1. health and fitness
2. disease

COAGGREGATION AND BIOFILM FORMATION BY
BACTERIA ISOLATED FROM CHRONIC WOUNDS
Thesis submitted in candidature for the degree of
MASTER OF PHILOSOPHY
BY
NGO MALEGUEL Epse KAMDEM JACQUELINE
April 2010
University of Wales Institute Cardiff
School of Health Sciences
Western Avenue, Cardiff, CF5 2YB
I
Declaration
This work has not previously been accepted in substance for any degree and is not being
concurrently submitted in candidature for any degree
Signed………………………………………………………… (Candidate)
Date…………………………………………………………....
Statement 1
This thesis is the result of my own investigation, except where otherwise stated. Other
sources are acknowledged by footnotes giving explicit references. A bibliography is
appended.
Signed………………………………………………………… (Candidate)
Date…………………………………………………………......
Statement 2
I hereby give consent for my dissertation, if accepted, to be available for photocopying
and for interlibrary loan, and for the title and summary to be available to outside
organisation.
Signed………………………………………………………… (Candidate)
Date………………………………………………………….......
II
Dedication
To my beloved, late Yannick, Operi and Lyne
III
Acknowledgement
My gratitude goes to God for his love and mercy throughout this journey.
I will like to thank my supervisor and my director of study respectively Professor Rose
Cooper and Adrian Peters for their valuable suggestions and the opportunity they gave
me to stretch out my imagination in order to accomplish this thesis. My appreciations
also go to Jenkins L, Rowland R, and Jones P of the Microbiology laboratory at UWIC
for their technical support. I also acknowledge my friends Sola, Gertrude, Judith and all
academic associate members for all the social activities we shared together while I study
in UWIC. To my lovely husband Mr Kamdem JC, my children Franck, Dan, Shalom
and Emma my parents Mr and Mrs Maleguel I will like to say thank you for always
being there for me.
IV
List of posters presented
Ngo MJ, Cooper RA, Peters A “Coaggregation and biofilms formation by bacteria
isolated from chronic Poster presented on the 2nd Annual Academic associates
Symposium, January 15th 2009 UWIC Llandaff Campus Cardiff, United Kingdom
Ngo MJ, Cooper RA, Peters A “The Possible role of biofilm in preventing chronic
wounds healing (an evaluation of a recent paper) ” Poster presented in the 9th Annual
Postgraduate KTP Colloquium, Gregynog Hall Newtown, Powys United Kingdom 1st 3rd July 2009
Oral presentations
2nd July 2009 ―Biofilm formation by bacteria isolated from chronic wounds‖ speaking
at the 9th Annual Postgraduate KTP Colloquium, Gregynog Hall Newtown, Powys
United Kingdom 1st -3rd July 2009
V
Declaration…………………………………………………………..............................
II
Dedication……………………………………………………........................................
III
Acknowledgements……………………………………………………….....................
IV
List of poster presented………………………………………………….......................
V
Table of contents…………………………………………………................................
VI
List of Figures………………………………………………………..............................
X
List of tables………………………………………………............................................
XI
Abstract...........................................................................................................................
XII
CHAPTER 1: INTRODUCTION
1.1 Background...............................................................................................................
1
1.2 Biofilms.....................................................................................................................
3
1.2.1 Biofilm formation……………………………………………………...................
4
1.2.1.1 Attachment phase...............................................................................................
5
1.2.1.2 Microcolony formation.......................................................................................
5
1.2.1.3 Detachment phase................................................................................................
6
1.2.1.4 Mature biofilm....................................................................................................
7
1.2.1.5 Quorum-sensing …………………………………………………...................
8
1.2.2 Biofilms in humans……………………………………………………..............
10
1.2.2.1 Biofilms in human diseases…………………………………………...............
11
1.2.2.2 Biofilms in wounds............................................................................................
13
1.3 Wounds.....................................................................................................................
14
1.3.1 Wound healing........................................................................................................
15
1.3.2 Acute wounds........................................................................................................
17
1.3.3 Chronic wounds.....................................................................................................
18
1.3.3.1 Diabetic foot ulcer...............................................................................................
19
VI
1.3.3.2 Venous leg ulcers................................................................................................
19
1.3.3.3 Pressure ulcer.......................................................................................................
20
1.4 The role of biofilms in chronic wounds....................................................................
21
1.4.1 Biofilm defence mechanisms.................................................................................
23
1.5 Coaggregation............................................................................................................
26
1.5.1 Coaggregation and the development of polymicrobial biofilms………...............
27
1.6 Network theory............................................................................................
29
1.6.1 Introduction to network theory...............................................................................
29
1.6.2 Barabasi- Albert model...........................................................................................
30
1.6.3 Node, hubs, links....................................................................................................
30
1.6.4 Network terminology…………………………………………………..................
31
1.7 Antimicrobial agent for biofilms...............................................................................
31
1.7.1 Restricted penetration.............................................................................................
32
1.7.2 Nutrient limitation, altered microenvironment/stress respond, and slow
growth..............................................................................................................................
1.7.3 Adaptive Responses................................................................................................
33
34
1.7.4 Genetic Alteration..................................................................................................
34
1.8 Treatment of chronic wounds……………………………………………................
36
1.8.1 Alternative to antimicrobial therapy...................................................................................
37
1.9 Control of biofilms....................................................................................................
37
1.10 Aims and objectives.................................................................................................
41
CHAPTER 2:
METHODOLOGY AND EXPERIMENTS
2.1.1 Bacteria tested…………………………………………………………................
42
2.1.2 Determination of the total viable count..................................................................
42
2.1.3 Coaggregation assay...............................................................................................
43
2.1.3.1 Growth curve assays............................................................................................
43
2.1.3.2 Visual coaggregation assay.................................................................................
43
VII
2.1.4 Net work properties................................................................................................
44
2.1.5 Biofilms assay........................................................................................................
45
2.1.5.1 Biofilms formation in microtiter plates………………………………...............
45
2.1.5.2 Biofilm formation using mixed bacteria cultures derived from the same
wound………………………………………………………………..............................
2.1.5.3 Biofilms of mixed cultured without key organisms…………………................
46
2.1.5.4 Biofilms of mixed culture and deficiency in microbial species or the issue of
node lost………………...................................................................................................
2.1.5.5 Biofilm staining...................................................................................................
47
2.1.6 Data analysis...........................................................................................................
48
47
47
CHAPTER 3: RESULTS
3.1 Results.......................................................................................................................
49
3.1.1 Viable counts.........................................................................................................
49
3.1.2 Growth cuves assay................................................................................................
49
3.1.3 Visual coaggregation assay....................................................................................
50
3.1.4 Matrix obtained from constructed network............................................................
52
3.1.4.1 Matrix from wound 1..........................................................................................
53
3.1.4.2 Matrix from wound 2..........................................................................................
54
3.1.4.3 Matrix from wound 3...........................................................................................
55
3.1.4.4 Matrix from wound 4...........................................................................................
56
3.1.4.5 Matrix from wound 5..........................................................................................
57
3.1.5 Analysis of networks..............................................................................................
58
3.1.5.1 Network properties..............................................................................................
58
3.1.5.2 Effect of node loss...............................................................................................
60
3.1.6 Biofilms formation in microtitre plate....................................................................
60
3.1.6.1
Biofilm formation of individual bacteria..........................................................
60
3.1.6 .2 Screening for biofilm formation.........................................................................
61
VIII
3.1.6 .3 Biofilm formation with mixed cultures of bacterial isolates………..................
63
3.1.6.4 Biofilm of mixed culture without keys organisms…………………..................
65
3.1.6.5 Biofilm of mixed culture the issue of node lost……………………..................
69
CHAPTER 4: DISCUSSION
4.1 Discussion..................................................................................................................
71
4.1.2 Relationship between caoggregation ability and growth phase……….................
71
4.1.3 Coaggregation assay...............................................................................................
73
4.1.4 Microtiter plate assay.............................................................................................
74
4.1.5 Coaggregation, biofilm and the issue of chronic wound………………................
74
4.5.1.6 Biofilm and wound healing...............................................................................
76
4.1.7 Network theory and the issues of node loss...........................................................
77
4.1.8 Clinical implications...............................................................................................
78
4.1.9 Conclusion and limitations.....................................................................................
80
4.1.10 Further work.........................................................................................................
82
Appendix........................................................................................................... .............
84
References.......................................................................................................................
92
..
IX
List of Figures
Figure 1. Presentation of a biofilm as a physical barrier to antimicrobial penetration….....
25
Figure 2. Schematic illustration of the possible roles of coaggregation in the development
of multispecies biofilms ……………………………………………………………………
28
Figure 3. Biofilm defence mechanism…………………………………………………......
35
Figure 4. Diagram of a microtiter plate................................................................................
46
Figure 5. P. aeruginosa NCIB 8626 growth curve ……………………………
49
Figure 6. S aureus 7422 growth curve ………………………………………..…………
50
Figure 7. Visual coaggregation score for different pairs of strains…………………….......
50
Figure 8. Coaggregation network of wound 1…………………………………………….. 53
Figure 9. Coaggregation network in wound 2……………………………………….........
54
Figure 10. Coaggregation network in wound 3………………………………………........
55
Figure 11. Coaggregation network in wound 4……………………………………….........
56
Figure 12. Coaggregation network in wound 5………………………………………........ 57
Figure 13. Stained biofilms in a microtiter plate………………………………………......
60
Figure 14. Classification of the result of biofilms in microtiter plate into 4
categories……………………………………………………………………………………
62
X
List of tables
Table. 1 Classification of coaggregation score ………………….......................................
44
Table. 2. Coaggregation partnerships obtained from bacteria isolated from chronic
wounds………………………………………………………………………………….....
51
Table. 3. Coaggregation frequencies of selected bacteria…………………………….......
52
Table. 4. The network characteristic of chronic wound bacteria based on coaggregation
assay……………………………………………………………………………………….
59
Table. 5. Adherence of various single species of bacteria isolated from chronic wounds
in microtiter plate …………………………………………………………………………
63
Table. 6. Adherence of mixed cultures of bacteria isolated from chronic wounds in
microtiter plate /coaggregation score………………………………………………...........
64
Table. 7. Adherence of selected single species of bacteria isolated from chronic
wounds in microtiter plate/adherence of mixed culture……………………………...........
65
Table 8. Biofilm formation of bacteria isolated from patient 1 in mixed culture, and
without each respective node………………………………………………………...........
66
Table 9. Biofilm formation of bacteria isolated from patient 2 in the mixed culture, each
without one respective node………………………………………………………………
66
Table 10. Biofilm formation of bacteria isolated from patient 3 in the mixed culture,
each without one respective node.........................................................................................
67
Table 11. Biofilm formation of bacteria isolated from patient 1 in the mixed culture,
each without one respective node.........................................................................................
68
Table 12. Biofilm formation of bacteria isolated from patient 5 in the mixed culture,
each without one respective node.........................................................................................
68
Table. 13 Biofilm formation of paired cultured/ coaggregation partnership of bacteria
isolated from patient 5..........................................................................................................
