
E.
malodoratus and E. gallinarum
for those species [30].
Following these two pioneering studies most other novel species of enterococci reported
thereafter, involved the application of DNA-DNA reassociation, 16s rRNA gene
sequencing, Whole cell protein (WCP) analysis, Fatty acid methyl esters (FAME)
analysis and long-chain fatty acid analysis as genetic evidence for establishing any newer
species of enterococci, in conjunction with conventional biochemical and physiological
tests. Recently, authoritative journals have required that the sequence of the 16s rRNA
gene be deposited in the Genbank when reporting a new species [29,31,32].
To date, as of August 2004 [14] there are 28 species of enterococci proposed with
appropriate genetic evidences. They are E. faecalis, E. faecium [29], E, avium, E.
cu.\seliflavu.s, E. dlrrans.
E.
gallinorum,
E.
malodorarus
(301 which are the commonly
reported species. The other specles subsequently proposed with appropriate genetic
evidences as summarized recently by Caravalho et al. [14] are E. canis, E. hirae, E.
mundrii,
E. rufinosu.~. E. psc~udourrum, E. solirariur, E. cecorum, E . rolumhoe, E.
saccharo!viicus,
E.
dispar.
mora\mic3nsis,
E. rarri,
E.
slrlfitre~ts,E. asini.
E. porcinus,
phoc,~licrrlicolo,En~erococcussp.
E2, and
Enicrococcus
E. g i h w .
E.
~illorum.E. hoemoperoxidus,
E, palletls, E.
E.
seriolicida, E. flovescens, E.
Nov. CDC PNS-El, Enierococcus sp. Nov. CDC PNS-
sp. Nov. CDC PNS-E3. The Phylogenetic relationship among 28
species of enterococci reported to date, and other species of related genera is depicted in
Figure 1, which is based on comparative analysis of 16s rDNA sequences.
REVIEW OF LITERA TURE
Figure I. Phylogenetic relationship o f Enterococcus species and other species o f related
genera based on comparative analysis o f 1 6 s rDNA sequences
Dcndrogram rtpmduced with kind permission of Dr.Richard Facklam.
CDC.Atlanta. U.S.A [Id].
REVIEW OF LITERA rURE
3.1. Recla~slficationof some Enterococcus Species
Some of the previously proposed enterococcal species are not validated as new
Enrerococcus species based on the results of recent genetic studies conducted by the
same, or other investigators as depicted in a recent review [12]. E. seriolicida showed
homology to Locrococcus gan~ieaeby WCP analysis, rRNA gene sequence analysis and
DNA-DNA reassociation experiments, and henceforth, the former has been denominated
as L. ganieae currently. Similarly. E. solitarius was later proved to be more closely
related to Tcrra~enococcus halophilus than
Enrerococc~rs by
10
any other species of the genus
16s rRNA sequence analysis and DNA-DNA reassociation
experiments. Likewise E flave~cmswas shown to be closely related to E. casseli/lo~~us
based on DNA-DNA reassociation experiments and hencefonh denominated as E.
c~us,scli/7a1~u.\,
while same was the case of E porcinus designated later as E. ~~illorum
as
both showed homology by genetic studies [I21
4. HABITAT AND ENVIRONMENTAL SIGNIFICANCE OF ENTEROCOCCI
Enterococci occur in a remarkable array of environments, since they are able to grow and
survive under harsh conditions. They can be recovered from water. soil, food, and a
variety of animals, birds and insects 112. 131. In humans. the major habitat of enterococci
appears to be the gastrointestinal tract although iso!ated less frequently from other body
sites. Entemcocci are numerous in the large intestine where concentrations of 10' to 10'
bacteria per gram are typical. Several studies carried worldwide indicate that enterococci
are found in feces of most healthy adults as well infants, with E. faecalis as the
predominating species followed by E,foccium [33, 341. Enterococci though prevalent, are
less commonly isolated from other sites such as vagina, anterior urethra, skin, and other
portions of gastrointestinal tract including oropharynx and bile ducts. Several studies
have indicated that enterococci can be isolated from hospital environmental surfaces. but
their role in strain transmission remains debatable [I 2. 131.
RE VIEW OF LITERA TURE
Enterococci are considered to be good indicators of fecal contamination of food
and water since they are present in the feces of humans and warm-blooded animals. The
term "fecal enterococci" include four species: E. furcalis. E. ,faecium, E. durans and
E. hirae [IZ, 131. With their emergence as a nosocomial pathogen, environmental or
other nonhuman sources could contribute to the dissemination for antibiotic resistant
enterococci, which pose a therapeutic challenge. Apart in-vitro transfer, in-vivo transfer
of antibiotic resistance genes under natural conditions between E. ,faecalis in sewage
water treatment plants has been described [35] creatlng awareness about the role of
environmental strains in the dissemination of antibiotic resistance. While the significance
of enterococci In health care settings have been studied extensively, focus on
environmental enterococcal strains could help to complete epidemiological studies and to
determine the role of these strains in antibiotic resistance dissemination in community.
5. EPIDEMIOLOGY OF ENTEROCOCCI
Nosocomial infect~onshave become onc of the major health care problems globally,
attributing to the morbidity and mortality of hospitalized patients. "A nosocomial
rnfcction
IS
one for which there is no evidence that the infection was present or
~ncubat~ng
at the trme of hospital admission To he classified as an infection, the
condition musl be manifested as a clinical disease and not merely colonization. However,
an asymptomatic patlent may he considered infected :f pathogenic microorganisms are
found in a body fluid, or at a body site that is normally sterile. such as the cerebrospinal
fluid or b l o o d II I]. Furthermore, the emergence of drug resistance among many bacteria
has raised an unpraxdented level of concern, since hospitals serve to be the ideal
breeding ground for the development and spread of several multi-drug resistant bacteria.
Data from several studies shows. over the past decade there is a clear shift in prevalence
from gram-negative to gram-positive species as the predominant cause of nosocomial
infections, among which entemcocci has become one of the top three pathogens causing
various infections [I 1, 131.
RE VIEW OF LITERATURE
5.1. Nosocornill Epidemiology of Enterococei
The clinical microbiology laboratory in collaboration with several teams of the respective
hospital generally carries out the surveillance of nosocomial infections. Federal bodies
like the Center for Disease Control and prevention (CDC)-National Nosocomial
Infections Surveillance NNIS), or the bodies approved by them carry out nationwide and
international nosocomial surveillance progams through a network of laboratories
worldwide. While many regional bodies in different countries, replicate the functioning
of CDC's NNIS, according to their setup following the standard guidelines for
nosocomial infections surveillance [20. 36. 371. Data from most of these programs shows
that enterococci continues to be one of the top three pathogens causing different kinds of
nosocomial lnfect~onslike urinary tract infections (UTI), blood stream infections (BSI),
s k ~ nand soft tissue infections (SSTI) and other miscellaneous infections since last
decade, and this has been supported by several other independent studies carried out in
various laboratories worldwide.
Nosocomial surveillance studies conducted in different pans of the world by
various groups showed that enterococci ranked among the top three pathogens causing
nosocomial UTI [20, 38-40] In 1984 the NNIS listed Enterococcus as the third most
common cause of nosocomial infection. enterococci caused approximately 10% of all
such infections, including I4 7% of UTls and 7% of bacteremias [13], the fact which was
supported by Morrison and Wenzel [19], who conducted a comprehensive 10 years study
of nosocomial urinary tract infections (LITI) due to enterococci from 1975 through 1984
in a U.S hospital. and concluded that enlerococcus was the second most frequent cause of
nosocomial UTI with a crude mortality rate of IS%, and the proportion of nosocomial
UTls due to enterococci increased from 6% to 16%. Davies et el. [41] in their
epidemiological study of nosocomial UTls in ~ediatricpopulation showed E. coli (26%)
and Entcrococcus species (15%) as the predominant pathogens causing nosocomial UTI,
and showed catheter related infections accounted for 48% of all the nosocomial UTIs,
while secondary bactcremia occurred m l y , with an incidence of 2.9%. While in another
study Moulin et al. 1421 showed that E. coli (39%) and Enterococcus species (12.1%)
REVIEW OF LITER4TURE
were the etiogens of nosocomial UTI among pediatric age group in their retrospective
one-year study in a pediatric hospital. Bouza et al. [38, 391 conducted a study on behalf
of Co-operative group of the European Study Group on Nosocomial Infections to
estimate the incidence of nosocomial UTI in Europe. The results from 141 hospitals
spread over 25 European countries, which participated in the study showed Enrerococcus
species as the second common pathogen isolated next only to E. coli from nosocomial
UTI. The SENTRY study of nosocomial UTI showed the pathogen occurrence and
susceptibility profiles in different geographic locations. Seven pathogens accounted for
90% of all isolates, and Enterococcu.~species ranked second accounting to 13% next only
to E, coli (47%) [40]. Most of these studies showed that bladder catheterization, prior
antibiotic therapy as the common risk factors for acquiring nosocomial UTI.
Enterococc~were the third most common cause of nosocomial BSI in the SCOPE
Program of IWI, conducted in 41 U.S. hospitals. They accounted for 11.7% of all
~solatesreported, with E. fueculr.\ as the most common species (60%). followed by E.
fueci~trn(20%). Vancomyc~nresistance was observed in 36% of all participating medical
cenlcrs, with van-A and van-B genotypes equally prevalent [20]. The study conducted by
CDC's-NNIS to describe the epidemiology of nosocomial infections in combined
medical-surgical (MS) intensive care units (ICUs) between 1992 and 1998, revealed that
enterococci contributed to 11% of primary BSI's in the study group, and concluded these
nosocomial infections (BSI) in MS ICUs were almost associated with use of an invasive
device (431. The SENTRY surveillance program conducted in 1997 revealed, that
enterococci ranked second, next only to Staphylococci as a cause of nosocomial blood
stream infection (BSI) during a one-year study conducted in different laboratories
worldwide during 1997. They also demonstrated 22-76% of HLAR among enterococci
[37]. A study by Diekema et al. [44] to find the age-related trends in pathogen frequency
and antimicrobial susceptibility of BSI in North American medical centers, showed
enterococci as one of the common pathogen, while vancomycin resistance among
Enferococcus species predominated among nosocomial BSI in patients over SO yeam of
age. Another prospective surveillance study of nosocomial BSl's in pe&aaic patients at
49 hospitals during a 6-year period conducted by SCOPE, showed enterococci as the
REVIEW OF LITERATURE
second common cause of BSl's occurring predominantly in very young and/or critically
ill children, with vancomycin-resistance in 1% and 11% of E. faecalis and E. faecium
isolates respectively [45].
In six years study of surgical wound infection surveillance at a tertiary care
center, Weiss et al. [46] showed wagulase-negative Staphylococcus and Enterococcus as
the two most frequent isolates before and after antibiotic restriction policies were
implemented in their hospital. Vanwmycin resistance was exhibited by 2.4% of
enterococci identified between 1996 and 1998. While in another study conducted to find
the epidemiology of nosocomial infections in combined medical-surgical (MS) intensive
care units (ICUs), Enterococcu.\ was found to be the single most frequently reported
pathogen accounting to 17% in patients with surgical-site infections [43].
5.2. Molecular Epidemiology of Nosocomial Enterococci
Initially, enterococcal infections were suggested to have evolved from patient's own flora
considering the natural habitat of enterococci in humans, and due to lack of concrete
epidemiological markers. But with the advent of molecular epidemiological tools
researchers were able to present genetic evidences for exogenous acquisition of
enterococci mustly fmm nosocomial setup. In 1986 Zervos et al. [I81 reported the
nosocomial transm~ssionand exogenous acquisrtion of S. foecolic. for the first time. They
conducted a case-control study by comparing patients with gentamicin-susceptible and
resistant S. ,/aecalis infections between 1981 - 1984 and concluded that all highly resistant
strains appeared to be nosocomial since I2 cases were clustered on a surgical floor and in
a bum unit, using plasmid DNA content as an epidemiological marker. Subsequently in
another pmspective study of 100 patients hospitalized on the surgical and thoracic
intensive care units (ICU) and a general medical floor, the same laboratory showed
nosocomial acquisition and inter-hospital spread of gentamicin-resistant enterococci.
using plasmid DNA analysis as an epidemiologic marker. Their results showed that ten
patient's cultures grew colonies of gcntamicin-resistantenterococci-six after admission to
the ICU and four after hospitalization on the medical ward. Resistant entemocci were
REVIEW OF LITERA 7URE
isolated from the hands of hospital personnel and were frequently isolated from
environmental surfaces 1471.
Later Weems el al. [48]showed genetic homogeneity among few HLGR isolates
using a gentamicin-resistance gene probe, suggesting nosocomial transmission of
enterococci. Wells et al. [49] investigated an outbreak of infections due to betalactamase-producing, high-level gentamicin-resistant (HLGR) E. ,faecalis. Restriction
enzyme digests of total chromosomal DNA showed nearly identical panems for selected
isolates of beta-lactamase-producing HLGR E. /ueculis, suggesting dissemination
through the hospital of a single strain of E. .faacalis Thereafter, several nosocomial
epidemiologic studies utilized molecular epidemiological tools for determining the
genetic relatedness of the enterococci. and showed that the most likely way these resistant
enterococci spread among hospital patients were, via transient carriage on the hands of
hospital personnel, patient-to-patient and inter-hospital transmission of strains.
Molecular epidemiological tools like PFGE were highly instrumental in studying
the clonality of nosocomial infections caused by HLGR enterococci and VRE in many
hospitals. as shown by several independent studies [SO-551.Nourse et al. [56] using
PFGE and van-PCR suggested that environmental contamination played an important
role in patient-to-patient transmission of VRE in a pediatric oncology unit, and
interventions including implementation of infection control measures were associated
w~tha decreased incidence of gastm-intestinal colonization. While another study showed
that the molecular epidemiological tools like PFGE was highly useful for detecting the
endemic strains responsible for colonization, or infection over a prolonged time period
1531.Bopp et al. [52]analyzed 116 VRE isolates obtained from patients in six New York
State hospitals by PFGE, Plasmid profiling and PCR in a Molecular epidemiological
study, which revealed genetic heterogeneity among isolates from within each of the six
hospitals. They concluded that PFGE typing could show that nosocomial VRE
transmission had occurred in some hospitals. In another study, arbitrarily primed
polymerase chain taction (AP-PCR) was successfully used to evaluate cross infection
and a possible outbreak of E. faecalis UTI in a urology ward in the period 1982-1996.
REVIEW OF LITERATURE
They revealed that five of the nine isolates had the same pattern, which had caused the
outbreak of E. faecalis UTI and suggested that cross infection had occurred via urinary
catheters or by hand contact in that ward [54]. Thus the application of molecular tools
were shown to be highly useful for studying the nosocomial epidemiology of enterococci,
which in many instances unraveled the outbreak sources in hospital as well community
settings, that might have gone unmasked if failed to apply.
5.3. Epidemiology of Multidrug Resistant Enterococci
Enterococci show a remarkable ability to acquire and disseminate antibiotic resistance
genes by a variety of routes especially in a hospital setup, the property that has propelled
them to become a predominant nosocomial pathogen [I 3, 17. 571. Of prime importance
are the emergence of
resistance
to high-level gentamicin andlor streptomycin, ampicillin
and glycopeptides among nosocom~alenterococci, since exhibition of resistance to any
combination of the above-mentioned antimicrobials poses a great therapeutic challenge.
Various studies worldwide had shown that enterococci resistant to these antimicrobials
were responsible for several nosocomial outbreaks and clonal dissemination. Thus
epidemiological investigations of these drug resistant strains and appropriate infection
control procedures, helps in reducing the morbidity and mortality due to these multi-drug
resistant enterncocci among hospitalized patients.
Vancomycin resistance among enterococci has emerged to be one of the major
nosocomial problems to deal with. Since it's first description in enterococci in 1986.
there is a steady increase in vancomycin resistance among nosocomial enterococci
worldwide. From 1989 through 1993, the proportion of enterococcal isolates resistant to
vancomycin (VRE) reported to CDC's National Nosocomial Infections Surveillance
(NNIS) system increased from 0.3% to 7.9% [58]. Subsequent studies showed a 47%
increase of VRE from 1994 to 1998, with 26% of nosocomial enterococci exhibiting
vancomycin resistance [59]. While a surveillance data reported by the NNIS srjtem for
1993-1997 compared with January-November 1998, showed a marked increase (55%) in
VRE associated with nosocomial infections in ICU patients from U.S. hospitals [S].
REVIEW OF LITERATURE
Boyce et al. (601, in their study showed the incidence of ampicillin-resistant
enterococci (ARE) increased sevenfold at a university-affiliated hospital between 1986
and 1988. The plasmid and chromosomal DNA analysis of the ARE isolates revealed that
the increase was due to an epidemic of 19 nosocomial infections that yielded closely
related strains of E, jaecium, whereas the non-epidemic strains were identified as E.
/irocium. E, raflnosus. E. d u r a n ~ and
,
E. gallinarum, and concluded that the increase in
the incidence of ARE was due to the selection of various strains of resistant enterococci
by the use of imipenem. Hanhug et al. [61] described the first nosocomial outbreak of
ampicillin-resistant and vancomycin-resistant E../irecium in Norway during 1995 to 1996.
Their results revealed that 149 patients were infected with ARE, and isolates from 1 15
cases were genomically related to the outbreak strain, while four infections were caused
by a van-B type VRE that was genomically related to the ARE outbreak strain. They
concluded that one year after the onset of outbreak. VRE occurred in wards which had a
relatively high consumption of vancomycin, and the first nosocomial outbreak caused by
ARE observed in 1995 was still ongolng.
Another prospective study revealed that 9% of enterococcal clinical isolates were
ampicillin resistant, while ARE were not isolated from hospital personnel or
environmental surfaces, they were common in the rectal flora and could spread to the
urinary system to become an emerging clinical problem 1621. Torell et al. [63. 641
showed the incidence of ARE among enterococcal isolates at a University Hospital in
Sweden increased from 0.5% to 8.1 % beween 1991 and 1995. and suggested multifocal
origin of the majority of the infecting ARE strains. They concluded that. non-recognized
fecal colonization and silent spread of ARE among many patients over a prolonged time
period as a major reason for the increase of ARE infections in their hospital.