69
XI
Abstract
There is a growing recognition that biofilms are the principal cause of chronicity or
persistence in infections. Biofilms have been implicated in chronic wounds as a cause of
delayed healing. However, only few wound management strategies treat wounds with
the assumption that biofilm may be the cause of failure to heal. The fact is biofilms are
difficult to treat because of their resistance to antimicrobial agent. Biofilm formation is
known to be a three stage process that has been found in dental plaque to be influenced
by cell to cell recognition also called coaggregation. The main aim in this study was to
investigate the ability of bacteria isolated from chronic wounds to form biofim in vitro
and the possible role of coaggregation in the establishment of biofilm. 164 pairs of
clinical bacteria were tested for ability to coaggregate. Out of 71 isolates 58 (81.69%)
gave positive coaggregation score and 13 (18.3%) did not coaggregate. The presence of
biofilm was tested using 59 of the 71 bacteria and all isolates (100%) indicated an
ability to form biofilm in vitro with various degrees of adherence. 74.57% were strongly
adherent, 15.26% moderate, and 10.17% weakly adherent There was a significant
association between the ability of isolates to coaggregate and to form biofilm (p<0.05)
Using isolates that had been recovered from the same patients, matrices were
constructed from 5 patients to investigate the structure of the network. The probability
that a node will be connected was high (p>0.01) indicating that bacteria in chronic
wounds are highly connected to one another. Random and selected node removal from
the network revealed that bacteria in chronic wounds may be strongly dependent on one
another and favour a polymicrobial rather than a monospecies infection. Network
analysis also demonstrated coaggregation ability of some bacteria to act as pioneers in
the establishment of the biofilm and provided support for the idea that coaggregation
might influence biofilm formation in chronic wounds.
XII
Chapter 1
Introduction
1.1 Background
Chronic wounds have posed serious problems worldwide to medical practitioners and
patients they include Pressure ulcers (PU), diabetic foot ulcers (DFU), venous leg ulcers
(VLU) and ischaemic ulcers (James et al, 2008). It has been suggested that 15% of
diabetics are likely to develop lower extremity ulcers (Reiber, 1996) while 14-24% of
diabetics with foot ulcers might undergo amputation (James et al, 2008). It is found that
in the United States around 100,000 limb amputations are performed amongst diabetic
patients yearly (James et al, 2008). VLU are sometimes very painful and represent
about 1% of the world‘s population (Trent et al, 2005). Chronic wounds present an
enormous economic burden in the UK, costing 3 % of the total NHS (National Health
Service) expenditure during 2005-2006 (Posnett and Franks, 2008).
Wound microbiota is considered to be polymicrobial (Percival et al, 2004) and usually
comprising of a number of microorganisms with diverse nutritional requirements.
Chronic wounds are therefore environments of complex mixture of organisms that
produce various chemical substances. In diabetic and obese individuals deep wounds
sometimes lack enough oxygen as a result of poor perfusion. This situation favours the
proliferation of many bacteria including anaerobes that take advantage of the anaerobic
condition created by the consumption of oxygen by the obligate aerobes and facultative
bacteria (Thomas, 2008).
The impact of pathogenic organisms on chronic wounds has been extensively studied
and reviewed through various methods of evaluation especially their role in preventing
healing (Howell-Jones et al, 2005). Most microorganisms isolated from chronic wounds
1
are recovered with traditional culture methods. The use of modern diagnostic
technology such as molecular techniques allows the identification of viable but nonculturable organisms, that otherwise will be undetectable by cultural methods (Dowd et
al, 2008; Thomas, 2008). Using conventional techniques the number of isolates usually
recovered from wounds ranges from 1-8 with an average of 3 organisms per wound
(Hutchinson, 1994) and in another study the mean number of bacterial species per ulcer
was found to range from 1.6 up to 4.4 (Kontianen and Rinne, 1988; Tentolouris et al,
1999). Research has shown that 86% of ulcers with no clinical signs of infection
contained more than one bacterial species (Hansson et al, 1995). For most research S.
aureus and coagulase – negative staphylococci have been the predominant organisms
isolated from chronic wounds (Howell-Jones et al, 2005). P. aeruginosa is another
frequently identified organism in 7-33% of ulcers (Hansson, 1995; Bowler et al, 1999;
Schmidt, 2000). Other aerobic bacteria or facultative anaerobic bacteria isolated from
chronic wounds include S. epidermidis (Brook and Frazer, 1998), E. coli (Ge et al,
2002) Enterobact. cloacae (Hansson, 1995; Brook and Frazer, 1998; Schmidt, 2000),
Klebsiella species (Hansson, 1995; Brook and Frazer, 1998; Ge et al, 2002),
Streptococcus species, Enterococcus species, and Proteus species. Anaerobic organisms
frequently found include Peptostreptococcus species, Peptococcus, Clostridium species
(Bowler and Davies, 1999), Bacteroides species, Prevotella species as well as fungi like
Candida (Bowler, et al, 2001; Howell-Jones et al, 2005). Anaerobic bacteria have also
proved to have damaging effects in colonised and infected wounds (Howell-Jones et al,
2005).
Several patient factors contribute to delayed healing, such as underlying diseases,
nutritional status and as well as the presence of microorganisms. A typical
polymicrobial infection of a wound is a biofilm (Percival, 2004) and biofilm has been
incriminated in impaired wound healing (Bjarnsholt et al, 2008). Biofilms-base wound
2
care (BBWC) management strategies that suppress biofilms in wounds have been
designed and used with some degree of success (Wolcott, 2008). Factors that trigger the
formation of biofilm in patients are not well known. Many of the processes leading to
the development of biofilm, such as primary colonisation, the expression of
extracellular polymeric substances and gross phenotypic changes have been described
(Rickard et al, 1999).
However the role of coaggregation in the primary development of biofilm communities
remains unclear (Rickard et al, 2003a). Coaggregation is a process by which genetically
diverse bacteria become attached to one another by means of specific molecules
(Rickard et al, 2002). Several studies have shown that adhesion influences the
development of complex polymicrobial biofilms. Initially, it was thought to occur solely
between dental plaque bacteria but several studies have proved that coaggregation
mediates biofilm development (Kolenbrander, 2000; Jenkinson and Lamont 1997;
Rickard et al, 2003b). However there is no evidence to suggest that coaggregation
interaction enhances the development of biofilms in chronic wounds.
There are
increasing reports of coaggregation between bacteria from other biofilms communities
in several diverse habitats (Rickard, 2003a). In this study we evaluate the role of
coaggregation interaction in the primary development of biofilms in bacteria isolated
from chronic wounds will be evaluated. An interest in that comes in part from other
studies on the nature of these interactions that revealed that coaggregation could
influence biofilm formation and species diversity (Rickard et al, 2003b) and their
involvement in the integration of pathogens into biofilms (Rickard et al, 1999).
1.2 Biofilms
Biofilm is a community of microorganisms surrounded by the EPS secreted by them,
attached to either an abiotic or a biotic surface. More properly known as biofilm,
3
everyday examples are slimes which flourish in places wherever there is water such as
wash hand basins in the kitchen, on teeth, contact lenses and in the gut epithelium.
When biofilm community is large, it can be seen as slime coating the inside of water
pipes or plumbing lines (Coghlan, 1996). It has thought that 99 percent of all bacteria
live as biofilm form of which the majority are beneficial to mankind including sewage
treatment for removal of contaminants from water before it is discharged into rivers.
However, biofilms pose a lot of problems to mankind; these include corrosion of pipes,
in industries and in medicine obstructing water filters, infection of medical implants
thereby leading to rejection of the implants and causing diverse chronic diseases in
humans.
Understanding biofilms is a means by which complex issues associated with microbiota
can be unveiled and those involving pathogenic organisms can be resolved particularly
the health related problems. Over the years, studies regarding bacterial structure and
behaviour have used planktonic cells that are cultivated in liquid or solid media.
However, recent studies have shown that, naturally most bacteria are attached to
surfaces as sessile form especially in biofilm (Costerton, 2005). Now, while it is true
that many bacteria can exist in diverse forms (planktonic or sessile) state biofilm
formation is associated with large aggregates of these sessile bacteria (Watnick, 2000;
Costerton et al, 1978) where the bacteria adhere to surfaces by means of adhesins and
extracellular polysaccharides (EPS) produced by the organisms themselves to form
microcolonies.
1.2.1 Biofilm formation
A process whereby a biofilm forming organism transforms from a planktonic to a
sessile form and a community of organisms living in a polymeric matrix is derived.
4
Biofilm formation comprises of 3 main stages; reversible and irreversible attachment,
microcolony formation and detachment of biofilm.
1.2.1.1 Attachment phase
Surfaces and interfaces are important in biofilm formation because they facilitate the
acquisition of nutrients. There is always an initial attachment of a pioneer bacterium
onto the surface. Initial attachment is mainly dependent on electrostatic attraction and
physical forces, rather than the chemical attraction (Postollec et al, 2006). There are two
types of attachment during biofilm formation, the reversible and irreversible
attachments. Some of the bacteria that finally form biofilm become irreversibly attached
and produce a matrix material composed of polymeric sugars, protein and/or DNA
(Wolcott et al, 2008).
1.2.1.2 Microcolony formation
This matrix helps the bacterium to secure itself to the surface and helps to protect the
colonising microbiota from environmental and host stress. As the bacteria begin to grow
and multiply they form an aggregate of cells called a microcolony. Bacteria within the
microcolony continue to divide until a critical density of bacteria or quorum forms that
allows the microcolony to develop further. Biofilm bacteria continue to produce
extracellular polymeric substances (EPS), a sticky substance that hold the bacteria
within the biofilm together and attach the biofilm to a surface. In addition, the EPS trap
scarce nutrients as well as protect the bacteria from chemical substances such as
antibodies and antimicrobials. The bacteria within biofilm multiply and the daughter
cells extend outwards giving the biofilm a mushroom-like which Mittelman (1985)
termed spider - web structure. The EPS also acts as an ion-exchange system because it
consists of charged and neutral polysaccharide groups which trap iron and concentrate
5
trace nutrients from the surroundings. The population of the cells increase biofilm
growth and as the cells produce more EPS are produced hence the increasing the ion
exchange propensity of the biofilm. In a short period of time a biofilm is established
(Mayette, 1992). In a mature biofilm, the most of the content is water which is usually
(75-95%), and the organisms (5-25%) of the biofilm (Geesey, 1994). This composition
gives the slipperiness that is often observed in biofilm. The EPS also snares other types
of microbial cells through physical restraint due to its gelatinous and sticky nature
which also attracts by electrostatic interaction. Within a biofilm, there exist varying
degrees of nutritional availability due to differential diffusion gradients; therefore
different categories of colonizers are able to metabolize wastes from the metabolism of
the other groups within the biofilm. For example products such oxygen when utilised by
the anaerobes creates a favourable environment for the anaerobes to thrive (Coghlan,
1996). According to Borenstein (1994) these categories of organisms can be bacteria
and/or fungi and can cohabit conveniently and form a mature biofilm within a short
period of time.