5.4. Epidemiology of Enterococcnl Colonization
Bonten et al. [65], investigated colonization of patients and environmental contamination
with VRE in an endemic setting to assess the importance of different sources of
colonization. They revealed that persistent VRE colonization in the gastrointestinal tract
REVIEW OF LITERATURE
and on the skin. the presence of multiple-strain types of VRE, and environmental
contamination might contribute to the spread of VRE. However, they concluded that once
colonization pressure was high, it became the major variable affecting acquisition of
VRE. Oon el al. 1661 conducted a study from January to March 1997 at a 900-bed
teaching hospital in Singapore to determine the prevalence of intestinal colonization of
VRE in the patient population. Out of the total of 299 consecutive stool specimens
screened. VRE were detected in the stool of 35 patients (12.3%). This group consisted of
four isolntes with Van-B (one E. .fueculis and three E.,faecium) and 31 isolates with VanC (30 E, casselr/lu~v.sand one E, gallinurum). Except for isolates from the same patients,
PFGE patterns were diverse, suggesting that the VRE isolates were genotypically
d~fferentand possibly introduced from many sources. Thus epidemiological studies of
entemoccal colonizers exhibiting drug resistance in GIT, helps in initiating appropriate
eradication approaches to decrease the risk of VRE infections in humans. especially those
hospitalized [67].
5.5. Epidemiologv of Enterococcal Superinfections
I'nor use of ant~m~crobialagents lacking enterococcal activity has been quoted as an
~mportant factor in the development of enterococcal superinfections in hospitalized
patlents [13]. Several authors have shown that enterococcal superinfection and
colonization developed in 2-59.0 hospitalized patients, after therapy with broad-spectrum
antibiotic like moxalactam. aztreonam and ciprofloxacin. However. other disposing
factors like urinary cathetenzation and wound infections have also been shown to
contribute to this f a c ~[68.69].
5.6. Epidemiology of Nosocomirl Environmental Reservoirs of Enterococci
Hospital environment plays a major role in transmission of entemocci, mostly resistant
to lhe commonly used antimicrobials in hospitals. The versatile nature of enterococci
enables them thrive in adverse conditions like those of hospitals. Noskin et al. [70]
showed that VRE are capable of prolonged swvival on hands, gloves, and environmental
REVIEW OF LITERATURE
surfaces. Their study revealed that E. faecium isolates survived seven days on
countenops. 24 hours on bedrails, 60 minutes on telephones and 30 minutes on
stethoscopes. Freeman et al. [71] showed that nosocomial enterococci were resistance to
heat (upto 8 0 ' ~for one minute) which enables them to survive the temperatures and
holding times specified by the health authorities for the disinfection of used or infected
linen. Also they could withstand 150ppm available chlorine for five minutes, the
treatment suggested by the health authorities for the disinfection of heat labile materials,
which underscores the significance of enterococci to survive and disseminate in the
hospital environment.
Several studies have shown that health-care workers and environmental surfaces
in hospital settings were responsible for transmission and spread of gentamicin and
vancomycin resistant enterococci Freeman et al. in thelr study using Pyrolysis mass
spectrometry authenticated that nosocomial spread of enterococci had occurred via
fluidized microsphere since the decontamination process was inadequate [72]. While
some studies have shown thermometers. hlood pressure handcuffs, IV fluid pumps,
bedrails and linen as different sources of contamination with VRE contributing to their
spread in hospital scttings [65. 73, 741. U'hile many studies have shown concordance
between hospital environmen~al strains and the patient isolates often resistant lo
vancomycinigentamicin using molecular epidemiological tools [75-801. Most of these
studies have funher suggested and proved that appropriate infection control measures,
minimizes transmission of these multi-drug resistant enterococci in hospital settings.
5.7. Epidemiology of Community Reservoirs of E n t e r m c i
Several epidemiological studies conducted in human subjmts from community have
yielded entcrococci resistant to various antimicrobials like ampicillin, gentamicin and
vancomycin. Most of the human subjects from community who carried drug resistant
mterococci were previously hospitalized and had used antibiotics. Vancomycin
resistance among community subjects had been the focus of study by several authors
since laat decade, and most of the studies have concluded that previous hospitalization
RE VIEW OF LITERATURE
andl or prior use of vancomycin as the common factors for community dissemination of
vRE [&I-831.While other studies have quoted that transmission of resistant enterococci
or resistance genes takes place between humans and animals in the community. One
possible explanation they have given for the animal to human transmission was the use of
gly~opeptidegrowth promoters like Avoparcin in feed animals [84-871. This fact was
further authenticated by another study, which showed a decreased incidence of VRE
isolated from poultry meat and from fecal samples of humans in the community after
discontinuation of avoparcin usage in animal husbandry [88]. But most of these
epidemiological studies have been conducted in Europe during last decade, where there
were no restrictions for using Avoparcin as growth promoters in farm animals, unlike
other countries. But the impact of recent ban on avoparcin as growth promoter in
European countries has not been studied much to give a clear picture of this less studied
problem
6. CLASSIFICATION AND IDENTIFICATION O F ENTEROCOCCUS
6.1. Genus Definition and Metabolic Characteristics
The genus fiinti*rococcrr.s consists of gam-positive. facultative anaerobic organisms
assigned as chemo-organotrophs, that are ovoid in shape and may appear on smear in
short chams, pairs. or as single cells. They are strict fermenters since they lack a Krebs
cycle respiratory chain. Their metabolism is homofermentative and produce lactic acid as
an end product via. Embden-Meyerhof-Pamas pathway. Their predominant end product
of glucose fermentation is the L (+)- lactic acid enantiomer. Like streptococci, these
orpanisms do not have cytochrome enzymes and are thus catalase negative. although
some strains do produce pseudocatalase [12. 13. 27. 281. Most strains produce a cell wall
associated glycerol teichoic acid antigen-the streptococcal group D antigen, but the
detection rate varies depending on the extraction procedure and the quality of antisera
used, while some rract also with group Q antisera [89, 901 Hydrolysis of L-pyrrolidonyl3-naphthylamide (PYR) is a characteristic feature that is seen also with group-A
streptococci but not other streptococci. Most strains in the newly defined Enterococcus
RE VIEW OF LITERATURE
genus possess the characteristics summarized by Sherman in 1937 such as the ability to
grow in 6.5% NaCl and at pH 9.6, to grow at 10 and 4 5 ' ~ ,and, for the most part, to
survive at 6 0 ' ~ for 30 minutes. Although the above screening tests appeared to be
sufficient in the pas1 to identify enterococci presumptively, it is now recognized that other
less commonly encountered gram-positive cocci can also give a positive reaction in some
of these tests [90].For example, some cultures of Laciococcus, Aerococcus, Pediococcus,
and Leuconostoc spp. are bile-esculin positive or can grow in 6.5% salt or both. Strains of
Pcdiococcus and Leuconosioc spp. can be group D positive, and some lactococci and
aerococci are PYR positive; lactococci and aerococci, however, should not react with
group D antisera. The phylogenetic analysis based on 16s rRNA gene reveals that the
genus .Enrerocor.clr.c is more closely related to Vogococcus, Teiragcnococcus and
C'arnohacic~riurnthan they are to the genus Sireprococcus and Lociococcu.~[I 2,29,90].
6.2. Cultural Characteristics and Morphologj
Enterococci generally produce well-circumscribed. smooth. raised colonies about 1-2 mm
In d~ameler,although some variants may appear smaller on prlmary isolation media such
as blood agar. Recently mucold encapsulated strains of E. ,faeculis have been isolated
from unne specimens. on 5% sheep blood agar and Muller-Hinton agar plates, which
emphasizes the versatil~tyof the colony morphology of enterocwci. Hemolys~scan be
observed around enterococcal colonies if the agar contains horse. rabbi6 bovine. or
human erythrocytes, but sheep erythrocytes are largely refractory to the effects of the
cnterococcal hemolysin [26]. Some stralns of E. ,faecalis may be a-hemolytic on agar
containing rabbit, horse, or human blood but non-hemolyt~c on agar containing sheep
blood, while some strains of E. durata are p-hemolytic regardless of the type of blood
used. All other species are usually a-hemolytic or non-hemolytic. Strains that are ahemolytic arc actually non-hemolytic strains that produce peroxide. which acts on the
blood cells in the medium and results in 'greening' of the agar, and not merely due to the
production of a toxin by the strain. E. cossel~/lo~ws,
E. mundiii and E. sulJureu~produce a
yellow pigment on Trypticasc blood agar medium, which can be detected by using a
white conon swab to pick up growth and examining the swab for a yellow color [12, 131.
RE VIEW OF LITERA TURE
6.3. Laboratory Media for Isolation and Enumeration of Enterococci
A diversity of media has been described and proposed for the isolation and enumeration
of enterococci, owing to their importance in different foods, feeds, and clinical and
environmental samples. Because of their requirements for several vitamins and amino
acids, enterococci cannot be grown easily in synthetic media. Profuse and rapid growth is
only achieved if rich complex media such as Brain Hean Infusion (BHI) broth or
Trypticase Soy (TS) broth are used. A recent review by Domig et al. details different
media used for the enumeration and isolation of enterococci from various sources [27].
Although various selective media are used for isolating enterococci from different
sources, we focus mainly on media used for cltnical specimens. For primary isolation of
enterococci from clinical specimens any blood agar base containing 5% animal blood
suppons the growth. The hemolysis of enterococci depends on the type of blood used
along with the basal media. but predominant enterococcal species are usually a-hemolytic
or non-hemolytic. All enlerococci grow at 35 to 3 7 ' ~and do not require an atmosphere
containing increased levels of carbon dioxide [I 21
Bile esculin test was applied to differentiate between enterococci (group D
slreptococci) and non- group D streptococci (90). Bile-esculin azide (BEA) agar
(available with various commercial manufacturers) is an excellent primary isolation
media, for ennchrnent and isolation of enterococci fmm samples that might be
polynicrobial in nature containing gram-negative bacteria. The azide in the medium
inhibits the gram-negative bacteria and enterococci appear as black colonies by
may exhibit a similar colonial
hydrolysis of esculin. However, Listerio monoc:~'~opcne.~
morphology on this medium after 48 h of incubation. Most other bacteria either grow
weakly or appear as colonies of different shape. Media like Columbia colistin-nalidixic
acid agar (CNA) or phenylethyl alcohol agar (PEA) are used for successfi~lisolation of
enterococci. CNA is advantageous over PEA, since hemolytic reaction can be read from
CNA if supplemented with blood, but not from PEA [12]. The Cephalexin aztreonam
arabinose (CAA) agar allows the isolation of E.furcium from heavily contaminated sites.
and in comparison with CNA agar it can differentiate E. .foecium from E. .foecolis and
REVIEW OF LITERA TURE
E.
durans by its ability to ferment ~ b i n o s e[91]. While other commercially available
media like Kanamycin-aesculin-azide(KAA) medium, Membrane filter enterococcus
(ME) agar medium contains various antimicrobials which suppresseslinhibits the growth
of other microbes, but enhanceslfacilitates the gowth of enterococci [27].
With increasing incidence of antimicrobial resistance among enterococci.
isolationldetection of vancomycin resistant enterococci (VRE) and aminoglycoside
resistant enterccocci are of clinical significance. For the detection of VRE in different
specimens, numerous variations of media and isolation procedures have been published.
Most of them are variat~onsof selective media, which differ with regard to the antibiotic,
or their concentrations used (271. Willey and colleagues demonstrated that VRE could be
detected by using Muller Hinton agar (MHA) supplemented with varylng concentrations
of vancomycin (6-12 pglml) [92]. Swenson et al, in a multilaboratory evaluation
determined that BHI agar supplemented with 6 pgiml of vancomycin as an optimal
medium for detection of VRE [93]. Many clinical microbiology laboratories worldwide
are using this medium for detection of VRE
Several techniques and media have heen developed for isolating enterococci from
Inanimate surfaces in hospital environment. Premoistened swab is the most commonly
used method for surface swabbing in hospital environments that are placed in enrichment
broth and then plated appropriately for isolating enterococci 112. 271. Recently
commercial manufacturers have come up with agar imprint methods (Rhodac-imprint
method), where the agar surface is directly applied to the environmental surface to be
cultured and examined for the growth of enterococci. Unfortunately, despite the
availability of an array of med~ato isolate and enumerate enterococci, there is no single
medium which equally meets all requirements. since in most cases a pronounced
selectivity is only achieved if lower recovery rates are tolerated and vice versa.
Mortova, the performance of each method depends largely on the matrices of the
samples, and on the associated microflora.
REVIEW OF LITERA TURE
6.4 Conventional Species Identification Methods
Classic species identification of enterococci involves assays for a combination of
biochemical and morphological characteristics of the unknown organism. After KilpperBalz's 1291 newer classification of enterococci based on the molecular and
chemotaxonomic approaches, Facklam and Collins in 1989 [90] described an
identification scheme utilizing conventional biochemical and physiological tests for
speciation of enterococci. In subsequent years, other authors described tests involving the
cleavage of methyl-a-D-glucopyranoside, the susceptibility to efrotomycin, and the
fermentation of D-xylose (94, 951 for difrerentiation of Enrerococcus species. Thereafter,
several authors devised various algorithms with minimal number of biochemical tests for
ldentification and speciation ol'enterococci. However, most of the tests were based on. or
der~vedfrom the conventional identification schema devised by Facklam and Collins [12,
90. 961. Today even though a broad array of novel identification methods prevails, for
most clinical microbiological laboratories worldwide. the primary method of identifying
Crircro~.occ.~c.~
strains relies on phenotypic characterization based on the conventional
~dentificat~on
schema dev~sedby Facklam and Collins [90].
6.4.1. Facklam 's Conwntional Phenotyping Schema
In 1972, Facklam published a summary of 26 physiological tests to differentiate group D
streptococci,
and thereafter over the years enterococci has undergone dramatic taxonomic
changes 1291. However. in 1989 Facklam and Collins devised an array of biochemical
and physiological tests for proper identification of twelve Enrerococcus species
prevailing during that period. The grouping is based on key phenotypic tests, and does
not necessarily conform to the grouping by 16s rRNA sequencing or grouping by other
molecular techniques. The identification schema has been constantly updated by Facklam
and his colleagues to fit in all entemcoccal species reported to date [12, 14. 31. 901 and
the details of the schema are presented elaborately in the forth coming pages. Most of
their phenotyping studies w m performed on lactococci and enterococci isolated from
humans.
REVIEW OF LITERA TURE
As mentioned in the "Genus definition", any unknown catalase-negative grampositive coccus presumptively identified as Enterocnccus, are subjected to the
conventional phenotypic tests listed in Table I to identify the species as per standard
procedures. The five species E. cecorum. E. calumbue, E. pollens, E. succharoly~icusand
Enlcrococcus sp. Nov. CDC PNS-E3 are included even though they are PYRase-
negative, and may grow very poorly in broth containing 6.5% NaCl (except Enterococcus
sp. Nov. CDC PNS-E3) [12, 14, 311. The identification schema is based by separation of
the enterococcal species into five groups, based on acid formation in mannitol and
sorbose broths and hydrolysis of arginine. Although identification of enterococcal species
by conventional tests is not rapid most identification can be made after two days of
incubation, while some may require incubation of the tests up to ten days.
Group l
This gmup consists of E, u ~ , i ~ tE,n ~malodorutus.
.
E. raffinosus. E, pallens, E, gih*us,E.
pwudrw~.ium.E. .r~c.i.h~r(~!r.li~li.\
and Enteroc,occlc.~sp. Nov. CDC PNS-E3.
REVIEW OF LITERATURE
hilrpld l a m rc1om.c I 4 vllh rmmukrn
' h14h rmnnttot. SOP.
nmvr mc,.
arp,n,rr
I'I(* pl-l
I ~ a h (kI I
'
4 ~ amh,m*
4
sn1. *,m,t,,i
R ~ . raninos.
I
T I I . o (I.. tcliun~c.MOT. molll~r!.
s u m PYI'p r u \ n r M(;F ~ ~ \ I ~ . I ~ ~ I +W - ~u ( ~l %~. ~R~ ~~, ~~~ aI r c~ r -n
C'
i ~Ilr.slnlns
~\c
anpoS118vc
195 ~kunm. uc pa,tlrcl. WCLIII~I c,cqn~,mr ,rcur (.?a. 0rem8ns shou a h n l rmcllunr)
S(I(.
10
.
k m w r rhuu~ntwcxMon d.1. inm I>V.tntns
''[alrrmnnl~ol
p n l t ~ l &p
c
toItr)(uh~~jnl
RE VIEW OF LITERA 7URE
Group 11
This group consists of E, faecaiis, E. haemoperoxidus. E. ,fhecium. E. casseliJavus. E.
mundlii. E. gallinarum and Enterococcus sp. Nov. CDC PNS-E2.
(;roltp 111
This group consists of E, drrruns. E. Irrrcrc~.E, dirpur. E rutti and E. ~illorum(E. rillorurn
and E, porcincts constitute a single species as proven by genetic studies).
RE VIEW OF LITERA TURE
Group-IV
This group consists of E. o.~ini,E. sulfureus, E. cecorum. E. phoenicuiicolo and
Entcroc0ccU.Y Sp. NOV.CDC PNS-E2.
(iroup I'
According to the latest classification 1141, this group consists of E cunis. E. columhae,
and E, moru~'~i~n.v~s.
6.5. Commercial Rapid Species Identification Methods
The convcntional phenotypic identification of enterococci is rather time-consuming and
oRen ambiguous, hence based on the recent developments miniaturized test kits have
been introduced and used for rapid phenotypic differentiation of enterococci. Most of the
kits and strips allow an indicator-based determination of sugar utilization or show
REVIEW OF LITERATURE
reactions based on their specific micrhial enzymes. There are several publications
describing comparative studies using these devices, but we have tried to give an account
of only the recent updates of the commercial systems available, within the scope of our
review. The most commonly used testing systems, or kits usedltested and evaluated are
API 20 Strep (Bio-Merieux, France). AP1 50 CH and Rapid ID32 Strep (Bio-Merieux),
API Rapid ID 32 system, Crystal Gram-positive and Crystal Rapid Gram-positive kits,
Vitek Gram-Positive ldentification Card (Vitek-2 system), Microscan Gram-Positive
ldentification panel and Phene Plate (PhPlate Microplate Techniques, Sweden) [14, 31 1.