1.2.1.3 Detachment phase
At maturity, a biofilm microcolony attains a critical density of microbial population
when the signalling molecules released by the colonisers portray the overpopulation of
the matrix and the need for some members to disperse (Stoodley et al, 2002; Costerton
et al, 1978). Although quorum-sensing molecules control biofilm formation, it is also
important to biofilm detachment as a means of regulating biofilm population (Stoodley
et al, 2001; Davies et al, 1998). In response to the signalling molecules some biofilm
colonisers transform to planktonic form and disperse from the biofilm thereby reducing
the population within the matrix. These transformed planktonic cells have the ability to
later transform back to the planktonic form to restart another biofilm in different
6
location. An enzyme often produced by biofilm organism which aid dispersion is
alginate lysate which breaks the matrix and the organisms disperse to other locations to
initiate biofilm (Rice et al, 2005).
1.2.1.4 Mature biofilm
The mature biofilm is a fully functional system made up of different species or genera,
almost like a human community with a complex, metabolically cooperative interaction
with each organism living in an adapted niche. Within the biofilms there are circulatory
systems such as water channels for the transportation of nutrients for metabolic
processes of biofilm organisms and exchange and disposition of waste products (de
Beer, 1995). Mature biofilm are imaginatively described by Coghlan in 1996 as a slime
city which he described the cooperation amongst biofilm organisms in which he wrote
―… Pooling their biochemical resources to build a communal slime city, several species
of bacteria, each armed with different enzymes, can break down food supplies that no
single species could digest alone."
Biofilm can form within hours of colonisation by an organism or may take several
weeks depending on the particular organism and the environmental factors (Mittelman
1985). According to studies by researchers biofilm organisms such as P. aeruginosa
cells adhered to stainless steel within 30 seconds of exposure (Vanhaecke, 1990) and
was found to form biofilm
within 8 hour of infection in thermally injured mice
(Schaber et al, 2007). A study conducted on a chronic wound-isolated P. aeruginosa
showed that this bacterium formed biofilm in vitro within 10 hours (Harrison-Balestra
et al, 2003).
7
Biofilm formation may also depend on cell density which influences the quantity of
signalling molecules produced by the organisms within biofilm, P aeruginosa wild type
biofilm EPS accumulation rate was found to be more rapid than those of the quorumsensing-deficiency mutants (Pei-Ching and Ching-Tsan, 2002).
Although quorum
sensing molecules have been proved to influence the production of biofilm in some
bacteria such P. aeruginosa, methicillin resistant-resistant S. aureus, there is still doubt
of its absolute regulation on biofilm EPS production (Pei-Ching and Ching-Tsan, 2002).
A study conducted by a group of scientists (Schaber et al, 2007) actually showed that P.
aeruginosa produced biofilm on specific host tissues independent of quorum sensing
molecules. They went further to demonstrated in a burn wound of a mouse that acylated
homoserine lactones (AHLs) signalling molecules often produced by Gram negative
molecules was not required for biofilm formation by P. aeruginosa.
Biofilm production may also depend on the infection sites; recent research (Manango et
al, 2006) found that strains of MRSA recovered from patients with a nosocomial
infection site were more likely to produce strong biofilm adhesions to polystyrene than
were strains recovered from the nasal cavity or pharynx of asymptomatic carriers. This
finding suggests that strongly biofilm-producing MRSA strains are associated with
nosocomial infection such as surgical site infection and pneumonia.
1.2.1.5 Quorum-sensing
Microbiologists have proved that bacteria do communicate through a process known as
quorum sensing (Reading and Sperandio, 2005). Quorum sensing is a phenomenon
whereby microorganisms communicate amongst themselves through chemical
substances called cell signalling molecules or auto-inducers. It has been demonstrated
that production of signalling molecules accumulate in the medium as the bacteria
8
multiply and at a certain density (critical density) the molecules triggers the expression
of various genes (Sauer et al, 2002) such as production of toxins (Sperandio et al, 2007)
and biofilm formation (Parsek and Greenberg, 2000; Davies et al, 1998) thereby
coordinating their activities. There are various types of signalling molecules which are
often group related. Gram positive bacteria utilise short chain amino acids called
peptides (Dunny and Leonard, 1997) whereas Gram negative bacteria make use of acylhormoserine lactones (AHL) (Parsek and Greenberg, 2000).
It is now believed that these signalling molecules play an important role in diseases
caused by pathogenic bacteria in animals, plants and human (Fuqua and Greenberg,
1998). In human diseases such as biofilm related infections e.g. cystic fibrosis,
organism like P. aeruginosa has been implicated in chronic infections characterised by
biofilm (Grez and Prince, 2007). Several studies have shown a correlation between QS
molecules of P. aeruginosa (AHL) and ability to form biofilm (Davies et al, 1998). The
wild type of P. aeruginosa was found with twitching a form of mobility characterise by
the possession of type IV pili is proven to influence biofilm development (Mattack,
2002). Because this mobility helps P. aeruginosa to spread and attach to surface which
is the first step to biofilm formation, it is now known that P. aeruginosa ability to cause
human infection is directly linked to its virulence factors which are regulated by QS
molecules (Smith et al, 2002). This cell to cell communication depends on autoinducer
like AHLs (acyl homoserine lactone) that bind and activate transcriptional regulator and
induce gene transcription (Grambella et al 1991) this transcription occurs when the
extracellular concentration of the AHL increase in the medium due to increase in
bacterial density. Once the critical threshold is reach the message is transcribed into the
expression of multiple virulence genes and the production of more signalling molecules
(Gabelo et al, 1991). The production of these gene are not just important to P.
9
aeruginosa virulence but also its twitching mobility and the formation of biofilm that
act as a shield to bacteria protecting them from host defence and antimicrobial by
increasing resistance (Davies et al, 1998). Bacteria that produce QS molecule can also
express various toxin an example is enterohaemorragic E. coli (EHEC) 1157 or V.
fisheri, for these bacteria AHL have been involve in the production of toxin (Sperandia
et al, 2001). The use of QS inhibitors have proven to reduce virulence in bacterial
infection an example is furanone
which has shown to reduce QS system in P.
aeruginosa (Bjarnsholt et al, 2005; Hentzer, 2003).
1.2.2 Biofilms in humans
In human host, there are various surfaces available for attachment particularly the
surfaces that are exposed to the external environment such as the skin, mouth,
respiratory and gastrointestinal tract (GIT). These sites and other parts of the body
support the planktonic as well as the sessile populations, some of which are commensal
bacteria. Most of these surfaces are in constant renewal of cells and this prevents a
permanent attachment of bacteria thus preventing biofilm formation. The exception of
the situation is found with teeth, a situation that encourages formation of biofilm which
results in dental plaque (Newman and Wilson, 1999; Lamont and Jenkins on, 2000).
Dental plaque is the biofilm that has received most attention. It is recognized to
comprise more than 350 species of bacteria (Moon and Moore, 1994).
Despite the continual shedding of these body surfaces, establishment of biofilm is
inevitable on some parts of the body where there are slow rate of shedding. Anatomical
sites such the vagina has biofilm which is mostly composed of lactobacilli. Biofilm in
the mouth, gut, vagina and wound are not necessarily detrimental and may actually
provide protection against infection (Reid et al, 2001).
Advancement in medical
10
treatment has resulted in the use of implants and other devices such as catheters and
prostheses that often encourage the formation of biofilm (Khardori and Yassien, 1995;
Habash and Reid, 1998). The association of these devices with biofilm formation has
posed a lot of problems to the medical practitioners and the patients.
1.2.2.1 Biofilms in human diseases
Biofilms have been found to cause serious health related infections because of their
resistance to antibiotics and host defence mechanism and such diseases are often
persistent and difficult to effectively eradicate (Allison et al, 2000). The application of
modern diagnostic techniques such as scanning electron microscopic (SEM)
examination, Confocal laser scanning microscopic (CLSM) examination and molecular
techniques has broadened the scope of understanding of biofilm structure physiology
and activities. Body sites such as the skin, teeth, mouth, respiratory and GIT normally
support the growth of biofilm as commensal flora which does not always result in
infection. However the majority of human diseases associated with biofilms are usually
linked with the presence of some implantable devices (e.g. catheters, heart valves,
prostheses) or impairment of the host defence systems such as cystic fibrosis patients
(Costerton et al, 1999). It has been shown that 65% of human infections involve
biofilms (Potera, 1999).
Although biofilms are associated with diseases in man, they are also beneficial to the
host especially in playing a protective role. In the vagina, lactobacilli produce acids,
bacteriocins, hydrogen peroxide and biosurfactants (Reid, 2001) that prevents the
existence of other bacteria (Wang, 2000). Once the health equilibrium is disturbed
organisms like G. vaginalis take advantage to establish infection. Many physicians
believe that re-colonisation of the vagina with lactobacilli reverts the situation and
11
restores vagina health (McLean and Rosenstein, 2000).
The dental plaque which
usually comprises of streptococci and Actinomyces spp and other species is known to
protect against colonisation by other bacterial pathogens. Under certain circumstances,
some species within the biofilm can outnumber the others resulting in infection (Marsh
and Bradshaw, 1995; Kolenbrander, 2000) such as caries, gingivitis and periodontitis
(Rosan and Lamont, 2000; Lemont and Jenkinson, 2000 a). However, acute infections
that are readily treated with antibiotics are not considered as involving biofilms, unlike
the majority of chronic infections in mildly compromised individuals that involve
commensal or common environmental organisms (Costerson et al, 1999). Presence of
microorganisms on either the exterior or the interior surface is possible and infection
can be localized to the insertion site or disseminated to cause bacteraemia, endocarditis
or septic shock (Costerson et al, 1999). S. epidermidis is the most common causative
agent associated with implant infections (Rupp and Archer, 1994). Biofilms have being
implicated in osteomyelitis with S. aureus causing foreign body related infection, with a
swift development and exhibition of multiple antibiotic resistance, giving them the
ability to transform from an acute osteomyelitis to one that is persistent, chronic and
recurrent (Brany et al, 2008). Infections such as otitis mediae, specially the one with
effusion, was hypothesised to be non bacterial infection but studies have shown this
disease is a biofilm related with one or more bacteria species implicated. Biofilm in this
infection is believed to be the physical barrier that helps explain the ineffectiveness of
antibiotic and host defences against the infection (Hall-Stoodley et al, 2004). Chronic
bacterial prostatitis and prostatic calcifications are known to be cause by bacteria
biofilms (Mazzoli et al, 2009).
One of the most commonly acquired hospital infections, urinary tract infections (UTI) is
associated with biofilm formation is directly linked to indwelling catheters used for
12
treating the patients (Hatt et al, 2008). A recent study demonstrated that E. coli strains
likely causing relapses of UTI showed a greater ability to form biofilm and that these
features may allow the bacteria to capture iron in a stressful environment to persist and
to cause recurrent UTIs (Mazzoli et al, 2009). A study conducted with the use of
scanning electron microscopy in patients with continuous symptoms of chronic
sinusitis, despite prior appropriate medical and surgical management, revealed the
presence of biofilm on the sinus mucosa of patients infected with P. aeruginosa (Cryer
et al, 2004). Once more it is believed that bacteria biofilm in these patients‘ sinus
mucosa show the complex nature of biofilm. In clinical nephrology, biofilm influence
the development of kidney stones and affect dialysis systems, including peritoneal and
central nervous catheters. Biofilm also play a critical role in persistent and resistant
renal and urinary tract infection (Marcus et al, 2008).