However, the errors encountered in identification with these commercial systems or kits,
were related io misidentification of E. gallinarum and E. cas.srl~flavuswhich needed
supplemental tests for motility and pigmentation to improve their identification by a
considerable perceniage 1951
In summary most of the systems or kits could detect only a limited number of
enterococcal species, and fuflher rests were necessary for a higher level of identification.
Even though these rap~d ~dmtification systems have revolutionized microbial
~dcntificationIn clin~calmicrobiology laboratories, it is still out of reach for most of the
clln~calmicmbiological laboratories In developing countnes. ldentification of novel and
unusual species has become unreliable with commercial systemu'k~tsw ~ t hthe increase in
\he number of enterococcal
species
species
and phenotyp~c similarities among enterococcal
114. 3 1. 97). Funhennore no commercial kit includes the whole set of tests used
In conventional testtng schema for tdent~ficattonof Dlterococcus species [96]
6.6. Molecular Pbenotyping Methods for Species Identification
Phenotyping or bioiyping, meaning the detection of carbohydrate fermentation and
enzyme pattern, can be regtuded as a traditional way of microbial differentiation.
According to merit devclopmen~s,alternative procedures have been sought to simplify
to speedup Uuse methods that are alternatively termed as "Molecular phenomyping"
since the procad-
used. helps in identifying and exploiting the phenotypic
charactniaics of the organism, unlike genotypic methods which exploits the genetic
REVIEW OF LITERATURE
content (DNA)of the organism. An ovefiiew of various methods available for speciation
of entemuxci, are reviewed briefly in the following sections.
6.6.1. Whole Cell Prorein (WCP) Fingerprinting By SDS-PA GE
The comparison of whole-cell protein panems obtained by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) under well-defined conditions has been
shown to be reliable method for identification and classification of enterococci, especially
those of unusual or atypical enterococcal species [98]. Results of some studies suggest a
h~ghcorrelation between similarity in protein patterns and DNA-DNA homology values.
Teixeira et a!. analyzed E. ,fuecium strains of atypical phenotype and showed that the
protein profiles were similar to those of typical stra~nsand easily dist~nguishablefrom
those of other enteroccxcal specles [97] Preliminary studies from our laboratory have
shown that atyp~cal h~ochemlcal variant strains of E fuccium. E, fuecu1i.c and E.
~ ~ a c . t ~ ~ l i f l exhibited
u~~us,
the same whole-cell protein profiles, as do typical strains, apart
from minor qualitative or quantitative d~fferencesIn the WCP profiles among strains
I V V ] Studies show that E. ~ u l l ~ n u n tand
m E, cusscliflu~~u.\strains that are very closely
related by 16s rRNA gene sequencing arc clearly separated by WCP profiling, while
many novel enterococcal s p s l e s have been proposed based on WCP profiles and other
DNA-DNA hybridization methods [ 14. 3 I]. With the advent of gel documentation
systems and software programs, the analys~sof proteln fingerprints have become much
caster, and as evident fmm current literature this technique is being followed by many
laboratorin worldwide
6.6.2. Olhcr Molecvlor Phenotyping methods
While several clinical micmbiological laboratories are using WCP profiling for
identifying or authenticating the taxonomic status of enterococcl, some researchers have
tned other novel mahods for identification and speciation of entemcocci. A recent
review summarizfi, other less commonly used methods for this purpose [28]. Pyrolysis
mass spectrometry (pyMS). Vibrational spectroscopic methods and Proton magnetic
resonance spatroscopy (1" MRS) have been used f o ~ rapid identification of
Slreptococnu; species and En,ercroccu.c species, and the results were compared and
REVIEW OF LITERATURE
confinned by 16 S rRNA sequencing and species-specific PCR [28]. While, Multilocus
enzyme electrophoresis (MLEE), Long-chain fatty acid analysis and Fatty acid methyl
esters (FAME) analysis were used as an adjunct to other methods, for the description of
the novel enterococcal species [3 I]. However, as of now their applications are restricted
to experimental studies with medical isolates, or food isolates.
6.7. Other Biotyping Methods
There are several other biotyping methods for characterization and identification of the
mterococcal species although less frequently used, besides the above-described
conventional biochemical phenotyping. or molecular phenotyping methods.
6.7.1. Srrotyping
The histop of serotyping dates back to 1933. when Lancefield reported that p-hemolytic
kcal streptoci~ciare typical for pnssesslng the group D antigen. With the advent of
no\,el techn~ques.the Polyphasic taxonomy has combined chemotaxonomic and genetic
techniques w ~ t hless emphasls on serolog~calcnteria for classification 1281. Not all the
strams produce a cell wall assoc~atedglycerol teichoic acid antigen-the streptococcal
group D antigen 112. 141. Moreover the detection rate banes depending on the extraction
procedure and the qual~tyof antisera used, while some strains react also with group Q
antisera 1891.
6.7.2. Anrimicrobial Susceptibility Testing
Enterc~occiare intrinsically
resistant
to a large number of antimicrobial agents. The
VanC phenotype (low-level resistance to vancomycin. susceptible to teicoplanin) is an
Inherent property of E g u l l i n u ~ mand E. ~ u . v s i ~ l ~ ( l uThis
~ ~ ~propeny
~v.
is not transferable
and is related to the p-nce
of species-specific genes vane-l and vanC-2 respectively.
which m a k s this propaty an excellent criterion for their species identification [12, 171.
The susceptibility or rcgistance 10 Efmtomycin-an elfamycin dnrg is another typical
attribute that was applied in the identification scheme of entemocci proposed by
Cawalho ct al, [94].
REVIEW OF LITERATURE
6.7.3. Bacteriocin Typing
The production of inhibitory substances that are effective agalnst closely related species
IS
an interesting phenomenon among certain bacterial groups. In 1963, Brock and his
colleagues were the first to describe the inhibitory substances produced by enterococci as
entermins [loo, 1011. Later some studies explored the ability of bacteriocin to type
enterococci from clinical specimens. Pompei and his colleagues exploited and evaluated
the potential taxonomic value of the bacteriolytic activity of enterococci. The detection of
enrerococcal lyo-groups was proved to be as reliable for species identification as
conventional methods. and the bacteriolytic pattern groups correlated well with species
groups phylogenetically determined [102]. But studies on bacteriocins from enterococci
in clinical setup are fbcused towards their role in bacterial pathogenicity although few in
number.
6.8. Molecular Genotyping Methods For Species Identification
Since last decade, there has k e n a rem men do us advancement in the field of clinical
inlcrobiology with advent of molecular techniques. Many novel genotypic techniques
~argetingthe nucleic acid material- DNA or RNA of the organism were used mostly for
laxonomic purposes in special laboratones in late 80's and early 90's. However since mid
90's. the application of molecular techn~ques for the identification of Enterococclrs
species has expanded dramatically, and a brief review of vanous methods potentially
adaptable for use in microbiology laboratones are summarized in the follow~ngsections.
6.8.1. SpecicsSpcciifir PCR Assays
Several variants of
PCR based nucleic acid amplification assays prevail for species
~dentificationof enterococci followed by clinical microbiology laboratories. A Multiplex
PCR assay was developed by Dutka-Malen et al. (151 to amplify internal portions of the
"ddl" and the vancomycin resistance (van) genes: vanA, vanB, vanCl and vanC21 3.
Since the vancomyin resistant genes "vanA" and "vanB" could be found in E. faecalis
RS
well E. faecium, the "ddl" genes for these two species were used for species
identifica~ion. v a g l and V a n c ~ 3arr specific for E, gallinarum and E. cassel~/7a1ws
REVIEW OF LITERA 7URE
respectively and thus identification of these genes, were equivalent to species
identification. Thus a total of six primer sets were used to identify four species of
clinically relevant enterococci. This multiplex PCR showed an excellent agreement with
other phenotypic identification methods for species identification. Subsequently, several
authors worldwide used this method for species identification of enterococci from
diflerent clinical specimens with minor modifications [61. 103. 1041. Kariyama et al.
[lo51 presented primers specific for E. jueca1i.s. which were modified based on the
information obtained from Dutka-Malen et al. (151. Enterococcus protein A (efaA) genes
specific for E. faeco1i.s and E. juecium were amplified by PCR and probed with
corresponding sequences in immunoblots without any cross-reactions with other
enterococcal species 11061. Amplification of the pEM1225 gene was repofled to be a
species-specific PCR assay for identification of E. .fuecrum using a nucleotide sequence
that is strongly conserved in this species as a target for the PCR. (1071. Species-specific
pninen for E. dttrun.\ and E, hirue targeting the "ddl" gene were also developed and
validated by PCR assays [108].
6.8.2. Mi.~cellaneousPCR Based assays
In a Randomly amplified polymorphic DNA (RAPD)-PCR (also called as Arbitrarily
primed (AP)-PCR) amplificat~onof random segments of genomlc DNA of enterococci
was d~rectedby using a single oligonucleotide pnmer of arbitrary sequence, which
d~splayedpolymorphism by the lengths of the amplified sequences obtained. However,
only few ditTerent primers were proved to be reasonably successful for species
icientificat~onof e n t m o c c i [109. IIO]. A Broad range (BR)-PCR assay was developed
lo
ampl~fya ponlon of 16s rRNA gcne. which pro\,ed to be species-specific only for six
species of enterococci (1091. Tyrrell and colleagues successfully identified eleven
enterococcel species by an Intergenic spacer (ITS)-PCR using primers to amplify rRNA
spacer regions. They performed gel electmphoresis analysis of the PCR products after
restriction digestion without sequencing the PCR product [I 1I]. Amplification and
sequencing of he Dala:D-ale ligases (ddl) genes was performed to identify all the
spaies tested except for E. mundtii and E ruJ7nusu.c [I 121. Squence analysis of the
small-subunil ribosom]
rjbonucleic acid-16S rRNA gene for identification of novel
REVIEW OF LITERATURE
Lnrerococcus species was performed alon'gwith conventional phenotyping and analysis of
whole-cell protein profiles [14, 311. However, none of these PCR based assays were
successful in identifying all enterococcal species.
6.8.3. Other Nucleic acid based mefhods
A commercially available non-radioactive DNA probe- Accuprobe system (Gen-Probe,
San Diego, USA) has been shown to be successful to verify the identification of majority
of enterococcal species of human origin by many clinical microbiology laboratories. This
non-radioactive DNA probe was based on a DNA oligomer having a structure
complementary to a segment of enterococcal rRNA 112, 14. 311. Even with the growing
range of molecular techniques available to identify, characterize and type enterococci, the
cho~ceof selecting the appropriate technique depends on the ~ndividual'sneed and their
setup.
7. GENETICS OF ENTEROCOCCI
The fr~quencyand spectrum of multidrug resistant enterococcal infections have Increased
ucirldwide dunng the last two decades This Increase has been attributed to a combination
of versatile genetic characteristics of enterncoccl, the selective pressure of antimicrobial
use. and envirnnmental changes that enhance the transmission of resistant organisms. We
will briefly review the unique and versatile genetic mechanisms present in enterococci in
the following secttons.
7.1. Gene Transfer Mccbanisms in Enterococcus
In entcmcocci primarily, acquirtd resistance to antibiotics occurs by horizontal transfer
of resistance genes located on various t y p s of mobile DNA elements (plasmids and
transpsons) and, secondarily
10 a
lesser extent by mutations on the bacterial genome.
The horizontal transfer of genetic elements involves the phenomenon of "conjugation"
driven by a unique genetic mechanism present only in enterococci. Although in-vitm
studies on eIcctrotrmsfomation o r E. ,/aat.o/is were tried (1 131, natural gene transfer
RE VIEW OF LITERATURE
mechanisms like transformation or transduction that are common in other gram-positive
"rganisms are not well established in enterococci. Hence, "conjugation" remains to be the
unique genetic mechanism present in enterococci for gene transfer.
7.2. Role of Mobile Genetic Elements
7.2.1 Plasmids
The extra chromosomal mobile genetic elements like plasmids play a major role in
transfening the genetic determinants encoding antibiotic resistance and virulence in
enterococci in a hospital setup. Although different classes of plasmids have been
described in enterococci. the conjugative (pheromone-responsive) plasmids. and the
broad-host range plasmids are of clinical significance. The presence of plasmid DNA in
S
fuaculis was first reported in 1972, subsequently Jacob and Hobbs [I 141 presented
cv~dencefor direct involvement of plasmid DNA in the conjugal transfer of multiple drug
resistance from S. fat~culrs. Thereafter, several conjugative or non-conjugative but
mohilimble plasmids In group-D-streptococci were discovered and characterized. Most
of the plasmids reporred encode determinants for resistance to antibiotics or UV light,
hemolysin and bacterioc~n.Some of the medically significant plasmids, which have been
characterized in detail. are plasmid pAMBl encoding eqzhromycin resistance [I IS].
plasmid pADl encoding hemolysin-bacteriocin, plasm~dpAD2 encoding erythromycin,
streptomycin and kanamycin resistance [I 161, plasmid pPDI, plasmid pCF-I0 encoding
tetracycline [ 6 ] ,plasmid pIP800 encoding gentamicin. chloramphenicol and kanamycin
resistance [I 171 and plasmid pAM373 [I 18).
The pAMBl is a conjugative plasmid that determines eqzhromycin resistance.
This resistance is representative of the so-called MLS phenotype (i.e., resistance to
macrolida, lincosamides, and streptogramin B). Plasmid pAMPl was originally
identified in S. fueculjs strain DS5 [I IS], which exhibited a broad host range with its
tmsferability into nine different species of streptococci, as well to Lactobacillus casei.
S t a p h y l o c o c ~ourcus,
~~~
and
Bacillus subtilis [ 6 ] .The salient feature of this p u p of
REVIEW OF LITERATURE
plasmids is that they exhibit transferability at relatively lower frequencies only on solid
surfaces such as filter matings, and not in broth matings.
The pheromone responsive plasmids like pADI, pPDI, pCFlO and pAM373 on
the other hand, transfers eficiently at a very high frequency (10'
-
per donor cell) in
hroth matings. These plasmids confer mating response to small sex pheromones secreted
by potential plasmid-free recipient cells. This mating signal induces synthesis of a surface
aggregation substance by the donor cell, which elicits formation of mating aggregates and
plasmid transfer between the donor and recipient cells (6, 1181. Recent studies reveal that
these pheromone responsive plasmids give rise to aggregation response when exposed to
sub-inhibitory concentrations of antibiotics used in treating enterococcal infections [I 191.
Apan from these two major types of plasmids. several other conjugative plasmids
have been reported from different laboratories and named accordingly. Of these, a
pl~eromone Independent plasmid pMGl encoding gentamicin resistance has been
described to exhibit transferability at a higher frequency during hroth matings from Japan
which ~ndicatesthere exists an alternate gene transfer system. which differs from the
pheromone- induced plasmid transfer system [120].
7.2.2. Transposons
Transposons are gene sequences that can move from one locat~onon the chromosome to
another, the genes required for this movement being contained within the Transposon
(Tn). All transposon classes have heen described in enterococci. of which the conjugative
transposons are exclusively found in E. faecalis predominating the other classes [6. 12 I].
Hodel-Christian and Murray identified a 4.7 kb composite transposon, designated Tn5281
In E. ,fuecalis which conferred HLGR by virtue of the 2 kb bi-functional gene
aac(6')+aph(2") flanked by 1.35 kb IS elements, designated IS256 [122]. Rice et al. [I231
reported a 26 kb composite transposon Tn5384 in E. ,foeculis. which conferred HLGR
and erylhromycin resistance. A simple transposon Tn1546 belonging to Tn3 family,
Possessing inverted repeats and encoding van-A type glycopeptide resistance was
described in E. ,ficcium. Later the same group described a composite transposon Tn1547,
RE VIEW OF LITERATURE
flanked by IS16 and IS256-like elements conferring vancomycin resistance in E. faecalis
[124].
While the former two classes of transposons jump between cells by the process
transposition, the conjugative transposons described predominantly in E. fueculis
doesn't jump, rather move between genomes through intercellular contact between
entemcocci. Some of the well-characterized conjugative transposons in enterococci are
the resistance transposons Tn916, Tn917 (61. Tn1546 like transposon [ I 251 and Tn1549
11261. Tn916 and Tn917 were identified originally in a clinical isolate of E, faecalis
DS16. ' h 9 16 is chromosome borne, conferring tetracycline resistance and has been
shown to insert into several sltes in different conjugative hemolysin plasmids like pADl
and pAMyl (61. while Tn IS49 encodes van-B type glycopeptide resistance in
E~lrc.rococc~rs
[ 1261.
7.3. Conjugative Gene Transfer Mechanisms in Enterococcus
The s~gnificanceof Enrcrococcus as an ascendant nosocomial pathogen lies in its
propensity lo acquire
resistance
to multiple antibiotics, used to treat enterococcal
~nfect~ons.
by a varie~yof conjugative mechanisms. The following sectlon briefly reviews
the different conjugative gene transfer mechanisms described in enterococci.
7.3.1. Pheromone-Inducible Plasmid Transfer in Enterococcus
The bacterial sex phemmone mediated conjugatton was described exclusively three
decades back only in the gmup-D-streptococci [127], as depicted in Figure 2. In a
generally accepted model of this system, plasmid free strains of E, faecalis typically
secrete into the culture medium a number of different small peptide sex pheromones
specific for different types of plasmids. When a potential donor cell containing a
pheromone-responsive plasmid (generally encoding antibiotic resistance) comes into
contact with its corresponding pheromone, transcription of a gene on the plasmid is
turned on, resulting in the synthesis of a sticky substance called aggregation substance
( 4 s ) on ils surface, which can pmmote attachment to recipients via a complementary
REVIEW OF LITERATURE
receptor called enterococcal binding substance (EBS). The close cell-to-cell contact
resulting from AS-EBS binding facilitated by random collision of cells, allows for
subsequent formation of some son of mating channels between the cells enabling the
pheromone-responsive plasmids to transfer from the donor to the recipient (mostly
plasmid free strains) bacterium. Since a cell-free filtrate of a recipient also elicits an
aggregation (clumping) response when it is mixed with donors, this substance has been
referred to as clumping-inducing agent (CIA). During in-~itroexperiments in test tubes,
clumps of cells actually fall to the bottom of the tube, resulting in a visible aggregate.