1.2.2. Biofilms in wounds
Biofilms are found widely in nature and have been extensively studied in other human
diseases for many years but the study of biofilms in wounds is a relatively new
development. Biofilms are slowly becoming recognised as a cause of wound infection
(Wolcott et al, 2008; James et al, 2008). Their presence is often associated with chronic
wounds but they can also be involved in acute wound infection. Biofilms may be
unperturbed by antimicrobial or neutrophil attack and can survive a relatively harsh
environment, resisting attempts at removal. Acknowledged as a potential cause of
delayed healing it may explain sometime disappointing response obtained from
traditional treatment of chronic wounds. In the past chronic infections due to biofilm
had been viewed as benign by clinicians and have not attended to biofilm infections
with utmost importance. However they are in fact insidious and persistent in nature
13
resulting in death as much as those of heart diseases or cancer, including those
implicated in wound infection (Wolcott et al, 2008).
1.3 Wounds
A wound occurs when the tissue is damaged and this could be a skin break, muscle
tears, burn or bone fracture. This could also be the result of various acts such as
gunshot, fall, or surgical procedure, diseases, or underlying conditions.
Wounds can be classified into two majors groups: elective and accidental wounds.
Electives wound are made out of deliberate choice under these are surgical incision,
amputation, drug injection, piercing and tattoo. Elective wound are generally acute and
when managed by trained medical personnel on many occasions they follow the normal
course of healing. Accidental wounds are unintended and may occur as a result of
infections diseases with direct effect on the skin such as chicken pox, shingles, and
impetigo. They may also arise as a consequence of ageing related heath issues, such as
pressure sores or a comorbidity like diabetes which when coupled with ageing gives rise
to increased morbidity and mortality. Some examples are pressure sores and diabetic
foot ulcers. Accidental wounds may also occur as a consequence of injury this could be
burns, cuts, bites, grazes, or punctures. Open wounds are those in which the underlying
tissues are exposed, whereas in closed wounds the skins are intact but the underlying
structures have been traumatised.
Wounds are often categorised according to the
manner in which the skin or tissue is affected, these include abrasion, incision,
laceration, punctures, avulsion, amputation, contusions and missile wounds. Some
wounds are the combination of one or more of these basics types.
A number of factors may increase the risk of wound infection which include the size
and shape of the wound, the position, presence of foreign bodies and the local vascular
14
state of the wound eg absence of oxygen in deep wound. Another risk factor is the
patient susceptibility. This depends on age, nutritional status, immuno-competency,
illness and drug therapy. The risk factors may also be increased by the population of
microorganisms‘ involved, individual bacterial virulence or a combination effect of both
the number and virulence. From the clinical point of view wounds are generally
classified into acute and chronic wounds. Acute wounds, either traumatic or surgical
follow the healing process at a predictable rate whereas the non-healing wounds will not
progress in a predictable manner but becomes chronic.
1.3.1 Wound healing
When the skin becomes compromised, the normal wound healing process resolves the
injury. In order to understand why an acute wound could end up being a chronic wound
lies in unveiling the process of wound healing. Understanding wound healing process
will provide insight for evaluating the stage at which healing stops and the factor that
may have contributed to the stalling of the process.
Wound healing is a complex process by which the body repairs the damaged tissue or
organ. When an injury to the skin occurs, the development of inflammatory response
by the body immune system is the first step. A number of cells of the immune system
are involved and these include platelets that form a blood clot, the white blood cells that
engulf the microorganisms, as well as the mesenchymal cells that develop into
fibroblasts.
In healthy individuals, healing occurs in three stages; inflammatory,
proliferative and remodelling phases respectively (Clarke, 1995). Clot formation is an
important step within the inflammatory phase because the clot reduces or stops bleeding
and causes the secretion of cytokines at the site of injury (Doughty, 2007).
15
The inflammatory phase is immediate and last 2-5 days with vasoconstriction, platelet
aggregation and thromboplastin forming clot which prepares the wound for the
initiation of healing. During the inflammatory phase, phagocytosis of microorganisms
and removal of debris from the wound are very vital to the healing process. This phase
ensures that the impediments to repair of damage tissues are removed in order to
achieve optimisation of reconstruction of matrix and epithelisation.
Then the proliferative phase takes over with the formation of an outer layer during this
process when epithelial cells migrate across the wound. The fibroblasts turn out
extracellular matrix which leads to the formation of granulation tissue while the
endothelial cells are involved in the formation of blood vessels (Kane, 2007). The
proliferative phase last 2 days to 3 weeks and is characterized by extensive growth of
epithelial cells, fibroblast deposition of collagen fibres, and continuous restoration of
blood vessels (Kane, 2007).
Finally, the remodelling or maturation phase may occur within 3 weeks of injury but
may last up to 2 years. At this stage collagen fibres are produced, blood vessels are
restored to normal, the outer layer is formed, and the epidermis is re-established to its
former state. The simple description of wound healing described above appears to
simple and straight forward but the truth is that this process of wound healing is
extremely complex, making use of numerous growth factors, integrins, enzymes, and
various types of cells (Nancy, 2009).
The process of wound healing described above may be hindered and even stop as a
result of various factors. Some of these factors may be specific to the individual‘s state
of health such as autoimmune diseases while some occurs across all individual groups.
These factors include reduced blood perfusion to the tissue, malformation of collagen
particularly in immunocompromised individuals (Nancy, 2009). These barriers
16
(perfusion, reduced oxygen pressure, malnutrition, systemic diseases and other
problems associated with poor healing wounds) particularly encourage proliferation of
microorganisms and this varies from wound to wound. The devitalised tissue often
encourage the formation of biofilm which has been linked to failure of wounds to heal
(Bjarnsholt et al, 2008; Rhoads et al, 2007) which offers the explanation for the cellular
and biochemical similarity of wounds often associated with biofilm (Rhoads et al,
2007). Recently, the evidence for presence of biofilm in wounds is increasing (Malic et
al, 2009; James et al, 2008; Ngo et al, 2007). Clinical evidence has shown that healing
of chronic wounds improved when biofilm eradication approach of treatment was
employed in patients (Wolcott, 2008).
1.3.2 Acute wounds
Clinically wounds are classified into acute and chronic forms. Acute wounds are those
that heal within a reasonable time frame after the formation of injury whereas the
chronic wounds fail to heal or break down after healing. Acute wounds have a wide
range of causes which often affect the healing process and consequently the duration of
healing. Although they may occur due to accidents (Leaper et al, 1998) or any traumatic
circumstance when a force exceeds the strength of the skin or the underlying supporting
tissues, the healing of such wounds might take longer to heal unlike surgical wounds
which are created through incision and are sutured to heal by primary intention.
The main symptoms of an acute wound are localized pain and bleeding. Specific
symptoms depend on the types and nature of the injury; for a wound created by missile
such as a gun shot, bleeding may be profuse and therefore the repair process would take
a longer period. Management of a severe traumatic wound initially involves emergency
procedures that could be resuscitation and restoration of the circulation to the affected
limb/area. The blood supply must be optimised, any dead tissue removed as this can act
17
as a target point for bacteria. With acute wound adequate pain control is essential. If
pain is not controlled adequately it can decrease oxygen uptake, increase mortality and
morbidity, affect mobility and induce chronicity.
1.3.3 Chronic wounds
Chronic wounds are wounds that do not heal over expected period of time irrespective
of the cause. All chronic wounds begin as acute wounds. In general, any that does not
heal within 3 months is termed a chronic wound (Mustoe, 2005). Some chronic wounds
often remain in an inflammatory state and not follow the normal pattern of the healing
process.
There are various reasons why a wound may not follow the normal healing progress due
to certain factors. Such factors include age which has a major impact on healing
because of decrease in inflammatory response and physiological processes such as
blood circulation, reduction in collagen formation, basement membrane degeneration
(Doughty et al, 2007). Malnutrition prevents wound healing by decreasing collagen
production and other proteins needed for wound repair. Bacteria in high concentrations
produce toxic end products and compete with cells in the granulation tissue for available
nutrients in the wound bed (Evans, 2005). Stress has also been implicated in the
impaired healing process. Studies have shown that decreased wound healing is
associated with psychological stressors such as pain and noise (Kane, 2007).
Although any wound can become chronic, most chronic wounds are diabetic foot ulcer
(DFU), venous leg ulcers (VLU) and pressure ulcer (PU). In these categories of wound
certain barriers tend to present more severely and more frequently in one category of
chronic wound than the others. In the diabetic foot ulcers, for instance, barriers to
healing are related to neuropathy and consist of poor perfusion, white blood cell
18
dysfunction, poor nutrition and pressure issues. However venous leg ulcer may also
have pressure issues as well as poor perfusion (Khanolkar et al, 2008)
1.3.3.1 Diabetic foot ulcer
In the United States, diabetic ulcers account for most foot and leg amputations (James,
2008). In a year, 2% of patients with diabetes develop foot ulcers with an incidence of,
costing an average of $7,000-$10,000 each (James, 2008). The neuropathic impairment
of musculoskeletal system as well as leukocyte dysfunction and peripheral vascular
disease complicating often complicate the incidence of chronic wounds (Marston et al,
2006). Most diabetic foot disease is prone to amputation, usually associated with
significant morbidity and mortality (Khanorlkar, 2008). Currently, there are
approximately 100,000 limb amputations performed in the United States every year; it
is estimated that more than a million people with diabetes require limb amputation each
year globally, an indication that amputation is perform worldwide every 30s (Jeffcoate
and Bakker, 2005).
The probability of an individual with diabetes developing foot ulcer is reported to be as
high as 25 % (Singh et al, 2005). (Khanolkar et al, 2008). The resultant effect of
peripheral neuropathy causes decreased sensation that can result in trauma to the foot.
Removal of underlying cause of diabetic ulcer will prevent rapid progression of
colonised wounds to infection.
1.3.3.2 Venous leg ulcers
An ulcer in the lower limb or an area of damaged skin below the knee that takes longer
to heal is a venous ulcer. Venous leg ulcers could pose an enormous challenge to both
patients and health services (Moffatt et al, 1992; Ruckley, 1997; Posnett and Francks,
2008). Venous leg ulcers are caused as a result of failure of valves within one or more
19
veins of the legs resulting in congestion. However other factors that contribute to
venous leg ulcers include arterial disease, obesity, trauma, immobility, vasculitis and
diabetes (Simon et al, 2004). In the United Kingdom
about 3% of the total National
Health Service expenditure (£2.3-3.1 billion during 2005-2006) was spent on 200,000
patients with chronic wounds (Posnett and Franks, 2008). Around 80-85% of all leg
ulcers are venous leg ulcers and 54 % of patients have venous ulcers for more than one
year (Kane, 2007).
VLU is associated with age and less common amongst individuals who are less than
45years old but increases with age. Studies have shown that one in every 50 persons
over the age of 80 is affected by venous leg ulcers (Kane, 2007) particularly in the
obese. Infection is a common complication, and also recurrence after healing.