Once the recipient cell has acquired this particular plasmid, the synthesis of the
corresponding sex pheromone is shut off to prevent self-clumping. This system of
conjugation, which occurs primarily in E. faecalis, is highly efficient and results in
transfer of plasm~dsin both filter and broth matings [6. 17, 127, 1281.
Figure 2. Pheromone-responsive conjugative system of E. fueculi~.
Pheromone A released from the potential rec~pientcell (right) interacts with plasmid A in
the potential donor cell (len) to induce synthesis of aggregation substance. Attachment of
aggregation substance to binding substance causes the cells to clump into visible
aggregates. Once the pheromone-responsive plasmid A has transferred from donor to
m i p ~ e ncell.
t synthesis of pheromone A is shut off.
Figure reproduced with kind permission of Dr. B.E. Murray, MD.,
University of Texas Houston-Medical School, Houston. Texas. U.S.A [I 71
REVIEW OF LITERATURE
Several studies worldwide have shown that clumping response was exhibited
predominantly by the antibiotic resistant E. faecalis from hospitalized patients possessing
pheromone responsive plasmids, and proved that these plasmids encoding antibiotic
resistance transferred between the experimental strains in-virro 1129-1351. Further these
pheromone
responsive
enterococci
producing
hemolysin-bacteriociniaggregation
substance have been shown to enhance the pathogenicity of enterococci [136, 1371.
7.3.2. Pheromone Independent Plasmid Transfer
AS
reviewed previously, some broad host-range plasmids can transfer between species of
enterococci and other gram-positive organisms such as streptococci and staphylococci.
The coexisting pheromone responsive plasmids can greatly increase the transfer
frequency of these plasmlds even though these plasmids transfer at a lesser transfer
frequency on sol~dsurfaces like filter matings (1381. Recently, Ike and his colleagues
have described an alternate conjugative plasmid transfer system, which was "pheromone
independent" but efliciently transferred the antibiotic resistant plasmid pMGl during
hroth matlngs [120, 125. 1391.
7.3.3. Transposon mediated Conjugation (Conjugative Transposons)
Several conjugative transposons reported to be prevalent in enterococci play a major role
In dissemination of antibiotic resistance genes to many different species. Studies confirm
that such transposons may have evolved from a Tn916 ancestor; the first reported
conjugative transposon in enterococci 1122, 1251. Several transposons encode
vancomycin and gentamic~nresistance in nosocomial enterococci, thereby enhancing
global dissemination of these resistant traits. All these studies imply that the genetics of
cntertxoccl is much complex and versatile than perceived currently, and underscores
their significance and impact in healthcare settings.
REVIEW OF LITERATURE
8 . ENTEROCOCCAL INFECTIONS
g , ~ Types
.
and their Clinical Significnnce
~nterococci lead commensalism in the gastrointestinal tract of humans and several
mammals. The transfonnation of this commensal into an opportunistic pathogen in health
care settings remains enigmatic to a larger extent. Even though enterocwcal
pathogenicity has been addressed a century back still several aspects of this nosocomial
pathogen remains unraveled. Their intrinsic resistance to multiple antibiotics, and unique
genetic mechanisms allows them to survive and proliferate in patients receiving
antimicrobial chemotherapy. This accounts for their ability to cause superinfections in
patients receiving a number of broad-spectrum antimicrobial agents [140]. Although E.
/uc~culi.r and
E.
fuecitrrn account for more than 95% clinical infections [13]. other
enterncoccal species are repnrted to cause clinical infections rarely [16. 1411. Recent
studies show that enterococci are the third most common pathogen isolated from
blondstream infections, the single most frequently reported type of pathogen in surgicalsite infections in intensive care units. and the second most common nosocomial pathogen
In the US [20] underscoring their clinical significance. The imbalance within the host
system (human body) disrupts the commensalism and makes it an opportunistic pathogen.
Funher, several predisposing factors along with the virulence factors, facilitates
successful evasion of the host defense leading to an array of clinical infections by
en~erncocci[26] Although the aspects of multidrug resistance and virulence needs to be
addressed concomitantly to elaborate their role in enterocwcal infections, this section
rrviews the array of infections caused by enterococci.
8.I.I. Urinary noo infedions
Urinary tract infections (UTI) a n the most common of all infections caused by
entemocci panicularly in hospitalized patients. It continues to be the second most
common nosocomial uropathogen accounting to an average 12-15% of nosocomial UTI
nexl only to E. cnli since two decades, as depicted by several studies conducted
worldwide [13. 19, 38-40, 1421. Although E. colr has been topplng the list since decades,
REVIEW OF LITERATURE
the frequency of it's isolation has declined over time from 35.6% in 1996, to 32.5% in
1998 and thereafter to 26.6% in 2001, on the other hand enterococci were isolated at an
increasing frequency over the years from 11.8% in 1996, to 15.3% in 1998, and thereafter
22.0% in 2001 which authenticates it's emergence as a significant nosocomial
uropathogen [ 1431.
The increased prevalence of nosocomial enterococcal UTI is probably the result
of many disposing factors like increasing use of catheterization and broad-spectrum
antibiotics mostly among patients of older age group as shown by several studies [38. 39,
42, 1441, but lesser prevalent (< 5%) among younger age group where catheter related
infections accounted for upto 48% of all the nosocomial UTI and secondaly bacteremia
and cerebral palsy occurring rarely [41, 421. The crude mortality rate due to nosocomial
(;TI remains to be approximately 18% where enterococci play a major role [19]. Further,
reviews show that enterococci can cause prostatitis and perinephric abscess in addition to
uncomplicated cystitis or pyelonephntis 113. 681, Some studies had shown that some
\
~mlcncefactors like "enterococcal surface protein-esp" increase the pathogenicity of the
urinary enterococcal lsolates thereby altering the outcome of the infection [145].
Apart fmm independent laboratories, several official bodies (SENTRY, SCOPE)
across the globe conduct multicenter antimicrobial Surveillance Programs to assess the
change in the bacterial profile and pattern of antibiotic resistance of nosocomial urinary
tract infections, which leads to more effective prescribing practices for entemcoccal UTI.
119, 38-40, 1431. Results of most of these studies show that ciprofloxacin and
nttrofurantoin remains to be ideal antibiotics for treating enterococcal UT1, although
some studies raise a concern on this option 11461. Fraimow et al. [I471 had proven that
some E, faecalis isolates requires vancomycin for growth, a phenomenon which could
have evolved due continuous exposure to high concentrations of vancomycin in the urine,
that narrows down the treatment options in case of multidrug resistance by enterococci.
Thus by and large cnterococci continues to play a a major role in the nosocomial UTI.
REVIEW OF LITERATURE
8.1.2. Bacterernb
The bacteremia exists in two fonns in hospitalized patients: enterococcal bacteremia
without endocarditis, and bacteremia with endocarditis. The management and mortality
of
either form of enterococcal bacteremia to a larger extent depends on the antimicrobial
susceptibility profile of the isolate, apart from other predisposingirisk [actors. Several
studies over these years elucidates that enterococci remains one of the top threenosocomial bloodstream pathogen till date since a decade [20, 21, 371. Although E.
/uc~culisremains the predominant species causing enterococcal bacteremia followed by E.
/accium, other species like E. orium. E. cusseliflu~~us,
E. durons, E. gullinorum and E.
ruffinoslls have been reponed to cause bacteremia although very rarely [90, 141, 148-
IS I ] Only 2% cases of enterococcal bacteremia are associated with endocarditis, which
are mostly community acquired [68]. However. few studies depicts that 8-32% of
enterococcal bacteremia associated with endocarditis were predominantly community
acqu~red,but their case-selection criteria were totally different [23, 241.
Although high-level gentamicin resistant bacteremia has been described since two
decades, several studies depict the emergence of vancomycin resistant enterococcal
bacteremia among imrnunocompromised hosp~talized patients since last decade in U.S
and several European countries [I 8.20. 1.521. But the frequency of vancomycin resistant
enterococci causing systemic infections in developing countries like India is not very
high and alarming, as in west [153. 1541 A prolonged hospital stay by patients with
severe underlying diseases and life threatening conditions remains to be the most
common riskipredisposing factor associated with bacteremia as depicted by several
studies. The diseases and conditions generally include cancer. diabetes mellitus, chronic
renal failure, major trauma, surgical procedures and urinary and vascular catheterization
(22-24. 150. 155- 1571. While patients who had previously received broad-spectrum
cephalosporins, gcntamicin/vanwmycin are at greater risk of acquiring enterococcal
bacteremia [36. 44. 47, 155. 158, 1591. Mostly enterococcal bacteremia tends to be
polymicrobial isolated along with staphylococci and enteric gram-negative bacilli. The
infection by enterococci mainly occurs in the form of urinary tract infection and
REVIEW OF LITERATURE
secondary bactmemia mostly related to abdominal and soft tissue infections, with E.
/aL.ca/isas a predominant species in all foci [22,47].
Mortality associated with enterococcal bacteremia remains high, since it occurs
p e r a l l y in debilitated patients. However, it is hard to define the exact role of bacteremia
in the fatal outcome of these patients, as several other complicating factors too are
accountable in these cases. Several studies worldwide show that mortality ranges upto
50% in most cases, although there are exceptions with antimicrobial resistance and
virulence determinants of enterococci playing a sipificant role in the outcome (152,
1601. In a comparative study mortality rates of 38.3% and 30.5% were shown for patients
w ~ t henterococcal bacteremia due to strains with and without high-level resistance to
gentamicin respectively [149]. Another study depicted that 34% patients with serious
underlying illness like bums, malignancy or granulocytopenia, diabetes and renal failure
died with enterococcal bacteremia [23], while Malone et al. 1241 showed a mortality rate
of 44% and found that male sex and a fatal underlying disease were significantly
associated with increased mortality. Huycke et al. (1611 showed that patients with
hemolytic, high-level gentamicin-resistant E. farcu1r.s bacteremia showed a five fold
Increased ( 3 1%) mortality rate within 3 weeks of bacteremla compared with patients with
non-hcmoly~c,gentamicin-susceptible strains in a cohort study. Thus the mortality rates
on a whole depend on the cohort group studied. apart from other influencing factors.
8.1.3. Endocarditis
Infective endocarditis remalns to be the most senous of all infections caused by
enterococci with a higher monality rate. Wtcrococctr.~remains third most common cause
and account for approximately 5-20% of all cases in infective endocarditis, but may vary
accordingly [13. 162-1641, Several studies have documented E. faecalis as the
predominant species causing enterococcal endocarditis, although other species too have
been reponed to cause this disease. Among isolates sent to the Saeptococcus laboratory
at the Centers for Disease Control, Atlanta, endocarditis was the diagnosis given for
Patients h r n whom E, avium. E. cosseli/lo~us. E, durans. E. gallinamm, and E.
ra/linmus w e n isolated, apart from E. ,foecolis and
E. ,faecium [go]. Although most cases
REVIEW OF LITERATURE
are predominantly community acquired, recent studies show a steep increase in the
percentage of enterococcal endocarditis acquifed in a hospital setup [165-1671.
While several factors have been depicted as predisposing factors for enterococcal
endocarditis, the male patients of older age with prior urinary tract infection or urinary
instrumentation and bacteremia are more prone to this disease when compared with their
female counterpart. as reviewed [13, 1621. Several virulence determinants and
antimicrobial (gentamicin) resistance in enterococci were shown to predict the outcome
of this disease by altering the mortality rate [129, 165, 1671. Hence early diagnosis of
enterococcal endocarditis is very imponant to initiate appropriate antimicrobial therapy
depending on the susceptibility profile of the isolate based on the blood culture report.
8.1.4. Neonutul infections
The emergence of enterococci among the neonates as a significant cause of nosocomial
infections has been depicted by many studies since last decade. The results of a recent
point prevalence survey of nosocomial lnfect~onsin 29 Pediatric Prevention Network
NlCll conducted by CDC in U.S. has shown that enterococci ranks second only to
coagulase-negat~ve staphylococci as the leading cause of nosocomial infections of
nennates in ICU [168]. On the other hand, the emergence of vancomycin resistant
entcrcxocci (VRE) In neonatal infections as shown by other recent studies poses a
lherapeutic challenge and emphasizes the urgency for more effective prevention
~nterventions[ 169-171 1.
Sepsis remains to be most commonly reported from neonates, with focal
infections including meningitis. scalp abscess and pneumonia in this group [169-1781.
The most common predisposing factors for neonatal enterococcal infections as reported
by recent studies are nosocomial acquisition due to prolonged hospital stay, long-term
antibiotic therapy, low birth weight, pre-term birth, central intra-vascular catheters,
prolonged ventilation and patients receiving total parenteral nutrition [172, 174, 1751.
Few reports show that cntcrococci causes other neonatal infections like urinary tract
infection accounting to 15% [4]], and conjunctivitis and peritonitis although less
REVIEW OF LITERATURE
frequently [13]. While the mortality rates of neonatal enterococcal infections, especially
those of sepsis ranges on an average from' 8-18% with higher rates for necrotizing
enterocolitis-associated infection [172, 1731. While appropriate infection control
measures have shown to reduce the incidence of neonatal enterowccal colonization and
infections from 67% to 7%. which emphasizes the impact of good infection control
p c t i c e [l71]. Most of the neonatal enterococcal infections especially those of sepsis,
respond well to appropriate antimicrobial therapy according to the susceptibility pattern
of the isolate.
8.1.5. Intra-iabdominal and pelvic infections
The clinical significance of enterococci in cases of bacteremia and super-infections in
selected patient populations has been well established. But, being a resident flora of the
GI tract in humans. their role as primary pathogens in polymicrobial intra-abdominal and
pelv~cinfections remains controversial. Although the clinical significance of enterococci
In intra-abdominal and pelvic infections remains murky, several reports depict their role
In peritonitis. intra-abdominal or pelvic abscess, surgical site infection, suppurative
thrombophlebitis. acute salpingit~s. and endometntis [179-1841. Although surgical
drainage remains the cornerstone of therapy for enterococcal infections involving a
d~scretefocus, in the circumstances typified by the compromised surgical patient, specific
antibacterial therapy directed against enterococci based on their susceptibility profile
clearly reduces the mortality rate [179, 18 I].
8.1.6. Skin and sop tissue infections
Enterococc~ rank the second most common gram-positive pathogen, and third most
common isolate associated with skin and soft tissue infections (SSTI) in hospitalized
patients next only to S. uureuv and E. coli [185-1871. These studies conclude that
enterococci account for approximalely 8-1 0% of SSTls in hospitalized patients, although
there are variations in different geographical areas. Like in intra-abdominal and pelvic
infections, the role of cnterococci remains controversial in SSTIs since they are
frequently isolated Fmm mixed cultures with staphylococci and gram-negative bacilli in
suwcal and bum wound infections, decubitus ulcers, and diabetic foot infections. An
REVIEW OF LITERA 77JRE
e n t e r ~ ~bacteremia
~ ~ a l accompanying any of these infections authenticates the clinical
,ignificance of enterococci in SSTls [13. 22,' 1861. While multidmg resistance among
e n t e r o c ~ ~varies
~ i geographically, vancomycin-resistant enterococci among SSTls are
uncommon outside the USA (17.5%) and Italy (7.4%) and the susceptibility profile of
enteroco~~i
need to be considered when selecting therapy for treating enterococcal SSTls,
since empirical therapeutic options like broad spectrum cephalosporins are ineffective
against enterococci [I 851.
8.1.7. Miscellaneous Infections
The involvement of enterococci in other infections appears although exceedingly rarely.
Enterc~occalmeningitis remains to be more common in neonates than adults [173, 1781.
Although rarely reported it generally seems to be related to an underlying disorder which
rends to be a complication of bacteremia in patients with endocarditis, or severe
~mmunodeficiencysyndromes [13. 1771. The reports of respiratory tract infections like
pneumonia and lung abscess shows that it appears In hospitalized patients who were
severely debilitated and ventilated, rece~vedprolonged antimicrobial therapy coupled
w~thenteric feedlng [ 173). Enterncocc~were rarely reported to cause other infections like
otitis, osteomyelitis, and septic arthritis although their role tn these infections remains to
he murky [188, 1891. While most of the cases are treatable with appropriate antibiotic
therapy. the emergence of multidmg resistant enterococci has narrowed down the
treatment options.
8.2. Management of Enterococcal Infections
Enterncocci are intrinsically resistant to many antimicmbial agents and readily acquire
additional resistances, which have propelled them to become a prominent nosocomial
pathogen [I 1, 131. Although the treatment of enterococcal infections depends on several
factors, the major factor to be considered is the antimicmbial susceptibility panem of the
isolate for choosing optimal therapeutic regimens for the treatment of infections caused
by muttidrug resistant strains. The standard treatment since several decades, for serious
infections like mtcrococcal endocarditis and meningitis. tends to be a bactericidal
REVIEW OF LITERATURE
antibiotic
therapy,
which
includes
a
cell-wall
active
agent
(penicillidvancomycin) and an aminoglycoside (gentamicid streptomycin) if the isolate
is susceptible to both the antibiotics, while bacteriostatic agents are sufficient to treat
most other enterococcal infections. Treatment of infections caused by strains resistant to
beta-lactams, glycopeptides and aminoglycosides has become problematic due to the
l~mited number of therapeutic options. No medical therapy is reliably effective for
endocarditis caused by strains resistant to all cell wall-active antibiotics and all
aminoglycosides [I 1. 13, 140, 1901. Although recently introduced novel antimicrobial
agents such as linezolid and quinupristiddalfopristin are effective in managing multidrug
resistant (including vancomycin) enterococci, their activity that is mainly bacteriostatic
raises a concern at times [191].
Ciprofloxacin and norfloxacin have been used successfully for enterococcal UTI,
but the organism's susceptibility is marginal for treating systemic infections with this
drug [ I I . 13. 140. 1901. Most of the serious infections, caused by VRE were successfully
treated with newer antimicrobials like linezolid and quinupristinidalfopristin, as
monotherapy, or In combination with other antimicrobials [57, 1911. While Murray 1571
has proposed a schema (Figure 3). depicting the possible antimicrobial-drug regimens for
thc treatment of clinically important ~nfectionscaused by enterococci resistant to both
ampicillin and vancomycln. which was based on a review of the literature relating to
VRE, personal observations and current clinical practice.
The colonization of the intestinal tract with VRE may predispose patients to
infections by this organism and may contribute lo its nosocomial spread. Thus anempts to
eradicate VRE from colonized patients were tried with different oral antimicrobial
regimens,
~ncludingbacitracin, novobiocin and a novel antimicrobial agent ramoplanin
that have has shown partial success [192]. In addition to systemic antibacterial therapy
and decolonization efforts, adequate debridement of devitalized tissues and drainage of
abscesses and fluid collections remains to be an integral p a l of appropriate management
of enterococcal infections [I 791.Thus, this enigmatic pathogen presents a stiff
therapeutic challenge ahead for health care professionals.
REVIEW OF WTERA TURE
Figure 3. Antimicrobial-drug regimens for the treatment of drug resistant
entnococcal infections [resistant to both Arnp~cill~n
and Vancomycin]
MC d m m p r l l n
M lb f d l a n n g
.Olnupnmln d m l b p n a n
.l*ld.r Invr*).lan
bnad'd
u e t n t m c n qumupmmd . l b p n l l n rnpnlln
bpmwcnLYplaB
H g h do" amprclln nr
.
W b n .r.W IW+n
p s n e m n n or smparyan
c
m l w l m r o l l l r Idk*n,,(
11 .ndOC-d~ln
.r\rnp~#l!n ~ n ~ p m m
,,I t u ~ h g h h, w a n .
y.nl.mnn
u .lnptomvn
l u d y rrelght h sv-b
uothbh, k be rurrtub re!*lurrd!1t+
.C-r.*a*
an tmslol,otu~
.o.~L +I
.Cons&,
rabb l p l r r m r n t
Flow-chart reproduced with kind permission of Dr. B.E. Murray. MD.,
University of Texas Houston-Medical School. Houston. Texas, U.S.A [57]
9. PATHOGENESIS AND VIRULENCE OF ENTEROCOCCI
9.1. Pathogenesis of Enterococci
Antimicrobial resistance has been cited as one of the major reasons for evolution of
entmocci as a prominent nosocomial pathogen, however there are evidences for
entemcoccal infections a century back even before the introduction of antibiotics [13].
This raises a concern about the versatility of entemocci to cause infections even without
antimicrobial resistance. The pathogenicity of enterococci was addressed a century back-
REVIEW OF LITERATLIRE
in 1899, by MacCallum and Hastings, who isolated an organism from a case of acute
endocarditis, and designated it Micrococcus' zymogenes based on its fermentative
properties. It was also found to be lethal when injected intraperitoneally in white mice,
and capable of producing endocarditis in a canine model. In subsequent years, studies and
reports had shown that this "fecal streptococci" was responsible for infections like
endocarditis, UTI, and wound infections [13,26].
The difficulty in treating enterococcal infections has driven several researchers to
bcus their efforts in studying the factors that undermine the commensalistic relationship,
since these factors in one-way or other contributes to the establishment of the organism in
a particular ecological niche. Infection occurs once when the host-enterococci
equilibrium is disrupted aided by various factors. In order to infect, enterococci must first
be able to colonize, primarily by adhering to the host mucosal surfaces with the help of
adhesins and surface carbohydrates. After adherenceicolonization the organism must then
evade the host clearance to cause any infection and ultimately produces pathologic
changes in the host with the help of an array of (virulence) factors [26. 1931. While most
ol'the colonizers or the commensals. do not posses all the (virulence) factors needed for
successful evasion and subsequent infection process, those that posses emerge to become
a successful nosocomial pathogen facilitated by other predisposing factors. Our review
elaborates the incidence and role of various abovementioned virulence factors in the
pathogenesis of enterococcal ~nfectionsin the following section.
9.2. Virulence Factors of Enterococci
Although the pathogenicity of enterococci was described a century back, the significance
of' enterococcal virulence in several cases is still debated. Unlike other gram-positive
pathogens like S. aureus and S~reprococcusspecies, enterococci do not posses classic
virulence fac~ors.But their predominant role in nosocomial infections with their intrinsic
and a q u i d antimicrobial resistance had provoked researchers to study the role of
possible virulence factors in the pathogenicity of enterococci. A brief review of the
REVIEW OF LITERATURE
virulence factors proposed and studied in enterococci pertinent to human
infections, are elaborated in the following sections.
-
9.2. I. Aggregation Substance AS
Adhesive properties act as virulence factors in the pathogenesis of enterococcal infections
like UTI and endocarditis. Studies suggest that UTI strains showing the highest invasion
and adhesive potential invade the kidneys, cause bacteremia. and, after having expressed
the serum-dependent surface modification, colonize the heart [194]. Aggregation
substance (AS) remains to be the most well studied adhesin of enterococci. AS is a
pheromone-inducible surface protein that mediates binding of donor cells to plasmid free
recipients. and is essential for high-efficiency conjugation of sex pheromone plasmids
and also acts as a virulence factor during host infection. Several different functions have
been attributed to AS in addition to bacterial cell aggregation as reviewed previously 161.
The other major function exhibited by AS, is adherence of the E, fueculir isolates to host
trssues. In vitm studies have shown that AS mediates adhesion to various eukaryotic cell
surfaces, such as cultured pig renal tubular cells. and promotes internalization by cultured
human intestinal cells, suggesting that AS-expressing cells may likely form larger
aggregates in vivo than cells not expressing this trait [195-1981. While Hirt et al. [I371
showed that AS contributes adhesion to fibrin and increased cell surface hydrophobicity,
which may induce localization of cholesterol to phagosomes and prevent or delay fusion
with lysosomal vesicles. Furthermore, AS exhibits resistance to killing by
polyrnorphonuclear leukocytes and macrophages, thereby promoting intracellular
survival of E. ,faecalis inside neutrophils [198, 1991. AS was also studied in the rabbit
model of E. Jaeculis endocarditis and found to be associated with greater vegetation size
compared to vegetations caused by isogenic AS-defective strains, although these
infections were not observed to be lethal [200]. The same study has shown that most
cytolytic strains of E. ,faecalis also express AS, by which both the virulence factors may
well work synergistically in the host system [ZOO]. Jea et al. [I361 have shown that E.
fuecalis strains bearing AS were pathogenic in endophthalmitis models, while studies by
lsenmann
a al. 1201, 2021 have shown that interaction of fibronectin and aggregation
substance promotes adherence of E. (aecalis to human colon.
RE VIEW OF LITERATURE
Several researchers in different geographical locations worldwide have studietl
the incidence of the virulence factor "AS" among clin~calas well non-cli~~ical
isolates to
prove this factor contributes substantially to the increased pathogenicity of enterococcl
[25. 129, 203. 2041 Most of these studies revealed that AS was prcscnt or~lvamong 1
firL,~,trlis
and the prevalence varied between 20-60°/u based on the geopraplncal location a i
well the type of infections from which cnterowcci were isolated tlowcver the role of AS
in adhesion and virulence of enterococci remains debatable since a recenl study h~
Johnson el al [205] has shown that AS and binding substance are not inalor contributors
to urinary tract colonization in a mouse model ol'ascending uliobstr~lcted(!TI
-
9.2.2. Enterococcal Surface Protein Esp
A recently identified cell wall-associated protein of E. fuecolis called Enterococcal
surl'ace protein-Esp has been shown to act as an adhesin like aggregation substance,
which revcaled a significant enrichment in infection-derived E. foecalrs isolates. Funher
s~ructurnlanalysis of the gene revealed that Esp exhibited global organizational similarity
to the Rib and C alpha proteins of group B streptococci [206]. In their study Shankar et
al 12061 did not identify the Esp gene in any of non-E. fu~colisspecies. But later studies
showed that a subpopulation of epidemic vancomycin-resistant E. ,faecium isolates
contained a varlant of the Esp gene that was absent in all non-epidemic and animal
~sc~lates
12071 Following their study Esp variants, were reported in E, forcium clinical
~solatesfrom different parts of the world [208, 2091. Subsequently Shankar et al. 11451
using an animal model of ascending urinary tract infection showed, while Esp does not
influence histopathological changes associated with acute urinary tract infections, but
contributes to colonization and persistence of E, fuecolis at this site.
Later Esp was shown to be highly associated with the ability to form a biofilm at
abiotic surfaces [210]. The Esp induced biofilms have increased antimicrobial resistance
and are important in infections involving catheters [21 I]. But the result of a recent study
contradicts the earlier hypothesis. The authors demonstrated that in v i m biofilm
formation occurs, not only in the absence of Esp, but also in the absence of the entire
pathogenicity island that harbors the Esp coding sequence, and concluded that E. faecalis
REVIEW OF LITERATURE
forms complex biofilms by a process that is sensitive to environmental conditions and
does not require the Esp surface protein [212]: Another study showed that endocarditis
isolates of E. fuecali.~ produced biofilm significantly more often than non-endocarditis
isolates. Furthermore, their results showed that Esp was not required, but its presence was
associated with higher amounts of biofilm (2131.
Waar et al. [21 I] hqpothesized that Esp might be associated with colonization and
spread, because it was more frequently isolated from feces of healthy volunteers and
transplant patients, a fact, which was supported since an epidemic Esp gene-positive E.
/ueculi.s
strain was found among liver transplant patients. Shankar et al. [214] showed
that Esp along with another virulence factor cytolysin was a pan of a pathogenicity island
in E. /ueculis. Recently, Leavis et al. [215] had described a novel putative enterococcal
pathogenicity island in E. faccium for the first time, which was shown to be linked to the
variant Esp virulence gene of E. faetium and associated with epidemicity. This was
completely different from the Esp-containing pathogenicity island previously disclosed in
I: fuc~c.ulrs In another study, infection-derived E /uccium strains enriched with Esp had
~ncreasedability to adhere to Caco-2 cells and were less genet~callydiverse than Espnegative isolates. But the authors indicated that additional factors are of importance for
adhesion, since Esp-negative E. ,fuccium fecal isolates from healthy individuals adhered
significantly better than Esp-negative infection isolates [216].
Se\eral studies have shown that the "Esp" vilulencc Ihctor was pre\aIzii~arnoi;i_
?0-70% of enterococci and tllc prevalence rate varied accordilly to the geo@rapllic;~l
location, as well other contributing bctors, wlllle the prevalellce was pltdominant in I:
,htc.c.ulrs than in 1.. ,j~tec.rrrrnisolates I157. 2 17, 2 I X] Most of tlic L'SI) positive strains were
resistant to one or more antibiotics [2 11). 2201 Samc studies have shcjw11that Esp gclie
plays a major role in dissem~nation o f vancnmyci~l-resistant I:.
predominant epidemic strains harbored the Esp penc. whilc mc~stot'
f ~ r ~ - c ' r r l l r l .sitice
11o1l-c01dcrn1c
strains were Esp negative (220-2221. A recerit study bv Oancea et al (?2.;1.demonstratetl
in vitro conjugative transfer of the Esp gene among I*,: ,~wc~itmr
and 1,'. ,/irc,c.iilr.cfrom
clinical samples reemphasizing the ~i~nificattce
of Esp in nosocorninl settings
RE VIEW OF LITERATURE
9.2.3. Adhesin of Collagenfrom E. foecalis -Ace
~ce-adhesin is a 74kDa protein identified. in E. jaecalis, which is significantly
homologo~sto the collagen binding protein- Cna, of S. aureus [224]. Subsequent
characterization of the Ace adhesin revealed, that apart from mediating attachment to
~xtracellularmatrix (ECM) collagen proteins, the Ace proteins were produced during
e n t e r o c ~ ~ ~infections
al
and was confirmed since anti-Ace antibodies were detected in
four different molecular sizes in Western blots [225]. Recently researchers from the same
laboratory had identified a new collagen-binding adhesin of E. ,faecium- Acm, that
showed 62% similarity to the S. aureus collagen adhesin-Cna, than to the E. ,faecalis
collagen-binding adhesin-Ace. Their results demonstrate that Acm, which encodes a
potential virulence factor, is functional only in certain infection-derived clinical isolates
of E, fuecium, and suggest that Acm is the primary adhesin responsible for the ability of
E, fuc~ciumto bind collagen [226]. Even though Ace adhesin have proven role in adhesion
of ECM, further multicenter studies are needed to unravel their exact role in enterococcal
~nf'cctions.
9.2.4. Cyiolysin/Bacteriocin
After the initial adherence to host tissues, enterococci invade and cause systemic
lnfect~onsand modulates the host inflammatory responses with the help of potentially
tox~csecreted products causing direct tissue damage. The major factors well studied to
enact this role. are the secreted factors cytolysin and the gelatinase (zinc
metalloprotease), which contributes to the seventy of enterococczl infections [26, 227,
2281. The cytolysin expressed by some strains of E. ./a~colisis a unique bacterial toxin
that is distantly related to ]antibiotic bacteriocins, a family of small, post-translationally
modified antimicrobial peptides. In addition to toxin activities, the cytolysin of E.
lacca1i.s possesses bacteriocin activity against a broad range of gram-positive bacteria.
which may provide a selective advantage for E. faeculis strains expressing this trait. The
operon encoding the cytolysin and the bacteriocin, are either encoded on a plasmid, or
integrated into the chromosome [26, 2291.
REVIEW OF LITERATURE
Although the significance of enterococcal cytolysin remains high in a clinical
setup, it was the bacteriocin production by enterococci reported half century back [loo],
[hat triggered researchers to probe into the molecular basis of this property. The study by
rock et al. [ l o l l depicted that 50% of the E. fueculis strains expressed a bacteriocin
activity against gram-positive bacteria, but not gram-negative bacteria. They also
demonstrated that the cytolytic and bacteriolytic activities were simultaneously lost by
some E. ,/oeculis strains after exposure to UV irradiation, but both the properties were
simultaneously regained upon reversion and concluded that a single enterococcal product
is responsible for cytolytic and bacteriolytic activities [100, 101]. Bacteriocins although
lnlt~ally explored for their antagonistic effects in bacteriotherapy, attracted much
attention in subsequent years because of their potential use as food preservatives. Several
studies were carried on food and other non-clinical isolates of enterococci, to study the
slgn~ficanceof bacteriocin production and to assess their biotechnological potential as
bod and feed additives, and were found to possess the same [25.204.230].
On the other hand, the studies carried out in clinical settings focused more on the
siyl~ficancc of cytolysin property, while only few studies explored the role of
hacteriocins exclusively among clinical isolates of enterococci. An initial study had
shown that there was a relation between bacteriocin production and virulence of E.
luccu1i.c.
which was later confirmed by many other studies [231]. Galvez et al. [232]
round that among 90 enterococcl strains of human origin, 36 strains produced
hacteriocins. Later Libenin et al. [233] screened the clinical isoiates for bacteriocin
production, and concluded that hemolysinhacteriocin produced by enterococci could be
considered as a marker of pathogenicity. Del Campo et al. [I041 had shown that
hacteriocin was more among vancomycin-resistant enterococci, than vancomycinsusceptible isolates of diflerent origins. The preliminary results of our study showed that
screening of enterococcal isolates for bacteriocin production, may be useful to elucidate
the virulence of nosocomial enterococci, since the property confers an ecological
advantage for the producer strain [234].
REVIEW OF LITERA 77JRE
Unlike bacteriocins, the role of cytolysin in the pathogenesis of enterococcal
infections has been well established and several studies have shown that the pADIencoded cytolysin contributed to virulence in E. faecalis [26, 200, 2281. Even before the
of the cytolysin the vimlence of enterococci was studied in a mice model
as hemolysin, which depicted their pathogenicity. The same authors
showed a high incidence of hemolysin production by E. .farcalis strains associated with
human parenteral infections [US]. Diagnostically, this toxin causes a beta-hemolytic
reaction on human and horse blood agar, but does not hemolyze sheep blood agar, which
IS
frequently used in clinical microbiology laboratories. Various studies on enterococci
isolated from patients with different infections showed that cytolysinihemolysinl
bacteriocin occurred at a frequency of 10-60% 1129, 152, 160, 161, 235, 2361. Most of
~hesestudies emphasized that presence of these virulence traits played a role in the
pathogenicity of entemocci.
9.2.5. GelarinasdProtease
(ielatinases are proteases of enterococci, which were recognized a century back when the
proteolytic and the hemolytic activities were used to divide the Strcprococcus furculis
into subspecies. The proteolytic isolates were designated as S. J u e c u l ~var.
~ liqu<fuciens,
and hemolytic isolates as S. furculis var. hemo!vric,us, while the isolates exhibiting both
proteolytic and hemolytic properties as S,/ueculii var. zyrnoycnes. But all these isolates
were later grouped under the same species as E. ,fuecalir based on genetic evidences [13].
The protease production could be detected rapidly by using 3% gelatin or 1.5% skim milk
agar medium. The virulence of enterococcal gelatinase has been proven in animal models
1237, 2381, which was authenticated further by several studies on enterococcal gelatinase
from human infections [129, 1521. Several studies have shown that 45-60% of the E.
/ui~culisisolates fmm human infections produced gelatinase, whereas none of the non- E.
/oc,c,ulis isolates did. But most of these stud~esdid not address whether gelatinase affects
the severity of disease, as it does in animal models [160, 217.239-2411.
REVIEW OF LITERATURE
9.2.6, Miscellaneous Factors
Few other factors have been shown to contribute to the pathogenicity of enterococci.
E
,fuecolis antigen
- A (EfaA) contributes to the host tissue adhesion as well for species
identification of E. ,faeca/is isolates [106]. A capsular polysaccharide antigen (cps) from
clinical isolates of E. .faecolis and E. .foeciurn was shown to contribute to the pathogenesis
of enteroco~ci by enabling them to survive the phagocytosis within the host system.