1.3.3.3 Pressure ulcer
Pressure ulcers (PU) are the result of tissue damage due to excessive or constant long
term pressure on the skin. It is a common health issues that is generally underestimated
but it occurs mostly in patients and individuals living in institutionalized settings. In the
UK almost 4 - 10% of patients on admission develop one or more ulcers of which the
elderly are the most at risk with a high incidence rate of up to 79% (Lyder, 2003; Gary,
1990).
In fact areas affected by PU suffer reduced supply of oxygen and other vital
nutrients which often result in damage to tissue and consequently devitalisation and
necrosis (NPUAP and EPUAP, 2009). Although a lot of efforts are geared towards
preventing PU in individuals in institutionalized the rate of PU remains high.
According to Rickard and colleagues (2009) the incident rate can still be as high as
66%. PU has been observed as a high risk factor especially in terms of increased
morbidity and mortality due to the secondary problems that often develop as a result of
PU including chronic infection which are often life threatening and might lead to death
20
(Bluestein and Javaheri 2008; Yarkony, 1994). Although preventive strategies are put in
place for PU in patients staying long in hospital, PUs are inevitable and existing PUs are
usually treated with antibiotics to resolve infections. Despite all these procedures
biofilm infections can not be ruled out (Richard et al, 2009).
1.4 The role of biofilms in chronic wounds
Research over the past 20 years has shown that many chronic infections are the result of
biofilm mode of microbial growth (Costerton et al, 1999; Parsek and Singh 2003). It has
been observed that chronic wounds share the same characteristics of persistent
infections that are rarely resolved by immune defences and resistant to antimicrobial
therapy known to biofilm-related diseases (Parsek and Singh, 2003). The role of biofilm
in delaying healing has been hypothesized (Mertz, 2003) but direct evidence of their
presence in chronic wound was revealed through the work of James et al (2008). With
the use of both scanning electron microscopy and light microscopy debrided material
from 50 chronic wounds, biofilms were found in 30 out of 50 and in only 1 out of 16
acute wounds. This was a statistically significant difference that gave the first evidence
that biofilms may be present in the great proportion in chronic wounds (James et al,
2008). This study gave credit to a previous hypothesis that biofilm could be the key
factor in delay healing (Bjarnsholt, 2008) and that the presence of biofilm in a wound is
often the major reason for chronicity of wound infections (Percival et al, 2004).
Although in the laboratory bacterial species are grown as pure cultures for purposes of
investigation of diseases and pathogenicity, however in the hosts such as in wounds
they exists as biofilm where they exist in a polymicrobial community (Ruden et al,
1995). In some infections such as dental plaque and cystic fibrosis biofilm has been
widely investigated but in wounds the role of biofilm is yet to be investigated (Wolcott
21
et al, 2008). Wound colonisation is certain with some bacteria from the patients‘ flora,
that caused the wound or the environmental organisms which often do not lead to
infection in healthy individuals. However, chronic wounds microbial bioburden often
differs from those of acute wounds because of the virulence capabilities of such
organisms (Demling and Waterhouse, 2007). Such virulence capabilities have been
found in biofilm forming organisms such as P. aeruginosa which often complicates the
healing process of community such chronic wounds (Bjarnsholt et al, 2007; Wolcott et
al, 2008).
As a result inaccurate diagnosis of wound infection becomes complicated because of the
presence of biofilm in wounds which cannot be diagnosed by the routine culture with
which the most practitioners are familiar (Lindsay et al, 2006). In fact routine cultures
in the laboratory usually diagnose planktonic bacteria and antibiotic sensitivities are
performed for the purposes of treatment. The fact that biofilm phenotypes are different
from the planktonic counterparts has made the situation more worrisome as the biofilm
organisms remain undiagnosed and continue to flourish in wounds with attendant tissue
destruction and other complications which may be life threatening (Rhoads et al, 2008;
Wolcott et al, 2008; Corterton et al, 1999). Once the biofilm is established on the
surface of the host, regardless of the environment (e.g. sinus, gut or skin), the sessile
bacteria exhibit significantly different strategies known to enhance their survival within
the inherent biofilms community.
The polymicrobial nature of biofilm, coupled with the production of diverse virulence
factors by the organisms within biofilm and EPS which shield the organisms from the
host immune system and antimicrobial intervention, biofilm infection in wounds exhibit
a recalcitrant form of infection. The differences often observed between acute and
chronic infections could be best explained by the presence of pathogenic or undesirable
22
biofilm. Chronic infections follow a persistent undulating course with frequent
exacerbation (Costerton et al, 1999) and will generally respond incompletely to
systemic antibiotics and or immunosuppressant, often re-emerging once the systemic
antibiotics are withdrawn (Fux et al, 2005; Mandel et al, 2002). Hence chronic wounds
are often associated with impaired inflammatory response of the host because of
overwhelmed host immune response. Treatment strategies that will resolve the chronic
inflammation has to be employed otherwise biofilm infection becomes permanently
untreatable because the host immune system will not be able to clear the organism
(Thomas, 2008). The response of chronic infections to steroids is possibly indirect
evidence that the inflammation stimulates the production of plasma exudates in the area
of infection which is necessary for the biofilms to thrive.
Errors in diagnosis of wound infection might occur if the assumption that biofilms may
be implicated is not carefully explored (Bjarnsholt et al, 2008).
1.4.1 Biofilms defence mechanisms
An understanding of biofilm defence mechanisms is fundamentally important to help
deal with the issue of biofilm in infections. Research has shown that biofilm may be
totally unperturbed by activated macrophage, neutrophils, antibodies, complement, or
other host defence mechanisms (Leid et al, 2002; Flux et al, 2005). Within the host
system, the biofilm is able to highjack many of the host‗s components such as
fibrinogen (Masako et al, 2005), neutrophils, DNA and collagen to incorporate into its
protective matrix, making it impermeable and invincible to host attacks (Leid et al,
2002). They are also resistant to biocides, drying or other environmental stresses (Flux
et al, 2005) which further fortifies them against treatment strategies.
23
Host defence system is as robust as dynamic but is usually made redundant by the
presence of biofilm. Several cells as well as the components secreted by them are
involved in the host defence system. For example the toll- like receptors senses and
detects fragments of Gram negative bacteria (LPS) or Gram positive bacteria (teichoic
acid) and on identification, a potent immune response is generated for the production of
cytokines, tumour necrotic factor (TNF) , interleukins and other pro-inflammatory
agents. Although the effect of the inflammatory process is to produce a swarming of the
area with neutrophils and macrophages (Masako et al, 2005) to the site of infection but
when the defence is hampered or in any way delayed, individual bacteria will have the
ample opportunity to adapt to the biofilm system of growth. The high regulation of pro
inflammatory cytokine production characteristically found in chronic wounds may be
explained by the presence of biofilm infection leading to the production of exudates
from surrounding capillaries (Wolcott et al, 2008). These highly nutritious exudates
infiltrate through biofilms and provide nutrients to the resident microbiota of the
biofilm. The supply of nutrients helps to maintain biofilm security and sustainability,
stability and fitness of the biofilm (Wolcott et al, 2008).
Chronic wounds are ‗stuck‘ in a persistent inflammatory state and contain elevated
proinflammatory cytokines, high protease levels and excessive neutrophils (MacAuley,
2006). Although host factors such as abnormal white blood cell function, diabetes or
venous insufficiency contribute to the onset of a chronic wound (Dowd et al, 2008), it is
suggested that host dysfunction rarely prevents healing directly. Rather, the impotence
of the initial immune response allows bacteria to establish a biofilm community in the
wound. Several studies of in vitro biofilms have shown that sequential events that
include attachment to an available surface followed by growth and proliferation into a
specific architecture called microcolonies (Bjarnsholt et al, 2007). These microcolonies
24
are in a self-made matrix of EPS that offers protection to embbeded bacteria (Flux et al,
2005) Fig.1
Figure1. Presentation of a biofilm as a physical barrier to antimicrobial
penetration (Wolcott, 2008)
In this state bacteria are able to resist the effect of antimicrobials (Donlan et al, 2002). It
has been revealed that in most chronic wounds deep dermal tissues harbour multiple
species of opportunistic pathogens (Gjodsbol et al, 2006) confirming their role in
wound healing. Gjodsbol et al, 2006 also found that P. aeruginosa –infected wounds
appeared significantly larger in terms of area than those without P. aeruginosa
(Gjodsbol et al, 2006; Madsen et al, 1996; Halbert, 1992). The presence of P.
aeruginosa seemed to delay or even prevent the healing process (Madsen et al, 1996).
Research has shown that despite relevant and appropriate treatment, many chronic
wounds will not heal or will take a long period of time to do so (Bjarnsholt, 2007). The
presence of bacteria in biofilms, eg P .aeruginosa, the inability of the immune system
such as the polymorphonuclear neutrophils (PMNs) to eliminate biofilm remains a
strong hypothesis of why chronic wounds fail to heal (Bjarnsholt, 2007).
25
1.5 Coaggregation
Gibbon and Nygaard in 1970 observed that genetically distinct bacteria recovered from
dental plaque had an ability to get attached to one another as a result they named this
adherence coaggregation. Since then more than 1000 oral bacteria have shown this
ability to adhere to one another and numerous studies have given evidence that
coaggregation is essential to the development of dental plaque biofilm (Marsh, 2006)
Coaggregation is therefore known as a process where by genetically distinct bacteria
adhere to one another though specific molecules (Rickard et al, 2003b) Studies have
shown that this process occurs at the cell membrane receptors of the cells involved with
one protein called adhesion one cell type and a compatible complementary saccharine
receptor on the other cell (Kolenbrander, 2000 and 2002). Research have further
emphasized
that for adhesion to be initiated there must be an initial specific
recognition between a receptor and a potential legand (Jenkinson, 1994; Lamont and
Jenkinson, 2000b). Initially known to occur only in few biological environment, it is
now recognised as a widespread phenomenon that occurs in urogenital tract and other
human biofilms related infections order than dental plaque Reid et al., 1988), as well in
bacteria isolated potable-water and freshwater system (Buswell, 1997).
Coaggregation has been associated with biofilm in various habitats. In aquatic biofilms
(Rickard et al, 2003a), as well as human diseases involving biofilms such as dental
plaque coaggregation has been implicated at the early unset of biofilms establishment,
more important are recent suggestions that coaggregation may involve in the integration
of various species of bacteria in to biofilms (Handley et al, 2001). In the laboratory
macroscopic observations of coaggregation show a clumping or aggregate of cell when
different cells types are mixed together. Macroscopically the clump of cells formed
consists of a network of interacting cell types.
26
The recognition may be intra, inter or multigeneric in nature (Kolenbrander, 1989). In
all these types of coaggregation, the cells appear to be interacting dependently of other
cells in the population (Kolenbrander and Andersen, 1986). The viable as well as dead
cells coaggregate, indicating that interaction is dependent on the availability of surface
molecules and not on cell viability. Since the beginning of the 1980s, several authors
have described coaggregation between dental plaque bacteria (Rickard et al, 2003a).