Further, antibodies to this capsular polysaccharide (cps) were shown to have prophylactic
and therapeutic efficacy 12421. Other studies show that extracellular superoxide,
lipoteichoic acids, and few other cell wall components contribute to the pathogenicity of
enterococcal infections by modulating the host immunity [243, 2441. While all these
studies emphasize the significance of these virulence factors in enterococcal infections,
the possibility to explore these newer therapeutic targets would be a promising area to
look upon, since enterococci has become tough to crack due to their ruggedness to all
available antimicrobials currently.
10. ANTlMlCROBlAL RESISTANCE IN ENTEROCOCCI
The property of antimicrobial resistance has complemented the emergence of enterococci
as a predominant nosocomial pathogen since last two decades. The rapidity with which
enterococci exhibit resistance even to the recently introduced novel classes of antibiotics:
linezolid and quinupristinidalfopristin. along with glycopeptide resistance. has become a
major issue of serious concern. especially in nosocomial settings [17. 57. 2451. The
antimicrobial resistance in enterococci can be classified into two types:
I ) InherenUlntrinsic resistance and.
2) Acquired resistance
The details of both types of resistance are reviewed in the following sections.
10.1. lntrln~icResistance
The inherent or intrinsic antimicrobial resistance is a property that is present in all, or
most of the strains of enterococcal species. The genes for intrinsic resistance, like other
RE VIEW OF LITERA 77JRE
species characteristics, appear to reside mostly on the chromosome. These intrinsic traits
expressed by enterncocci enable them to exhibit'resistance to p-lactams, especially semisynthetic penicillinase-resistant penicillins, cephalosporins and aztreonam, as well to low
levels of aminoglycosides and clindamycin [13, 57. 681. The reports of treatment failure
with penicillin in enterococcal endocarditis were reported soon after the introduction of
penicillin in the early 1940s. since MIC of E. faeculis and E. faecium were generally
between 2 to 8 pglml and 16 to 32 pgiml respectively, which were at least 10 to 100
times greater than those for most streptococci proving that enterococci were considerably
less susceptible to penicillins than streptococci [57]. Later, studies showed with evidence
that the intrinsic resistance of enterococci to b-lactams (penicillins) is a characteristic
feature of these organisms and it appeared due to low affinity of the pen~cillin-binding
proteins [246-2481 Most of these studies have shown that the MIC of penicillins was only
bacteriostatic on enterococci. and very higher concentrations (>I00 pg/ml) of p-lactams
(penicillins) were required in order to be bactericidal. While none of the cephalosporins
tested show activity against enterococci at least in-vivo, making them ineffective in
treatlng enterococcal Infections [ 13, 571.
Entemcocci exhibit intrinsic resistance to low-levels of aminoglycosides and
clindamycin, with MlCs for various aminoglycosides ranging between 8 to 250 pg/ml
generally, although occasional exceptions were also reported. The low-level
am~noglycosideresistance among enterococci appeared to be due to low uptake of these
agents by enterococci, which is associated with the proteins involved in electron
transpon. While some E. ,faccium strains were reported to show higher MICs for specific
group of aminoglycosides than E. ,faeculis, and the combinations of penicillin plus these
aminoglycosides fail to show synergism against those E. ,faecium strains. [13. 57, 140,
249, 2501. Although intrinsic resistance is exhibited for low-levels of aminoglycosides,
high-level resistance is of more clinical significance and tends to be acquired and are
generally encoded by more than one plasmid [251].
The intrinsic resistance to the glycopeptides- vancomyciniteicoplanin is exhibited
by few species of entemcocci. The genes encoding the VanC type of vancomycin
REVIEW OF LITERATURE
resistance are endogenous, species-specific components of E. gallinamm encoding vanCI gene and E. casseliflovus/E. flavescens encoding vanC-21vanC-3 genes respectively
which enables them to exhibit low levels of vancomycin resistance. The MICs are much
lower when compared to other types of acquired inducible vancomycin resistances,
ranging from 2 to 32 and 0.5 to 1 pg/ml for vancomycin and teicoplanin respectively [13,
571. While some reviews report that wild tqpe strains of enterococci exhibit intrinsic low
level resistance to fluoroquinolones, clindamycin, lincomycin, and trimethoprimsulfamethoxazole (TMPISMX) due to reduced drug accumulation either by decreasing
the drug uptake or Increasing eflux of the drug [I 31.
10.2. Acquired Resistance
The most important type of antimicrobial resistance exhibited by enterococci is the
acquired resistance either due to a mutation in the existing DNA (that occurs less
frequently), or by acquisition of new DNA, such as plasmids or transposons (that occurs
very commonly in enterococci). The conjugative gene-transfer system plays a major role
In acqulsltlon of plasmids or transposons encoding resistances to different antibiotics as
rev~ewedpreviously. Enterococci exhibit acquired resistance to an array of ant~microbials
as reviewed in the following sections [I 3, 571.
10.2.1. Resistance to Chloramphenicol, MLS, Tetracycline and FIouroquinolones
The transferable chloramphenicol resistance encoded by plasmid was reported in E.
luecalis, and upto 40% of clinical isolates of enterococci exhibited chloramphenicol
resistance mediated by chloramphenicol acetyltransferase [252]. Two decades back, in
1974 Clewell et al. [I 151 first demonstrated that a broad host range conjugative plasmid
pAMBl encoded erythromycin resistance in a S. ,fuecalis strain, which was a
representative of the so-called MLS phenotype (i.e.. resistance to macrolides,
lincosarnides. and streptogramin 8 ) . Some studies showed that erythromycin resistance
exists on different determinants (ermB gene) canied by Tn917 that is widespread in
human and animal isolates of enterococci [6]. The strains exhibiting MLS phenotype, in
REVIEW OF LITERATURE
addition to erythromycin resistance confers high-level resistance to clindamycin [6]
leading to serious therapeutic problems.
Although less significant, studies have shown that different genes like tetL, tetM,
tetN tetO and tetS encoded by plasmidsiconjugative transposon (Tn916) exhibited
tetracycline resistance. These genes mediate tetracycline resistance either by active efflux
of tetracycline from cells, or by a mechanism that protects the ribosomes from inhibition
by tetracycline [6, 131. While studies have depicted that some E. jaecalis and E. ,faecium
isolates exhibit flouroquinolone resistance by mutations on the genes encoding DNA
gyase and topoisomerase IV leading to alteration of the target site [253]. The versatility
of enterococci has been authenticated further, since some clinical isolates of enterococci
exhibit resistance to the recently discovered novel class of antibiotic called
oxa7olidinones (linezolid) by mutations of the target site [245], which has created a
serlous impact in management of vancomycin resistant enterococcal infections for which
this newer antibiotic was reserved.
10.2.2. Acquired Aminoglycoside Resistance
Apart from intrinsic (low-level) resistance to aminoglycosides, many clinical enterococci
were shown to exhibit acquired high-level resistance to aminoglycosides (streptomycin
and kanamycin) three decades back, with MlCs generally >2.000 pg/ml, and
concomitantly resistant to synergism with cell-wall active agents (penicillins) [249, 2501.
Subsequently Jacob et al. [ I 141 demonstrated that high-level resistance to streptomycin
and kanamycin were carried by conjugative plasmids, after which the acquired
aminoglycoside resistance was proven to be transferable by many other reports. and
shown to be mediated by both streptomycin adenylyltransferase and aminoglycoside
phosphotransferase [I 171. In 1979 Horodniceanu et al. reported the first high-level
plasmid-borne resistance to gentamicin in three strains of S~reptococcvs.faecalis
subspecies zymogenes, which were also resistant to kanamycin, sisomicin, netilmicin, and
tobramycin, to macrolide antibiotics, chloramphenicol. and tetracycline, and were able to
g
canied by them [254]. Since then several
transfer the plasmids encoding d ~ resistance
REVIEW OF LITERATLIRE
studies were conducted to reveal the genetics of the aminoglycoside resistance in
enterococci.
Generally, aminoglycoside resistance in gram-positive cocci like enterococci is
due to synthesis of enzymes, which modify the antibiotics [8]. Table 2 depicts various
aminoglycoside resistance genes prevalent in enterococci and their susceptibility profiles
for synergistic activity [8]. High-level resistance (HLR) to kanamycin (without
gentamicin) is a fairly common trait and is due to the production of a 3'-
phosphotransferase-APH(3')-III.This enzyme is important because it also eliminates
synergism between cell-wall active agents and amikacin through phosphorylation of the
3'-hydroxyl group, although it does not necessarily confer HLR to amikacin. Ferretti et al.
12.551 have shown that HLR to gentamicin results from the bifunctional protein (AAC(6')-
IbAPH(2")-I), encoded by a single gene with two active sites, one with 6'acetyltransferase activity and the other, 2"-phosphotransferase activity. The combination
of these activities results in HLR or resistance to synergism for all commercially
available aminoglycosides ~ncluding gentamicin, sisom~cin, netilmicin, tobramycin,
kanamyc~n,and amikacin, except streptomycin, which is not modified by this enzyme.
However, HLR to streptomycin may be due to streptomycin adenylyltransferases:
ANT(6')-la or ANT(3")-la, which can coexist with the gene(s) for HLR to other
aminoglycosides, or can either be due to ribosomal resistance [256]. Eliopoulos et al.
12561 showed streptomycin MlCs of 4.000 to 16,000 pgiml for strains with streptomycin
adenylyltransferase. while MlCs were up to 128,000 p g m l for strains with ribosomal
resistance.
Further, the bifunctional enzyme AAC(6')-IIAPH(2")-I does not modify
spcctinomycin too. but this agent, which is not a true aminoglycoside, is not generally
bactericidal against enterococci and does not appear to show synergism with cellwall
active agents [8, 171. Studies show that the bifunctional aac(6")-le-aph(2")-la is no longer
the only aminoglycoside resistance gene in enterococci, since, new aminoglycoside
resistance genes aph(2")-ic [257], aph(2")-Id and aph(2")-lb [8] are known to encode
resistance to gentamicin, and a new approach for detecting resistance to aminoglycoside
synergism may be required in these cases.
REVIEW OF LITERATURE
Table 2. Susceptibility profiles of genes that mediate resistance to aminoglycoside
synergism in enterococci.
Aminoglycoside antibiotics
Resistance gene GentamicinTobrrmycin Amikacin bnamycin Netilmicin DibckacinStreptomycin Arbekacin
u ~ ~ ( 6 ~ - I ~ - ~ p h f ?R " l ~ l ~
R
R
R
R
R
S
S"
uphf?l-lh
R
R
S
R
R
R
S
S
uph/?l-k
R
R
S
R
S
S
S
S
uph(?'i-Id
R
R
S
R
R
R
S
S
aphf37-lllu
S
S
R
R
S
S
S
S
UUL /c('i-i~
s
R
s
N-I
s
N-I
onrf3'i-lu
S
S
S
S
R
S
un1/4?-lo
S
R
R
S
S
s
s
un~~fi'i-~u
S
R
~
l
R
S
~
S
I
RS
NT- not tested; R- resistant to synergism; S- susceptible to synergism
a
Fony percent of ~solatestested susceptible to synergism
Table adapted from Chow (81.
Thus acquired aminoglycoside resistance In enterococci gains more clinical
s~gnificance,since several stud~escarried out over the last two decades shows that many
of the strains have the ability to transfer especially the gentamicin resistance to other
srrains, even though different types of aminoglycoside resistance genes are prevalent
among enteroccxci [ I 31.
10.2.3. Beta-Lacemuse and Nan-Beta-Lacramase mediated Penicillin Resistance
Beta-lactamase mediated penicillin resistance in enterococci was first described in 1983.
It was encoded by a transferable pheromone responsive plasmid, and was constitutively
produced and cell bound (2581. Thereafter several reports of beta-lactarnase producing
Enlerococcus
were published, although most of these isolates were From the United
States [259, 2601. While Rice et a]. [ X I ] showed that some E.,faecalisstrains exhibited
chromosoma~ mediated beta-lactamase production, which also encoded HLR to
,
s
REVIEW OF LITERATURE
gentamicin. The beta-lactamase production goes undetected by routine laboratory
tests such as MIC or disk diffusion testing due to the relatively low levels
of' beta-lactamase produced constitutively by enterococci. Hence a specific betalactamase test such as the chromogenic cephalosporin based methods is recommended
[13, 1901. While low-level penicillin resistance has been reported to be an intrinsic
property of E. faecium due to low-affinity of penicillin binding proteins-PBP [246, 2471,
some studies reported strains with much higher levels of penicillin resistance that were
beta-lactamase negative 117, 621. Recently. a novel p-lactam resistance mechanism was
proposed in E. .faecium due to a bypass of DD-transpeptidation, which did not involve
PBP (DD-transpeptidases) [262]. Thus enterococci adapt newer mechanisms for
overcoming the action of p-lactams, to combat them
10.2.4. Acquired Glycopepiide Resistance
The most worrisome resistance trait to emerge in enterococci is the resistance to
~ancomycin.Since the first report of Vancomycin resistant enterococci (VRE) in 1988 by
littley ct al. 12631 in England, and Leclercq et al. [264] in France, which was shown to be
plasmid mediated and transferable. a large body of research has been stimulated
worldwide to elucidate the genetics, epidemiology and causal factors responsible for
emergence of vancomycin resistance in enterococci. Five types of vancomycin resistance
have been reported in enterococci: VanA, VanB, VanC, VanD, and VanE as shown in
Table 3 [57. 2651. The mechanism of resistance has been best characterized for the vanA
cluster of seven genes found on the transposable (mobile) genetic element Tn1546 as
shown in Figure 4. In the presence of an inducer like vancomycin, transcription of the
genes necessary for resistance to vancomycin is activated as a result of the interactions of
a sensory kinase and a response regulator. The transcribed genes are translated into
enzymes, some of which make cell-wall precursors ending in D-alanyl-D-lactate (D-AlaD-Lac), to which vancomycin binds with very low affinity. Others prevent synthesis of,
or modify endogenous cell-wall precursors ending in D-alanyl-D-alanine (D-ala-D-Ala),
to which vancomycin binds with high affinity. All but one of the genes in the vanA
clusters have homologues in van9 gene clusters, that in turn have a unique gene not
found in the vanA clusters.
REVIEW OF LITERATURE
hyrrds,. h ~ w n
.~.*=d
q r
e j r,., ,I ,
,
,
b
,k,rrd
-d
L! N, D l
a.
?,
lh , 0 6 4
ID
d
len.*'l
.hruum,r o,
'h * c m ,
L I
ma..
+
&
.".I
.
t. .UI
nh
n.'
1 .,a
...
< L
,U. D t r
21114
0.5
.
I
,
ul
.,,*,rri*
,,"*.,l,".
d d ~ r r . ~r m
) wburr
r , n l u , m I**\,...,
make cell
w
1.1
pnc
lnhibnaon O( wlC
"a
,l
smthu'
h~ah
aIf#nllvlor vane
Mns-n
-tan
. n l . ~s
n.t h
Vancornyc~n-hubccpt~hle
crrtertroccl r)nthcz,c c c l l - u a l l prccursorr ending i n 1)-Ala-1)-Ala.
which. aner t~iln,locution from the cytapla\rn to the cell surlkcc. bind \unconrycin n i t h high
atlinil). ollue hound. t h e w p r c c u r w r s cunnol pnntcrputc I n cell-wall s ~ n t h c s i s .
Vunconrycin-rc*istunt enlerwoccl. I n the prcrcncc of'sn inducer lihc vmcomycin. pencrate
precursors w i l h d i l k r e n l tertnlnl (I>-Alu-1)-l.ilc, 1)-Alu. or IJ-Alu-1)-Scr). which have l o w
aninit) l o r vunconiycin and thus C . U c(>nt~nue.
~
i n large pun. l o hr used l o s y n t h r s i ~ ecell w a l l
A l u dcnotcs alnnyl or slunine. and X luctulc fi,r VanA. Vunt), und V a n n types ofresi5rancc and
r c r i n r Ibr VUII~' und V u n l typch
?c#rSdr~4~**hklrdpa\hd#ld&.S.&.Muc$yr~.
~ ~ ~ . t T u a r r H u r ( n - ~ ~ , U J A ~ Y l u r o y ~ .
64
RE VIEW OF LITERATURE
Less is known about VanD24 or VanE23 types of resistance, but the genes for
types A, 8, D, and E all appears to be acquired. Jn contrast, the genes encoding the VanC
type of vancomycin resistance are endogenouslintrinsic, species-specific components of
6. gallinarum (vanC-I ) and E. cassel~~uvuslE.
flovescens (vanC-2ivanC-3) respectively
as reviewed previously [57, 2511. Recently McKessar et al. [266] has described another
mechanism of vancomycin resistance in some E. .faecalis isolates encoded by VanG gene,
with MIC ranges from 12-16 pdml and theteicoplanin MIC < 0.5 pg/ml.
In-vitro studies have shown the transferability of vancomycin resistance gene
(vanA) from enterococci to S.aureus, S. epidermidis, and other gram-positive organisms
via plasmid-mediated conjugation [131, 2671, which created panic among health care
professionals. As anticipated, the first documented case of infection caused by
vancomycln resistant S. oureus-VRSA with MIC >I28 pgiml was reported in a patient
from Michigan in the United States in June 2002 [268]. Subsequent molecular genetic
studies have authenticated that the vancomycin
resistance
was transferred from
enterococci to S. alrreus. Thereafter several reports of VRSA were published from U S ,
Japan and other countries, although not at the pace of VRE. Thus the ability of
enterococci to acquire and transfer newer traits has raised serious concern for
~mplementingstringent infection control measures to prevent the spread of VRE in health
care settings, span from appropriate use of antimicrobials.
10.3. Laboratory Detection of Antimicrobial Resistance in Enterococci
The diversity of the emerging antimicrobial resistance traits among enterococcal isolates,
and occasionally the intrinsic resistance exhibited by certain species of enterncocci to
some antimicrobials, creates an additional need for accurate identification at the species
level and continuous surveillance of the resistance characteristics [17, 57, 2511. On the
other hand, several of the specific drug resistance characteristics acquired by enterococcal
strains are not adequately detected by the routine susceptibility tests most commonly used
in the clinical microbiology laboratories, so they require modifications of the usual
procedures, or to use the most accurate methods recommended for the recognition of
RE VIEW OF LITERATURE
resistance in enterococci. Hence, early detection of these strains would be of great value
to the selection of the more appropriate antimicr~bialtherapy for the treatment of serious
e n t e r o ~ ~ infections
~~al
leading to better patient management, and to minimize treatment
failure and spread of antimicrobial resistance. Although the National Committee for
Clinical Laboratory Standards (NCCLS) has published the standard susceptibility testing
for enterococci [190], different laboratories have adapted several modifications
of this standard method. In summary laboratory detection of antimicrobial resistance In
enterococci can be performed by two methods I) Phenotypic identification as per NCCLS
guidelines, and 2) Genotypic identification of antimicrobial resistance. A brief review of
both the methods would be presented in the following sections.
10.3.1. Phenotypic Methods for Detecting Antimicrobial Resistance-NCCLS Guidelines
Smcr several years, controversy and confusion exist regarding antimicrobial
susceptibility testing of enterococcal isolates, particularly about the reliability of
phenotypic methods for detection of HLR to aminoglycosides and resistance to
vancomycin [269]. Updated guidelines for the selection of antimicrobial agents are
followed for routine testing and repofling, as well as performance, and interpretative
criteria of susceptibility testing for enterococci have been published by the NCCLS [I901
and are summarized in Table 4 and 5. Although NCCLS guidelines is regarded as the
standard for selection of the agents to be tested, it has been emphasized by several studies
that the selection can vary depending on the antibiotic usage pattern by the clinicians in
any particular hospital setting [I 31. The choice of testing depends on the need, although
dlsk diffusion testing is performed more widely than dilution testing. While other testing
methods like E-test (PDM epsilometer. AB Biodisk, Sweden) have been used in some
studies for quantitative antimicrobial susceptibility testing of enterococci, where a
preformed antimicrobial gradient from a plastic coated strip diffises into an agar medium
inoculated with the test organism. The MIC is read directly from a scale on the strip at the
point where the ellipse of growth inhibition intercepts the strip. Furthermore. phenotypic
antimicrobial testing is ansidered very helpful for routine detection of resistance in
enterococci since they do not discriminate the resistance markers rather detect them
qualitatively irrespective of their mechanism of action.
RE VIEW OF LITERATURE
Table 4. Interpretivecriteriaafor disk diffusion and d~lutionsusceptibility testingbfor Enreroroccus
species, according to NCC1.S recommendations [I901
Disk diffusion teatsd
Report
group'
Antimicrobial
agent
Zone dinmeter
(mm)
Disk
content
--____
S
Penic~llins
Pcnicill~n
lounits > I S
117
Ampicillin
lop9
Streptogramins
Quinoprist~nl
dalfopr~stinr
15pg
219
Glycopcptides
r17
30pg
Vancomyc~n
:I4
30pg
Te~coplan~n
An~inoglycoside\-H1.R
Gentamicin
120pg
'10
Streptomycin
300pg
r10
Macrol~des
Etyhromycin
15pg
123
Tetracyclines
.I9
30pg
Ietracycl~ne
1 6
30pg
Doxycycl~ne
219
0
Minocycl~ne
Anuaniyc~n\
Kifampin
5pg
220
Phcnicol\
Chloramphenicol
30pg
218
Fluoroqu~nolones
:21
Spg
C~pmfloxacin
117
5pg
Levofloracin
:I7
IOpg
Norfloxacin
Nitrofuranto~ns
N~trofurantoin
300pg
517
Fosfomycins
Fosfomyc~n
?OOpg
?I6
A
H
C
I1
.-
I
Dilution tests'
MIC ( ~ d m l )
-
-
I
R
-
.
~ 1 6
>I6
'1
2
14
15-16 514
11-13 510
9
9
8-16
16
t32
Z32
7.9
7-9
56
-4
5500
-
>SO0
14-22 :I3
50.5
1-4
>8
15.18 514
13-15 r I ?
15-I8 514
:il
54
8
R
8
216
116
816
17-19 ,-I6
51
2
14
13-17 112
4
16
232
16-20 515
14-16 513
13-16 112
51
52
14
?
4
8
r4
28
216
15-16 114
532
64
?I28
13-15 512
34
I28
256
.
-
R
S
514
516
58
16-18 C15
9
-
1
4
.
.
.
.
.
.
.
p
'Interpretative cntena for susceptibility testing : S, susceptible. I.~ntermediate;R, resistant.
h~encral
comment. Ccphalosponns, aminoglycos~des
(except for high level resistance screening).
clindamycin, and Tnmethopnm~sulfamethoxazolemay appcar active In vitro but are not effect~ve
clinically, and ~solatesshould not be reported as susceptible.
'Report group (ant~microbials):group A recommended for inclusion in routine primary testing panel:
group B cl~nicallyimportant agents particularly for nosocomial infections & may warrant pnmar). testing:
testing i f situation demands. viz., for endem~c!epidemic
group C .alternative agents that may
. requlrc
.
mulidrug resistant st&ins; group 11 - primarily used for treating unnary tract infections;
d~onditionsfor disk diffus~ontesting: med~um.MHA; inoculum. 0.5 McFarlandturbidity standard (growth
method or d~rectcolony suspens~on);incubation, 3S°C. amb~entair. 16 to 18 h (24 h for vancomycin);
quality control: S, aureus ATCC 25923 ( E faecal~sATCC 29212 & E,faecalis ATCC 51299 for HLAR)
"onditions for dilution testing: medium. MHA for agar dilution or cation ad~ustcdMuelier Hinton broth
for broth dilution; incubation, 35'~. ambient air, 16 to 20 h (24 h for vancomycin): quality control:
E faecalis ATCC 29212 (E faecalis ATCC 51299 for HLAR and vancomycin resistance screening).
'For reporting against vancomycin-resistantE .forcium
-
-
REVIEW OF LlTERA TURE
a. Detection of High-Lewl Aminoglycoside Resistance
The agar dilutionhroth dilution screening method and the disk diffision screening
for detection of HLR to aminoglycosides are generally used to predict the
effect between an aminoglycoside and a cell wall-active agent [190, 2691.
several laboratories are routinely using these methods worldwide successfully, and the
details of the performance and interpretation of the tests are as shown in Table 5.
Gentamicin and streptomycin are the two agents that are tested by several laboratories on
a
routine basis, since, enterococcal isolates that are resistant to gentamicin can also be
considered resistant to other aminoglycosides except streptomycin, although exceptions
arise very rarely where strains susceptible to gentamicin may be resistant to kanamycin
and amikacin. In such instances, susceptibility testing for these alternate aminoglycosides
may help in choosing the ideal synergistic combination for therapy 12691.
b. Detection of Vancomycin Resistance
Since the first report of vancomycin-resistant enterococci (VRE) [263, 2641, it has
become a serious therapeutic problem. With newer details regarding the genetics and
clin~calsignificance of vancomycin resistance emerging constantly [13], the laboratory
diagnostic procedures and interpretive criteria of vancomycin resistance in enterococcl
keeps changing. or is subjected to modifications. Many methods commonly used by
clinical laboratories, including the conventional disk diffusion method and automated
systems, have had problems in detecting enterococcal strains with low to moderate levels
of vancomycin resistance in enterococci [270]. While most of the modifications and
recommendations for disk diffusion testing and agar dilution screening for detecting
vancomycin resistance suggested by various studies 193, 2711 were further evaluated and
adopted by NCCLS as shown in Tables 4 and 5.
c. Detection of B - L a c m Resistonce
The routine antimicrobial susceptibility tests like disk diffision or dilution methods
perform well in detecting the usual resistance due to changes in PBPs. But the resistance
due to the production of klactamase by enterococcal strains is not detected by these tests,
since an inoculum 100-fold greater (10' cfuiml) than routinely recommended be
REVIEW OF LITER' TURE
necessary for demonstrating resistance mediated by p-lactamase. Hence, in the absence of
pgp-mediated resistance, p-lactamase producing enterococci should be considered
resistant to penicillin, ampicillin. and the ureidopenicillins, since several reports of
p-
lactamase positive enterococci causing infections have been published [259, 2601. The
detection of $-lactamase production by enterococci can be detected by using a
chromogenic cephalosporinase based (nitrocefin) method as recommended by the
manufacturer.
10.3.2. Genotypic Methods for Detecting Antimicrobial Resistance in Enterococci
The genotypic methods can rapidly detect specific antimicrobial-drug resistance genes
and substantially contribute to the understanding of the spread and genetics of acquired
enterococcal resistance. But because of their high specificity and versatility of
antimicrobial resistance in enterococci [8. 17, 571, genotypic methods have their own
llmitations, since they will not detect antimicrobial resistance due to a mechanism that is
not included in the testing, as well as emerging resistance mechanisms. The high-level
gentamicln resistance is a good example to authenticate this fact, since they have more
than one genetic mechanism to exhibit resistance
[a]. But these problems too have been
part~allyovercome by using alternate procedures like multiplex-PCR, using which more
than one gene could be amplified at the same time [105, 2691. Several laboratories
worldwide are using the PCR based genotypic methods routinely. to identify various
aminoglycosideivancomycin
resistance
genes to aid in diagnostics, and to study the
genetics and epidemiology of acquired enterococcal resistance [20, 105.2691.
I I. EPIDEMIOLOGICAL TYPING METHODS FOR ENTEROCOCCI
Microbial typing is the first and foremosr step in establishing nosocomial epidemiological
sunreillance system after preliminary identification of the nosocomial pathogen The
results of the microbial typing depicts the degree of relatedness among the isolates
studied, which in turn helps in initiating and executing appropriate infection control
measures in health care settings. Microbial typing system can be categorized into two
groups
RE VIEW OF LITERATURE
1) Techniques those determine the phenotypic characteristics- phenotypic techniques
2) Techniques those characterize genetic determinants- genotypic techniques.
As reviewed in previous sections, the emergence and dissemination of multiple
antimicrobial resistance traits among enterococci, and the evidence supporting the
concept of exogenous acquisition of enterococcal infections have generated an additional
need for typing the isolates as a means of assisting infection control and epidemiological
studies both within and among different healthcare settings [13, 17, 571. Thus several
studies were initiated and being carried out, to study the epidemiology, and
outbreaWclonality analysis of drug resistant enterococci in nosocomial settings, as well in
community using appropriate techniques. A brief review of the different epidemiological
typing procedures followed by several laboratories worldwide is presented in the
followtny sections.
11.1. Conventional Phenotyping Methods
The conventional phenotyping techniques were used since 1960s for epidemiological
invest~gations of enterococcal infections based on phenotypic characteristics of
enterococci, even though they were not highly reproducible and sufficiently
d~scr~minatory.
The conventional/classic phenotypic typing methods used to investigate
the diversity among isolates ot nosocomial enterococci include biotyp~ng,antibiotyping,
serotyping, bacteriocin typing and bacteriophage typing, the details of which have been
rev~ewedpreviously (Section 6). Most of these typing techniques were used extensively
before the advent of molecular techniques for characterizing enterococci. But today
molecular techniques are used extensively along with biochemical techniques, for
epidemiological typing of enterococci [272].
Biochemical typing based on physiological characteristics has been used by
several studies for outbreak analysis as well, subtyping of enterococcal strains [272-2751.
While, several studies have utilized antimicrobial resistance as a trait (antibiotyping) for
e ~ i d e m i o l o ~ i ctyping
a~
of mterococci. along with biochemical typing [259, 276, 2771.
REVIEW OF LITERA TURE
Few laboratories have also used other typing systems like serotyping, bacteriocin typing
and bacteriophage typing, although less extensively [12, 131. Since biochemical typing
was showing a higher discriminatory index than other phenotyping techniques, automated
biochemical fingerprinting systems were introduced commercially for strain typing [272].
~ o s oft these phenotypic typing systems although yield usehl information, are generally
tlme-consuming, difficult to reproduce and interpret. Some of these techniques frequently
fail to adequately discriminate among strains because strains of several enterococcal
species do not usually show enough physiological variation to be useful [97], and for
some others the reagents are not readily available; therefore, they have limited value in
epidemiological studies. Thus today. most of the laboratories use phenotyping methods in
association wrth more recent genotyping techniques, for epidemiological investigation of
enterococcal isolates. which enables to track dissemination and evolution of multi-drug
resistant strains more efficiently in health-care settings.
11.2. Molecular Typing Methods
The applications of novel molecular genotyping tools have provided critical insights into
ep~demiological aspects of nosocomial enterococcal infections, especially in relation to
outbreaks due to strains that exhiblt clinically Important antimicrobial resistance. The
genotyping methods are comparatively more discriminative than other phenotype-based
typlng methods, since the approach focuses on genetlc determinants rather than merely
phenotypic characteristics. Over the last two decades several molecular techniques like
Plasmid and genomic DNA profiling. restriction enzyme analysis-REA of genomic and
plasmid DNA, chromosomal DNA restriction endonuclease analysis by pulsed-field gel
electrophoresis (PFGE) [278-2801, Ribotyping, Multilocus enzyme electrophoresis
(MLEE). and PCR-based typing methods such as, the random amplified polymorphic
DNA-RAPD-PCR assay, and repetitive element sequence REP-PCR have been used to
investigate the genetic relationship among enterococcal strains [281, 2821 . While few
studies have used DNA sequencing based tools especially to determine differences in the
nucleotides among specific resistance genes in entemocci. However results from a large
number of investigations over the last decade have indicated that restriction digestion of
REVIEW OF LITERATURE
genomic DNA by PFGE has an edge over other genotyping tools for typing enterococcal
and is being considered as a "gold standard tool for epidemiological typing of
n o ~ ~ c o m ienterococci.
al
Another recently introduced technique called amplified fragment
length polymorphism-AFLP analysis shows a similar power of discrimination when
compared with PFGE for typing drug resistant nosocomial enterococci [55, 2831. A brief
review of few important genotyping methods used widely for epidemiological typing of
enterococci is presented in the forth-coming sections.
11.2.I . Plasmid DNA Based Typing
The first report of plasmid DNA in 1972, encoding erythromycin and tetracycline
resistance in enterococci [6], initiated several researchers to probe into the molecular
basis of plasmid mediated d u g resistance among nosocomial enterococci. However in
1986, Zervos and his colleagues utilized plasmid DNA content as an epidemiological
marker and authenticated nosocomial transmission and exogenous acquisition of
gentamicin resistant S,jut~cu11.sfor the first time. Thereafter, several studies started
utilizing plasmid DNA profiling as an epidemiological tool for typing enterococci, and
were able to provide molecular evidences fbr dissemination of multidntg resistant
enterococci, withln, as well outside hospital settings [47,278. 284,2851.
The plasmids encoding resistance to ampicill~nand gentamicin were isolated from
outbreak strains, and checked for homogeneity by restriction digesting the plasmids,
which authenticated that few strains of enterococci were responsible for dissemination
within as well outside hospital over a wide geographical area [SI, 2861. Plasmid typing
has been used successfully by several authors in US, Japan and India, to study and prove
the dissemination of gentamicin resistant enterococci in hospital settings [135, 287,2881.
Few authors used plasmid typing for differentiating colonizers from pathogenic
enterococci [289], as well to study the dissemination of MLS enterococci [290]. While
most of the recent studies have shown that plasmid typing, when used along with PFGE
of chromosomal DNA gave more conclusive results for epidemiological typing of
enterococci [287, 2881.
REVIEW OF LITERATURE
11.2.2. Chromosomal DNA Analysis by Pulsed-Field Gel Electrophoresis (PFGE)
~ F G Eis presently considered as the "gold standard for the epidemiological analysis of
nosocomial enterococcal infections by many investigators. Although the PFGE technique
IS
more cumbersome and time-consuming, apart from high equipment costs, the
enterococcal strain typing results they yield, makes them currently the single most useful
and reliable typing method. Further, some authors have experienced much more difficulty
and inconsistency in the generation of clearly visible plasmid patterns with enterococci
than with other organisms such as E. coli and Shigelia species. In such instances PFGE
proves to have an edge over other techniques, and generates clarity in polymorphisms if
any. among enterococcal strains studied [279].
PFGE can be performed by two methods namely contour-clamped homogeneous
electric field (CHEF) electrophoresis, and field inversion gel electrophoresis (FIGE).
Whilc CHEF electrophoresis reveals better hand resolution over 250 kb more reliably,
FlGE reveals better band separation in the 50 to 200 kb range. Thus most studies use
CHEF electrophoresis extensively for PFGE, since polymorphisms are seen more among
hands > 250 kb [279]. More than a decade several studies have used PFGE extensively in
nosocomial epidemiology for studying the clonal relatedness among nosocomial and, or,
environmental enterococci, as well to track the dissemination of genetically related clones
[75, 110. 148.272,281,282,291.292].
Although PFGE has been more discriminatory than other typing methods as
shown in several studies, epidemiological interpretation of PFGE profiles was not clearcut always. The occurrence of genetic events, such as inversions, deletions and other
rearrangements of the chromosome. as well as insertion of transposons or mobile
elements, where shown to be associated with substantial changes in the PFGE profiles,
leading lo problems in clonality assessment and interpretations by several studies [293,
2941. Hence, most researchers preferred applying PFGE along with other phenotypic
and/or at least an additional genotypic technique, since the approach was highly helpful
for appropriate epidemiological interpretation [27. llO.28 I].
RE VIEW OF LITERA W R E
Several authors have used PCR based genotyping techniques like RAPD-PCR or
REP-PCR along with the gold standard techniqlle PFGE, for epidemiological typing of
nosocomial E. faecalis and E..faeciurn resistant to vancomycin, high-level gentamicin and
and ampicillin [I 10, 281, 282, 291, 2931. Thal et al. [295] utilized plasmid
content and hybridization analysis along with PFGE to trace the clonality of
glycopeptide-resistant E. ,faeciurn isolates collected from Michigan hospitals over a 6year period. Most of these studies have concluded that PFGE was highly powerful in
clustering the outbreak strains, which showed similarity in polymorphism profiles among
the clustered isolates of enterococci. PFGE analysis although used mostly for
epidemiological typing of nosocomial E, facco11.v and E, faecium, have also been shown
to be useful for typing other enterococcal species like E. raffinosus, and other unusual
spectes of enterococci [I481
Some studtes have shown that multicenter PFGE studies with centralized server
for interpretation will address epidemiological questions effectively. De Lencastre et al.