1.5.1 Coaggregation and the formation of polymicrobial biofilms
With recent discoveries on coaggregation of microorganisms in the ecosystem apart
from the dental plaque, it is becoming increasingly recognised that coaggregation is
essential in the formation of several species biofilm communities (Saginur et al, 2006;
Rickard et al, 2003b). The role of coaggregation in biofilms has been demonstrated in
other systems such as biofilms in fresh water and fast-flowing water systems (Pitt et al,
2003).
Coaggregation is a form of interaction that contributes to the development of biofilm in
two forms. The first form of adhesion is by single cells in a liquid medium recognizing
and attaching to a genetically distinct organism in a pre-existing biofilm, a phenomenon
known as coadhesion (Jenkinson, 1994; Bos et al, 1994; Busscher et al, 1995). An
example of this adhesion was demonstrated in oral streptococci where its vast repertoire
of adherence properties is known to be expressed at the cell –surface receptors
(Jenkinson, 1994; Lemont and Jenkinson, 1997). This has given streptococci the ability
to bind to human tissues components, epithelial cells and to other bacteria. The second
route of coaggregation is by individual planktonic organisms adhering in a suspension
with the resultant aggregate of microorganisms adhering to a pre-existing biofilm
27
(Rickard, 2003b). Coadhered cells can then become part of the biofilms community
(Fig.2).
Figure 2. Schematic illustration of the possible roles of coaggregation in the
development of multispecies biofilms (based on Rickard et al, 2003b). a) Primary
coloniser adheres to the surface directly on the substratum; (b)Adhere cells multiply; (c)
Single coadhere to pre-existing attached cell or to another cell in suspension
(coaggregation)then to pre-existing attached cells or a group of identical cells
(autoaggregation) adhere together then to the biofilm; (d) Multiple cells in the biofilm
(multispecies biofilm).
An example of this type of adhesion is common in dental plaque whereby Porphy.
gingivalis commonly associated with periodontal disease adhere to Strept. gordonii
(Lemont et al, 1994) and to other bacteria. In dental plaque studies have shown that F.
nucleatum acts as a bridging organism between the early colonizers and late colonizers
(Kolenbrabder et al, 1993). Studies in other environments like chronic wounds need to
be carried out to confirm or reject the above observations. Research over the past 20
years has indicated that chronic infections are the result of biofilms mode of microbial
28
growth (Trent et al, 2005). Factors that lead to bacteria biodiversity found in chronic
wounds remain unknown and the issue of coaggregation influencing bacterial diversity
in other environment could throw light on what happens in chronic wounds.
1.6 Network theory
1.6.1 Introduction to network theory
Several authors have proposed mathematical methods for studying networks, some
examples include: non – linear dynamics (Kaplan and Glass, 1995), and enion
probability analysis (Strevens, 2003). One of the most widely used of these methods is
network theory (Abert and Barabasi, 2002). Barabasi in the ―New Science of Networks‖
shows how everything is connected to everything else. The model provides a
description of how complex systems can evolve by self-organizing principles and how
they function. There are mathematical properties to these networks. There are three
known conditions for these properties to occur. For the network to be sustained the first
condition is the ability to grow, expand while constantly evolving and adapting.
Secondly preferential attachment to the most connected i.e. a node will prefer to link
with the most connected. The third condition has to do with competitive fitness which
means the ability of a node to be most attractive (Barabasi et al, 2002). Barabasi and
colleagues (2002) suggested another kind of network that has not received much
attention, the scale –free network, without average number of links. The characteristic
of scale-free network is that as the number of nodes decreases within connections, a
node simply declines. Net work constructed from bacteria coaggregation interactions in
dental biofilm (Kolenbrander, 1993) appears to possess several well connected
organisms such as Strept. oralis and F. nucleatum fit to this model.
29
1.6.2 Barabasi- Albert model
The Barabasi-Albert model also known as the BA model is an algorithm for creating
random scale–free networks using a preferential attachment mechanism (Albert and
Barabasi, 2002). Preferential attachment would indicate that a node is most likely to get
most new links when it is the most connected. Nodes with higher degree of connections
would be able to add more links to the network. The use of scale free networks are
widely utilised in natural and man-made systems in modern world such as the internet,
the World Wide Web (www), citation networks, face book, Google network and some
social networks. Although several networks are free networks meaning that they have
scale-free degree distributions, some are random graph model such as the ERDOSRemyi (ER) model and Watts-Strogatz (WS) mode do not exhibit scale-free.
1.6.3 Node, hubs, links
It is generally known that corporate systems emanate from small units. Networks of
units such as bacteria, chemical compounds, professional bodies and internet websites
all share important network properties (Boye, 2003). The various connections amongst
them are known as links. A node can be an individual, bacterium, a business, a web
page, or an amino acid while links may include internet connections neurotransmitters,
telephone lines letters, exchanges of substances, or any form of materials which can be
used to transfer information or adhesion interaction. The emergent free scale – network
(Power laws) principle can be likened to the system which operates in nature such as
those observed in living cells and in friendship, that is few nodes (20%) have most
links whereas most nodes (approximately 80%) would have just one or two links. The
node with numerous connections is a hub (Ball, 2002). Google.com which can be called
a hub has multi- billion of links. In science molecules of (adenosine triphosphate) ATP
30
linked with (adenosine diphosphate) ADPs and water are hubs in the cells; in dental
plaque Fusobacterium nucleatum network is a hub (Barabasi, 2002).
1.6.4 Network terminology
Network graphs or matrices are constructed from one node to another. Two nodes are
connected by a finite series of edges (arc). The size of a graph (M) is the number of
edges of which the graph is composed and n is the total number of nodes in a given
graph. There are basic mathematical terms used to characterize a graph and give the
most elementary information about these graphs. The clustering co-efficient (γ)
characterizes the extent to which a node adjacent to any node are also adjacent to each
other i.e. the probability that two nodes will be connected. A γ of one (1) implies the
graph consists of disconnected, but individually complete subgraphs, and γ of zero (0)
implies that no neighbour of any node (i) is adjacent with any other neighbour of i. The
average degree (k) of graph states how many neighbours each node has, and thus
quantifies the relationship between n and M. All graphs must have k<n. Another
network measure is the characteristic path length (L). It is the shortest distance between
one node and another. Therefore the characteristic path length of an entire graph would
be the median of the means of the shortest path length connecting each node to all other
nodes.
1.7 Antimicrobial agent for biofilms
Studies have shown that organisms living in biofilm have high resistance to antibiotics
compared with planktonic cells. In fact, the cells in a biofilm can develop up to 10-1000
times more resistance antimicrobial agents than the planktonic organisms (Gilbert et al,
2002; Prosser et al 1987). The characteristic property of biofilm is their inherent
resistance to antimicrobial agents and host immune defences (Gilbert et al, 1997). Since
31
organisms within biofilms are highly resistant to antimicrobial there is the need for
appropriate concentration of antibiotic in the system (systemic dosing levels) to
effectively treat biofilm infection. However most of the doses of the antibiotic
preparations being marketed are mainly targeted towards the planktonic phenotypes
which are usually ineffective against organisms in biofilm (Mah and O'Toole, 2001).
The resistance of organisms within biofilm to antimicrobials have been receiving
attention of recent and a lot of studies have been carried out towards unveiling the
mechanisms of resistance of these organisms within biofilm to various biocides. The 4
major mechanisms include (1) restricted penetration of antibiotic through the biofilm;
(2) nutrient limitation, altered microenvironment, and slow growth of biofilm cells; (3)
adaptive responses; and (4) genetic alteration to "persister" cells.
1.7.1 Restricted penetration
Biofilm organisms resist antimicrobial through the physical or chemical barriers to
antimicrobial penetration that are provided by the EPS in which the organisms are
embedded (Mah and O'Toole, 2001). Once the biofilm is formed, delivery of nutrients
to the cells is dependent on diffusion through the EPS. The substances in the EPS act as
a diffusion barrier, either by limiting the rate of molecule transport to the biofilm
interior or by chemically reacting with the molecules themselves (Dolan et al, 2002).
Biofilms are mostly water, so solutes of the size of antibiotics will readily diffuse
through the biofilm matrix (Rani et al, 2005). However, the negatively charged EPS
restricts permeation of positively charged molecules of antibiotics, such as
aminoglycosides, by chemical interaction or molecular binding (Lewis, 2001).
According to De Beer and colleagues (1994) the mathematical models indicated ideally
there should be no barrier to the diffusion of antibiotics into a biofilm; however studies
have shown that certain antimicrobials fail to penetrate biofilms. For example chloride,
32
disinfectant was found not to reach >20% of the bulk media‘s concentration within a
mixed K. pneumonia and P. aeruginosa biofilm (De Beer et al, 1994). Once the
antibiotic is bound onto the surface layers of the EPS, the availability of the
antimicrobial into the depths of the biofilm would be greatly reduced (Stewart et al,
2002). Microbial biofilms formed in conditioning films present an even greater
problem, because antimicrobials poorly penetrate fibrin or host protein complexes.
Some practitioners are of the opinion that EPS is not the only barrier that can delay
penetration of antimicrobial agent but a combination of multiple mechanisms determine
the overall antimicrobial resistant (Steward et al, 2002).
1.7.2 Nutrient limitation, altered microenvironment/stress respond,
and slow growth
When bacteria lack a particular nutrient, growth rate is slowed. Transition from
exponential phase to stationary or decline phase is usually associated with an increase in
resistance to antibiotics (Tuomanen et al, 1986). It is known that in a mature biofilm
low growth of bacteria has been observed (Brown et al, 1986) because of nutrient
limitation. The physiological changes can result in resistance of organisms within
biofilm to antimicrobial agents. Cells in the outer layers close to the flowing liquid have
ready access to nutrients and oxygen. These cells are metabolically active, normal in
size, and similar to planktonic organisms. Gilbert and colleagues (2002) in their study
examined growth-rate-related effect for planktonic cultures and biofilms of P.
aeruginosa, E. coli and S. epidermidis. It was observed that both the planktonic and
biofilm cells were resistant to ciprofloxacin. However, as growth rate was increased, the
planktonic cells became more susceptible to ciprofloxacin than the biofilm cells
indicating that some other properties other than slow growth were vital to the resistance
of biofilms to antimicrobial (Evans et al, 1991). Furthermore the near complete
33
consumption of oxygen and glucose in the surface layers creates anaerobic niches in the
depths of the biofilm where the cells down regulate into an extremely slow-growing or
nongrowing state in order to survive (Leid et al, 2002). Subsequently, those antibiotics
readily diffused through the biofilm are ineffective in killing the slow and non growing
cells in the anaerobic regions of the biofilm. The age of the biofilm also affects its
susceptibility to antibiotics. Older (10-day-old) biofilms are significantly more resistant
than 2-day-old biofilms (Rani et al, 2005). This emphasizes the need for prompt
diagnosis and treatment.
1.7.3 Adaptive Responses
Biofilm cells adapt to environmental fluctuations in temperature, pH, osmolarity, and
nutrient availability, through the expression of multiple stress-response genes (Huang et
al, 1998). The genetic alteration not only ensures survival in community living but also
affords protection from the host immune system, environmental toxins, and
antimicrobials (Steward, 2002). Antibiotic resistance is further enhanced by the
horizontal exchange of resistant plasmids. Plasmids are extrachromosomal circles of
DNA that may encode resistance to antimicrobial agents, including beta-lactams,
erythromycin,
aminoglycosides,
tetracycline,
glycopeptides,
trimethoprim,
and
sulfonamides (Donlan, 2002; Tenover, 1998). The high cell densities and greater
probability of contact between the cells in the biofilm clusters favour the higher rates of
horizontal transfer of plasmid DNA. This phenomenon has contributed to the dramatic
increase of antimicrobial resistance among nosocomial pathogens (Parsek et al, 2003).