[296] made an anempt to test the efficacy of a molecular surveillance network by
subjecting MRSA and vancomycin-resistant E, faccium genotypes for PFGE typing in six
hospitals in the metropolitan New York City area. The results of their study were
encouraging, and concluded that cooperative venture fnr molecular typing of enterococci
helps in rap~dtracking of drug resistant strain dissemination locally as well glohally.
Furthennore, recent advances In image processing systems assist in effective analysis and
interpretation of PFGE banding profiles, and to store the PFGE banding profiles in a
databank for comparative analysis using computational methods. This would provide an
efficient tracking system to assist hospitals, clinics, and chronic care facilities in
controlling the spread of multi-drug resistant enterococci locally, as well globally.
11.2.3. Other Gcnotyping Methods
Apan from plasmid DNA profiling and chromosomal DNA analysis by PFGE, several
other genotyping methods were used for epidemiological typing of enterococci. But most
of these methods were performed as an adjunct to the gold standard technique PFGE. As
reviewed above, several authors used RAPD-PCR for epidemiological typing of
REVIEW OF LITERA TURE
enterococci [I 10, 281, 282, 2911. Most of these studies concluded that even though
RAPD-PCR was simpler and easier to perform .when compared to PFGE, they were no
better than PFGE in typing enterococcal strains. But the RAPD-PCR results were able
enough to track the epidemiological panern of enterococci, although with limitations.
Ribotyping was used by Kuhn et al. [2721 for epidemiological typing of enterococci, as
an adjunct with PFGE. Recently, Krawczyk et al. [297] evaluated a novel method based
on amplification of DNA fragments surrounding rare restrictron sites (ADSRRS
fingerprinting) for typing strains of vancomycin-resistant E, fuecium. Another recent
technique, amplified fragment length polymorphism (AFLP) is reported more frequently
by several studies, for epidemiological typing of enterococci. Antonishyn et al. [ 5 5 ]
evaluated fluorescence-based amplified fragment length polymorphism analysis-f-AFLP
for molecular typing of vancomycin-resistant E. /ueclum in a hospital epidemiological
study and compared the results with PFGE. Jureen et al [283] conducted a comparative
analysis of AFLP and PFGE among ampicillin-resistant E. fuecium in a hospital outbreak
and subsequent endemicity. Most of these AFLP based studies produced patterns of
comparable discriminatory power while possessing some advantages over PFGE, like
less-time-consuming and, internal standards for typing nosocomial enterococci.
Although genotyping has an edge over other microbial typing methods, it can be
rlghtly concluded that no single "ideal" method can be used without clinical
epidemiologic investigation, but any of these techniques is helpful in providing focus to
infection control practitioners assessing possible outbreaks of nosocomial infection.
12. ENTEROCOCCUS- THE INDIAN PERSPECTIVE
Surveillance studies conducted by various bodies across the globe have ranked
enterococci among the top three pathogens causing nosocomial infections, leading to
substantial morbidity end mortality [S, 11. 371. Although surplus data regarding
nosocomial enterococci are available from United States and Europe, Asian countries
account for petite contribution regarding the significance and prevalence of this emerging
nosocomial pathogen. The incidence of entemoccal infections and species prevalent in
RE VIEW OF LITERA T b R E
India has not been investigated extensively and thoroughly, and as evident from literature
there is a paucity of information on the versatility of multidmg resistance among
enterococci from India. Hence, we will present a brief review regarding the significance
and prevalence of enterococci in lndia as evident from different stud~esconducted over
the years in the following section.
12.1. Non-Human Reservoirs of Enterococci
Even before the emergence of enterococci as a predominant nosocomial pathogen, their
significance outside the hospital setup was explored. The first Indian report regarding the
significance of enterococci although outside hospital, dates back to 1970, when Thapliyal
et al. studied the statistical correlations between total coliform and enterococcus counts in
water supply of Tarai region, and validated that enterococci can be used as a fecal
~ndicatoralong with E. colt in potable water [298]. Thereafter in 2001, Grover and
Thakur further authenticated in their study that drinking water samples contaminated with
E. ,Jueculi.s were unfit for consumption [299]. Saikia et al. [300] studied the prevalence of
Enren~coccusspecies and their antimicrobial susceptibility from the intestines of ducks in
.Assam. They isolated E , Juc,colis and E. Jaecium in ducklings less than eight weeks old,
and E. furcali.s. E.fiecium and E. yullrnarurn in duck more than eight weeks old. Further,
all the Enterococctr.~ species isolated were resistant to the macrolide and l~ncosamide
antibiotics, while chloramphenicol and gentamicin sulphate were the only antibiotics of
those tested, which were moderately effective. While Shakuntala et al. [301] showed that
G~tc~rococcus
species constituted 3 % of all bacteria isolated, in their study camed out to
find the prevalence of Streptococcus agaluctiae in milk from untreated healthy cows, and
those with mastitis in Manipur. But no studies from lndia have yet shown animal to
human transmission of enterococci as a cause of infection.
12.2. Significance of Nosocomial Enterococcal Infections
Although few reports prevail on the non-human reservoirs of enterococci, most of the
Studies conducted were focusing the significance of enterococci in human infections. In
RE VIEW OF LITER! W R E
1974 Arora and Tyagi isolated enterococci from apparently healthy young adults with
significant bacteriuria 13021. Thereafter, hardly.were any reports on enterococci from
human infections for more than a decade. In 1985 Mishra et al. [303] depicted that
enterococci plays a role in neonatal septicemia. Later in 1997 Bhat et al. 11741 showed
that neonatal bacteremia was caused by high-level aminoglycoside resistant-HLAR
e n t e r o c ~ ~ in
c i a tertiary care hospital in Mangalore. Of the total 41 strains of enterococci
lsolated 35 (85.4%) were E. fuecalis and six (14.6%) were E. foecium. A total of three
(8.6%) strains of E. /uecah.s and two (33.3%) E.,fueciurnstrains showed HLAR, but none
were vancomycin resistant
Nischal and Macaden [274] conducted a preliminary study on 50 isolates of
enterococci from various clin~calsamples. and performed biochemical speciation and
checked the hemolytic activity in enterococci using a panel of erythrocytes. They showed
that activity of the hemolysin was better against human A and 0 blood group
erythrocytes, and also against guinea pig and fowl erythrocytes at 37".
Chanopadhyay et
al. (3041 showed that E. {urculis appeared to be the commonest organism followed by
peptostreptococci and Stri~ptococcusi~iridon.5in root canal infections. In 2001 Hiremath
el al. [305] reported that Entc,rococc~.$causes chronic suppurative Otitis media (CSOM),
even though Pseudornonus uerupino.su and S. oureus were the predominant etiogens
isolated In 2003 Parvath~ and Appalaraju [306] in their one-year study showed a
prevalence rate of 3.5% enterococci from various clinical samples in a tertiary care
hospital in Coimbatore. The highest incidence was from isolates of urine (43%), pus
(40%), wound swabs (1 I%), and the least incidence were noted in discharges from fistula
(2%), blood cultures (2%) and peritoneal aspiration fluid (2%) while E. faeculls
accounted to 88% of the isolates. Recently. Viswanathan 13071 reported the first case of
enterococcal conjunctivitis from Mumbai (Bombay) in a diabetic patient, caused by E.
fuecu1i.s.
Many studies have depicted that enterococci plays a major role in Infective
endocarditis (IE). In 1996, Tripathi et al. 13081 in a prospective clinical and etiological
study of IE showed that S. oureus was the predominating organism (24%), while
RE VIEW OF LITERA TURE
EnterocOccUS was
shown to contribute to 8%. While Khanal et al. [309] in their study of
IE showed that high level gentamicin resistant enterococcus was a major etiological agent
of IE along with Staphylococci that added to the complexity of the problem. Garg et al.
[3 101 carried a study of IE and analyzed the clinical profiles and outcome in 192 Indian
patients over a period of ten-years (1992-2001) in a super specialty tertiary care referral
hospital in Lucknow. Their results revealed that enterococci contributed to 8.1%, while
~treptococclwas the commonest cause of IE accounting to 23.2% with high morbidity
and mortality rates. Most of these studies concluded that Rheumatic heart disease-RHD
was the commonest underlying heart disease present in approximately 50% of patients,
while blood cultures were positive in 50-70% of episodes approximately.
Gupta et al. [3 1 I] studied the uropathogenic strains from inpatient and outpatient
departments from April 1997 to March 1999 for their susceptibility profiles. Their results
revealed that E. farcalrs was the only gram-positive cocci causing UTI, apart from
predominant gram-negative bacilli. In a one and a half year study from a tertiary care
hospital in Mumbai the incidence of Enr~rococcusspecies in urinary tract infections was
Sound to be 7.3% with E. furculir as the predominant species isolated (87%) followed by
E.
fuecium
(10.8%) and
E. durans (2.14%) [312]. Desai et al. [I441 studied the
prevalence and distribution of various species of entemcocci in another tertiary care
hosp~talin Mumbai, by processing various clinical specimens of catheterized patients
with UTI. Enterococci were prevalent in 22% of the total specimens with Foley's
catheters and bum wounds to be the major site of isolation. They ident~fiedseven species
ofenterococci from a set of 202 isolates, with E.,/aecalis (49.5%) and E./aecium (35.6%)
predominating, while E. swum (9.4%). E. hrrae (2.4%), E. rqflinosus (1.9%) and one
isolate each of E. gallinamm and E. cusselij7oi~us were the other members of
Enrerococcus
species identified. Their study showed a high rate of colonization as
opposed to infection. UTI by enterococci due to catheterization was found in 8.9% of the
patients, due to high rate of colonization of Foley's catheters, and use of broad- spectrum
antibiotics.
REVIEW OF LITER?TURE
12.3. Role of Enterococci in Polymicrobial infections
The role of enterococci in polynicrobial infections still remains debatable as reviewed
although several experimental studies have shown that enterococci contribute
to the severity of the disease in case of polymicrobial infections like intra-abdominal and
wound infections, bums or abscesses, surgical site infections, and bacteremia. Sood et al.
13131 conducted a retrospective analysis of positive blood cultures obtained during the
pried of five years (from January 1991 to December 1995) in case of suspected
bacteremia from a tertiary care hospital in New Delhi. Their study showed that there was
an increase in bacteremia due to polynicrobial etiology, while the gram positive isolates
espec~ally that of E. faecalrs were multi-drug resistant which had prognostic and
therapeutic implications. Murthy et al. [314] showed that Enrcrococcus was isolated less
frequently from postoperative wound infections, while S. aureus (32%) and
P,\cudomonas species ( 2 1%)
were the commonest organisms. Later Vijaya et al. [315]
also allowed that En/erococcu.s was isolated less frequently from diabetic foot infections,
u h ~ l eS u1rrcw.s (33%) was found to be the most common isolate.
Revathi et al. [316] showed that E.,faeculis ranked fifth contributing to 8.5% next
only to P.\eudornonus species (36%), S. uureus (19%), Klehsiella species (15.5%) and
I'rorc~~isspecies
( I I%) in a five year (June 1993-June 1997) retrospective study of
bacteriological analysis of pus samples from bum patients at a Hospital in Delhi. While
Singh et al. (3 17) in continuation of the previous study from the same hospital showed
that the bacteriological trend remained the same over the next five years (July 1997 and
April 2002) except for the emergence of Acinctoha~,!rrspecies (9%). But the incidence of
antimicrobial resistance among the nosocomial pathogens isolated was markedly
increased, with concomitant resistance to penicillin and aminoglycosides exhibited by
61% of E. faecalis resulting in limitation of therapeutic options. Sharma et al. [318]
showed that enterococci play a role in peritoneal infection in patients undergoing acute
intermittent peritoneal dialysis (PD) in a prospective study carried (September 2000 to
February 2001). They concluded that in acute intermittent peritoneal dialysis the
incidence of bacterial infection was 30% with preponderance of gram-negative over
RE VIEW OF LITER' TURE
gam-positi~eorganisms, and organisms of fecal origin were commoner than those of
origin reemphasizing the role of fecal streptococci (enterococci) in intra-abdominal
~nfections.
12.4. Hospital Reservoirs of Enterococci
Since 1987 when Zervos et al. proved the exogenous acquisition of enterococci from
hospital environment [la], several studies started focusing in tracing the reservoirs of
enterococci in hospital environment for effective infection control. Fotedar et al. [319] in
their study showed that the housefly (Museo domestics) acted as a carrier of pathogenic
microorganisms including enterococci in hospital environment in NewDelhi. In primary
~solations,it was observed that the load of bacteria carried by the test group (surgical
ward) of llies was significantly more (P < 0.001) than for the control flies (residential
area). P. uenrgrnosu, E.,/ucculr.s and viridans streptococci were isolated only from the test
flies. whereas the isolation rate of S uurcus was sign~ficantlyhigher in test houseflies
than in the control houseflies and concluded that houseflies may act as vectors of
potentially pathogenic bacteria in a hospital environment. Chandrashekar et al. [I761
conducted a microbiolog~cal surveillance by sampling the equipments, cradles, other
inanimate objects and environmental surfaces to trace the reservoirs of nosocomial
pathogens In a neonatal intensive care unit-NICU. Their results showed a prevalence of
4.7% for Enreroec)cca~next only to A'leh.siellu pncumoniue (27.3%). E. coli (16.8%). S.
uurcus ( 1 1.7%). and S. cpidi~rmidisand P. aeruginosu (10.2%). Thereafter no reports
have been published. to show the reservoirs of enterococci in hospital environment from
lnd~a.
12.5. Antimicrobial Resistance among Enterococci
The property of antimicrobial resistance has enabled enterococci to emerge as a
Prominent nosocomial pathogen, with few studies from India highlighting this fact. In
1995 Mathai et a]. showed high-level aminoglycoside resistance among enterococci
13201. Later in 1997, Bhat et el. conducted a study to determine the drug resistance
REVIEW OF LITER4WRE
pattern of enterococci isolated from cases of neonatal bacteremia and showed that out of
41 strains of enterococci isolated 8.6% and 33.3% of E. ,fueculis and E. fiecium strains
showed high level aminoglycoside resistance-HLAR, while none exhibited
vancomycin resistance [174]. Although penicillin resistance has been quoted high among
e n t e r o c ~ ~in~ western
i
literature, Jesudason el al. [321] showed that only 10.2% of
enterococci from urine cultures exhibited penicillin resistance in a study conducted in a
tertiary care hospital in Vellore, between January and June 1996. In 1999 Agarwal et al.
[322] showed
concomitant
high-level
resistance to
penicillin
(HLPR) and
aminoglycosides in 16% enterococci at Nagpur. While low level vancomycin resistance
with MIC 516 pgiml was encountered only in 3.3% enterococci that were not associated
with HLPR or HLAR. Parvathi and Appala Raju [323] screened 100 isolates of E.
fircc,ulrs for beta-lactamase production and showed that 32% and 34% isolates were
posctive by acidometric method and cloverleaf technique respectively.
In 1999 Purva et al. reported the first case of vancomycin-resistant E, fueciurn
isolated from the blood culture of a patient with non-Hodgkins lymphoma from New
Delhl. India [324]. Thereafter, few more studies reported the prevalence of VRE in India,
although the resistance rates were very low when compared to those of U.S and European
studies. Parvathi and Appalaraju [323] in a one-year study showed that 95% of the 100
~solatesof enterococci were sensitive to vancomycin by disk-diffusion testing. Taneja et
al. [I531 showed that 5.5% of enterococci isolated from urinary specimens between
October, 2000 to April, 2001 in a tertiary care hospital exhibited vancomycin resistance.
Among the eight (5.5%) VRE obtained. five were E..fuecium, and one each of E, fuecalis,
E. cussellflu~~us
and E. pseudou~~ium
with MIC ranging from 8 to 32 kgiml.
Mathur et al. [I 541 showed the emergence of multi drug resistance among 444 E.
Jirecalis isolates in a one-year study at a tertiary care center of northern India. Their
results revealed that 26% and 66% of the isolates exhibited HLAR and ampicillin
resistance respectively, while 88% and 85% were resistant to ciprofloxacin and
erythromycin respectively. Vancomycin resistance-was found in five (1%) isolates, of
which four had van-A phenotype and one had van-B phenotype as evident from the
REVIEW OF LITERATURE
,,,~lts of MIC. Recently Karmarkar el al. [325] showed that 23% of the 52 enterococci
isolated were resistant to vancomycin with. an MIC > 4 pgiml, but sensitive to
teicoplanin, and underscored the importance of antimicrobial susceptibility testing of
e n t e r o c ~ ~Although
~i.
several studies have shown the molecular basis and epidemiology
of drug-resistant enterococci worldwide, no studies addressing this aspect are available
from lndia as evident from the available literature.
12.6. Need of the Hour for Extensive Multifaceted studies on Enterococci
Most of the lndian works published till August, 2004, have reported only the prevalence
and antimicrobial susceptibility pattern of enterococci over a small study period. The
emergence of resistance to penicillins, high-level aminoglycosides, and vancomycin
among enterococci in lndia is in high-rise as evident from recent literature. This diversity
and, in some cases, species specificity of emerging antimicrobial resistance traits among
enterococcal isolates emphasizes the importance of accurate identification at the species
level, and continuous surveillance and characterization of the antimicrobial resistance in
cnterococci. Studies show that the genetics of antimicrobial resistant mechanisms in
enterococcl are versatile and may vary geographically. which enables them to
disseminate and transfer the determinants rapidly in hospital environments: the breeding
ground for most of the antimicrobial resistant nosocomial bacteria. Hence multifaceted
studies focusing on the molecular basis of antimicrobial resistance, and clonal
rclationshlps among those drug resistant enterococci would help in projecting the
versatility of this emerging nosocomial pathogen from Indian sub-continent. The results
of such a study would be of immense value to set local. as well national standards for
therapeutic-prophylactic policies. and to strengthen the habits of appropriate and
jud~cious antimicrobial use in health care settings. Further, the lack of genetic
information regarding the antimicrobial resistance and epidemiology of enterococci from
Indian sub-continent has made it the "need of the hour" to probe into these aspects.
Hence multifaceted studiff probing these issues from lndian sub-continent would be a
valuable contribution, which can address the Indian perspective of Molecular
characteristics of nosocomial enterococci to the world.