1.7.4 Genetic Alteration
A biofilm-specific phenotype is thought to be induced within a subpopulation and this
will result in the expression of various mechanisms in order to resist the detrimental
34
effects of antimicrobial agents (Gilbert et al, 1997; Maria-Latran et al, 2000). When
cells attach to a surface, they will express a general biofilm phenotype (Cotter and
Stibitz, 2007). The majority of cells exposed at the outer regions of the biofilm are
frequent contact with antimicrobials therefore they are more susceptible to prolonged
exposure to antibiotics (Cotter and Stibitz, 2007). Most exposed cells in the biofilm die
rapidly on exposure to a cidal antibiotic such as ciprofloxacin that effectively kills slowgrowing cells. It is now thought that the presence of a subpopulation of "persister" cells
within the biofilm (Fig. 3) may be responsible for the profound resistance to complete
eradication of biofilm bacteria. Persisters are phenotypic form of wild-type cells that
remain dormant in the presence of antimicrobial and are usually tolerant to numerous
drugs (Keren, 2004). Persisters cells can be up to 0.1% to 10% of the cells in a biofilm
(Roberts and Steward, 2005). Their purpose is for the survival of the population of the
organisms on exposure to lethal agents such as antimicrobials. When antimicrobial
therapy is discontinued, the unaffected persister cells reverse the phenotype and become
metabolically viable to regrow the biofilm. Infection may reoccur at a later time or may
linger for months, years, or even a lifetime, or until the colonized device is removed
(Rorberts and Steward, 2005).
Figure 3. Biofilm defence mechanism (Wolcott, 2008)
35
1.8 Treatment of chronic wounds
Chronic wounds are those that fail to heal which may be due to various factors such as
infection, compromised immunity or underlying health conditions as well as poor
nutrition. When the reason is due to an underlying condition these conditions would
have to be resolved to aid healing. However in cases where infections have been
implicated appropriate procedures ought to be employed to assist wound healing. The
available treatments include debridement (surgical, enzymatic, and/or by dressing
changes) and antibiotics. Debridement removes dead or necrotic tissue which can be a
source of endotoxins that inhibit fibroblast and keratinocyte migration into the wound
(Stewart, 2002; Stewart et al, 2001). Foreign bodies must remove, as the presence of a
silk suture reduces the number of bacteria required to induce infection (Robson, 1999).
Biofilms is now known recognised to induce chronicity in wounds (Bjarnsholt et al,
2008). Therefore controlling biofilms in wounds as well as management of underlying
conditions such as pressure, poor perfusion or poor nutrition has been suggested as a
strategy that would enhance wound healing (Wolcott, et al 2008). Biofilm management
of chronic wounds is now based on multiple concurrent strategies specifically targeting
biofilm behaviour (Wolcott and Rhoads, 2008). It includes cleaning the wounds of any
undermined tissue, regular debridement, use of immunosupressants (to deprived the
biofilm of its nutritional source), but this also blocks the host‘s healing responses and
should be considered a last resort (Wolcott, et al 2008). Recent clinical evidence
(Wolcott and Ehrlich, 2008) has shown that biofilms are best managed through physical
disruption.
36
1.8.1 Alternative to antimicrobial therapy
The extensive use of antibiotics in treatment had encouraged the emergence of
resistance of microorganisms to antibiotics which has resulted in the failure of many
therapeutic interventions (Bowler, 2001). The persistent nature of chronic wounds poses
huge challenge to health practitioners as well as the patients because most of the
antibiotic treatment interventions often fail to provide the expected results. As a result
of the antimicrobial resistance by biofilm, organisms attempts have been made to
explore the possibility of using natural antibacterial substances as alternative means of
treating wounds infected with bacteria with multiple resistances to antibiotics, including
biofilm organisms. Alternative antimicrobials have been researched, proposed and used
with some degree of success. Among these naturally occurring compounds are honey,
tea tree oil, silver iron as well as maggot therapy. Patients with chronic wounds that
have undergone prolong antibiotic treatment are often willing to use alternative
treatment strategies.
1.9 Biofilms control
In order to control biofilm, several strategies have been developed depending on the
characteristics and environmental system in which the biofilm is found (Steward et al,
2001). The methods used include mechanical removal, use of antimicrobials, nutrient
deprivation and enzymes. In wound treatment strategies have been developed to control
biofilms to enhance wound healing.
Biofilm organisms are protected by the EPS against the action of biocides, it is essential
for the organisms to be exposed to the lethal concentration of the antibiotic hence
mechanical removal of biofilm form wound (debridement) is often used alongside
antibiotic treatment. The purpose is to destroy the physical integrity of the biofilm
matrix (Chen and Stewart, 2000) which serves as a barrier to the action of antibiotics.
37
Removal of biofilm by mechanical method has been the main way of controlling
biofilm in dentistry and in industry because it is the most effective method.
The patients are prone to contamination because the hospital environment serves as
reservoir for
bacterial pathogens. It has been established that biofilm bacteria cause
device related infections (Costerton et al, 2003) and two commonly associated
pathogens in nosocomial infections that regularly form biofilms, meticillin-resistant S.
aureus (MRSA) and multi-drug resistant isolates of P. aeruginosa have been linked to
device related infections. It is therefore important to control biofilm in the hospital. S.
aureus, the most isolated organism in nosocomial bacteraemia in the UK was found to
show 30 - 50% resistance to methicillin (Diekema et al, 2001; Fluit et al, 2001; EARSS,
2003).
P. aeruginosa is associated with hospital outbreaks and it is known to be drug resistant
hence its control is important (Costerton, 2003). Prevention and treatment are two
therapeutic approaches to control biofilm. The use of aseptic measures followed by
hospital adapted room design elements, with sink availability in various compartments
can help reduce hospital transmission of P. aeruginosa and other pathogens (Smith and
Hunter, 2008). MRSA is responsible for a number of infections ranging from
pneumonia, catheters related infection, skin and surgical sites, and prosthetic implant
(Diekema et al, 2001). P. aeruginosa is a pathogen found among immuno-compromised
patients particulary those in intensive care unit in paediatrics or in burns units, and is the
cause of chronic infection as in cystic fibrosis (Govan and Deretic, 1996; Cunha, 2001;
Gales et al., 2001; Lawrence, 2002; Rossolini & Mantengoli, 2005). Incorporation of
antimicrobial agents into and onto implant materials has been used with limited success.
The infections associated with biofilm may appear to respond to systemic antibiotics
because planktonic cell respond and symptoms are reduced, but the persistence of
adherent cells leads to recurrent episodes of infection. Control of biofilm from these
38
organisms is therefore vital since biofilms have such an enormous impact on healthcare.
A review paper on biofilm control suggested that the reduced growth rate of microbial
cell within established biofilm confers reduced susceptibility to antimicrobial agent,
which in turn contributes to persistence (Cooper and Okhiria, 2006). A study was
carried out on the effects of three hospital biocides (benzalkonium chloride (1% w/v),
chlorhexidine gluconate (4 % w/v) and triclosan (1% w/v) on two nosocomial pathogens
by Smith and Hunter (2008), MRSA and P. aeruginosa, in planktonic and biofilm
cultures. The results indicated that the minimum bactericidal concentration (MBC) of
the antimicrobials for planktonic populations of the 2 organisms were less than the
manufacturer‘s recommended concentrations whereas they were ineffective against the
biofilms at a recommenced concentration for use. In the study 0-11% of the MRSA
biofilm survived while about 80% of those in P. aeruginosa biofilms survived.
The composition of biofilm in terms of the matrix has played a major role in the
prevention of the immune system from removal of biofilm from the body (Fux et al,
2005; Leid et al, 2005). In wounds, removal of biofilm is performed is mainly achieved
by debridement (Steed et al, 1996) in order to allow adequate penetration of antibiotic
through the remaining microorganisms in the wounds. Combination of physical removal
of biofilm in wounds (debridement) in combination with antibiotics anti-biofilm
strategies with traditional topical and systemic strategies in infected and chronic wounds
are mostly used to control biofilm in order for the immune system to cope the infection.
Another way to control biofilm is to use substances that prevent biofilm formation. A
study on an anti-biofilm agent bovine lactoferrin showed that Pseudomonas in the
presence of bovine lactoferrin divided normally but could not form biofilm (Singh et al,
2002; Singh, 2004). Another substance xylitol which was found to exert anti-biofilm
effects in oral bacteria (Modesto and Drake, 2006) is incorporated in chewing gum for
inhibitory impact on dental plaque (biofilm) (Katsuyama et al, 2005). Similarly, since
39
quorum sensing controls biofilm formation, quorum sensing inhibitors have been used
to disrupt biofilm formation as a means of biofilm control (Gonzalez and Keshavan,
2006). Methods for promoting biofilm detachment by using chemical agents such as
enzymes or other agents have also been applied although no much of success has been
recorded (Chen and Stewart, 2000) because biofilms are composed of various
substances and each agent can not completely remove all the components of biofilm
(Joao et al, 2005). There are many challenges ahead in the biofilm, chronic wounds and
the issue of control that may bring about improvement in clinical practice. Therefore
more research relative to biofilm control and management strategies in chronic wounds
will be relevant.
40
1.10 Aims and objectives
The aim of this study is to investigate coaggregation as a means to gain insight into the
processes involed in biofilm formation by bacteria isolated from chronic wounds.
Objectives
To perform a coaggregation assay on bacterial strains isolated from chronic wounds.
To review the network properties and investigate how this may provide information on
the effect of both coagregation and biofilm formation in chronic wounds.
To observe the development of biofilm formed by mixed bacterial population isolated
from chronic wounds.
To investigate the extent of biofilm formed by a coaggregation network from bacteria
of several species and compare it to a biofilm from a single species.
To determine the effect on biofilm biomass when selected bacteria are omitted from a
community.
41
Chapter 2
2.1 Methodology and experiments
2.1.1 Bacteria tested
Clinical isolates used in this study were isolated from chronic wounds of patients
attending the outpatient clinic of University Hospital of Wales (appendix 1) Organisms
had been isolated, identified and stored at -700 C until required (Cooper et al, 2009). A
total of 71 bacteria isolated from 17 patients with chronic wounds were used for
coaggregation analysis and 59 of these bacteria were tested for their ability to form
biofilms. P. aeruginosa NCIB 8626 S. aureus 74022 (clinical isolate provided by Mr
Alan Paull of University Hospital of Wales) were used as reference organisms in this
study.
2.1.2 Determination of the total viable count (Miles and Misra surface
drop method)
Aseptic conditions were maintained throughout the procedure. All inocula for all
experiments were expected to have a concentration of less than 105 colony forming unit
(CFU) per milliliter of broth. An initial dilution of one in hundred of the 24 hour broth
culture was made by pipetting 300 µl overnight broth culture into 29.7 ml of tryptone
soya broth (TSB; Oxoid, Cambridge UK). Sterile eppendorf tubes were used for serial
dilution of the initial dilution. 180 µl of sterile ¼ strength ringer‘s solution (Oxoid) was
distributed into seven sterile eppendorf tubes. Twenty microlitre of the initial dilution
was added to the first eppendorf tube and mixed by mechanical means. 20 µl was
transferred from this eppendorf into the next serialy diluted onto eppendorf until a 10-7
dilution was obtained. 10 µl of each dilution was plated in duplicate onto Petri dishes
containing nutrient agar (Oxoid, Cambridge UK). Petri dishes were incubated at 37 0C
for 24 hours in a gaseous atmosphere. Colonies of bacteria that grew on the plate were
42
then counted using a colony counter and the original CFU/ml calculated for each
inoculum.
2.1.3 Coaggregation assay
2.1.3.1 Growth curve assays
In other to determine the appropriate harvest times (stationary phase and mid stationary
Phase) of bacteria used in this study, two growth curves were constructed using a
culture each of P. aeruginosa NCIB 8626 and a clinical isolate of S. aureus (74022)
respectively (Rickard et al, 2002). An initial dilution of 1/1000 was made of an
overnight nutrient broth culture of the test micoorganism. One ml of this diluted
inoculum was pipetted into 99 ml TSB at time 0 and flasks were incubated at 370C in a
shaking water bath (100 rpm). At known intervals (0, 3, 6, 9, 12, 15, 18 and 24 hours)
the optical density (O.D) at 550 nm was determined using a spectrophotometer (CE
1010 Cecil, UK).
2.1.3.2 Visual coaggregation assay (Cisar et al, 1979)
Prior to the visual coaggregation assay test organisms were grown in TSB at 370C on a
rotary shaker set at 200 rpm. Organisms were harvested at two different time intervals
during early stationary phase and mid stationary phase using the growth curves prepared
above. The early stationary phase of P. aeruginosa NCIB 8626 was between 12 and 15
hours, and so this period was also used to harvest Gram negative bacteria. The mid
stationary phase of S. aureus that occurred between the 15 to 21 hours was used to
harvest the Gram positive organisms. Cells were harvested from batch cultures and
concentrated by centrifugation for 12.5 min at 3000 x g, then washed three times with
centrifugation in between in sterile de-ionised water and re-suspended to an OD.at 650
43
nm of 1.5. Pairs of test organisms were then mixed in equal volumes (200 µl) at room
temperature in 6 x 50 millimetre (mm) silica Durham tubes (Scientific Lab Supplies,
Nottingham, UK). Mixtures of organisms isolated from the same wound were vortexed
for 10 seconds and rolled gently for about 30s, then allowed to stand at room
temperature. The degree of coaggregation of each pair was scored using a semiquantitative assay originally described by Cisar et al (1979). The scoring criteria can be
seen in Table 1. Control tubes for each isolate alone were included to assess
autoaggregation (self–aggregation). Where present, autoaggregation was scored using
the same criteria and that score was deducted from the coaggregation score of a mixed
(paired) culture. Scores were reproducible after growth in batch culture for a set perid of
time, and three bacthes of all cultures were tested separately to confirm the
reproducibility of coaggregation.
Table 1. Classification of coaggregation score by Cisar et al (1979)
Score
Interpretation
‗0‘
no coaggregate in suspension
‗ 1‘
small uniform aggregates in a turbid suspension
‗2‘
easily visible coaggregate in turbid suspension
‗3‘
clearly visible coaggregate which settle leaving a clear supernatant
‗4‘
large flocs of coaggregate
that settle almost instantaneously leaving a clear
supernatant
2.1.4 Net work properties
Adjacency and connectivity matrices were constructed from selected data. The
following formulae were used to calculate various network properties:
44
n is the number of node or element, in this case the number of bacteria Size of graph
(M) – this is the number of edges of which the graph is composed (connections between
nodes/bacteria in the graph as a whole).
The average degree of graph (k) = 2M/n k, indicate how many neighbours each node
has, and thus qualified the relationship between n and M. All graphs must have k<n.
C = connectedness = M/n2
The characteristic path length (the shortest distance between one node and another (L) =
(∑di,j/n)/n where d = distance between node i and j
The clustering co-efficient (γ) = (number of realized links/ number of total possible
links) averaged over all n. This is the number of nodes connected to a node which is
also connected to another other.
2.1.5 Biofilms assay (Christensen et al, 1985)
2.1.5.1 Biofilms formation in microtiter plates
This protocol was devised by Christensen et al (1985). Organisms were removed from
the
-700C freezer, thawed at room temperature and a loopful plated onto nutrient agar
plates and incubated overnight at 370C. After being checked for the purity of the culture
and if an axenic culture was seen (ie no contamination), one colony was inoculated into
10 ml TSB and incubated for 18 hours at 370C without shaking. A one in hundred
dilution of each culture was prepared using aseptic technique as described above. Each
of the diluted inocula was mixed gently by inversion.
45
Figure 4. Diagram of a microtiter plate (from Commons.wimedia.org/wiki/File:
Microtiter_plate.JPG)
Microtiter plates (Fig 4.) were inoculated with (200 µl) of diluted inoculum per well,
leaving outer rows blank, except that wells in column 11 were inoculated with sterile
TSB alone to act as sterility control. Six wells for each diluted culture were prepared.
After inoculation, the diluted inoculum was immediately streaked onto a nutrient agar
(NA) as a purity plate. Each microtiter plate and each sterility plate was covered and
incubated at 370C for 24 hours. The purity of the cultures was checked and if there was
no contamination (i.e. an axenic culture was seen), microtitre plates were stained as
described below. For some of the inocula a total viable count (TVC) was performed to
establish the number of bacteria (to give an idea of the inoculum size). Reference
organisms were treated the same as the test organisms.
2.1.5.2 Biofilm formation using mixed bacteria cultures derived from
the same wound
After an initial dilution of one in hundred (as described above on 2.1.5.1) a pooled
culture of several bacteria from a single wound was obtained by mixing 100 µl aliquots
in sterile tubes, and then mixed gently. The mixed bacterial inoculum was inoculated
into a sterile microtiter plate as above and incubated at 370C for 24 hours.
46
2.1.5.3 Biofilms of mixed cultured without key organisms (bacteria
showing highest score of coaggregation)
For this assay the same protocol described above (2.1.5.1) was used except that the
organism with the highest coaggregation partnership was not included in the mixture.
2.1.5.4 Biofilms of mixed culture and deficiency in microbial species or
the issue of node lost
Mixed cultures were performed as above (2.1.5.1) except that in this assay one
bacterium from the same wound was omitted from the mixture. Several tests were done
and a different organism was omitted at each time to evaluate the effect of node loss
(absence of a given bacterium) on biofilm formation and on each network.
2.1.5.5 Biofilm staining
After 24 h at 370C the supernatant broth from each microtitre plate was removed into a
discard jar with a micropipette and the plate and each well washed three times with
sterile phosphate buffer saline (PBS; Oxoid Cambridge UK) ) to remove bacteria that
were not adhered to the wells. Biofilms adhered to the wells were then fixed with 2.5%
w/v glutaraldehyde for 5 minutes. Glutaraldehyde was removed after 5 minute into a
toxic waste bottle in a fume cupboard. Wells were washed three times with the use of
plate washer with PBS and stained with 0.25% w/v crystal violet for 5 minutes and
washed again with PBS. Plates were dried by blotting vigorously on paper towels. The
dye present in each well (stained biofilms) was solubilised, with 200 µl of 1:1
ethanol/acetone mixture. Absorbance was read spectrophotometrically at 570 nm using
plate reader (Dynex).
47
The extent of biofilm present was related to the optical density of crystal violet
extracted from adherent bacteria using a classification of weak, average, strong biofilms
former. The cut-off OD (ODc) for this microtiter –plate test was defined as three
standards deviation above the mean OD of the negative control. Strains were classified
as follows:
OD < ODc non adherent
ODc < OD < 2 x ODc, weakly adherent
2 x ODc < OD < 4 x ODc = moderately adherent
4 x ODc < OD = strongly adherent
2.1.6 Data analysis
Optical density readings from the microtitre plate were input to Minitab v 15. Data were
analyzed by one way ANOVA. Results were reported as being significant, where p <
0.05. The degree of coaggregation was analyzed by Pearson Chi2 test in Minitab v.15.
Significance was reported with p<0.05. (Appendix 4).
The network properties were analyzed using the network properties theory (Barabasi,
2002). Selected matrices were constructed (network) and the following were calculated:
The size of the graph (M), the average degree of graph (K), the connectedness (C) the
characteristic path length (L), and the clustering co-efficient (γ).
48
Chapter 3
3.1Results
3.1.1 Viable counts
All sample inocula were found to be less than 105 colony forming units (CFU) per
millilitre of broth.
3.1.2 Growth curves assay
Two growth curves were constructed by measurements of time versus optical density.
Immediately prior to coaggregation assay Gram negative cells were harvested between
the 12th and the 15thhour, which corresponds to early stationary phase of P. aeruginosa
NCIB 8626 (Fig 5.), and from the 15 th to the 21st hour (mid stationary) for S. aureus
7422 (Fig 6.).
Figure 5. P. aeruginosa NCIB 8626 growth curve (arrows indicate the early
stationary phase)
49
Figure 6. S aureus 7422 growth cure (arrows indicate mid stationary phase)
3.1.3 Visual coaggregation assay
In order to detect all possible coaggregation partnerships between strains, harvested
cells that have been isolated from the same wound were arranged in pair wise
combinations and tested for visual coaggregation. Scores ranged from 0 to 4 were
obtained (Fig 7. a and b).
Coaggregation Scores
1
3
4
4
3
2
0
3
4
4 0 0
0
(b)
( a)
Figure 7. Visual coaggregation score for different pairs of strains
50
Autoaggregation was observed in 3 instances in each of one culture of C.striatum, A.
heamolyticum and P.mirabilis.
A total of 164 pairs were tested (appendix 1). Of the 71 isolates, 58 were able to form a
coaggregation parternership (81.7 %) and 13 (18.3%) did not coaggregate with another
bacterium (Table 2). The degrees of coaggregation were analyzed by Pearson Chi2 test
in Minitab v.15. Significance was reported with p<0.05 (Appendix 4).
The degree of coaggregation between each pair was scored using a semi quantitative
assay originally describes by Cisar
(Cisar et al, 1979). The scoring criteria were
described on page 44. Bacteria tested showed variation in their ability to form a
coaggregation partnership, but 58 of the 71 strains tested coaggregated with one or more
partner strain (Table 2). This result showed that coaggregation is a common
phenomenon among bacteria isolated from chronic wounds.
Table 2. Coaggregation partnerships obtained from bacteria isolated from chronic
wounds.
Organism
Number tested
Coaggreg.
12
%
Coaggreg.
86
Non
Coaggreg
2
Coaggreg.
pairs
27
S. aureus
14
C. striatum
12
9
75
3
27
P. aeruginosa
9
8
89
1
18
E. faecalis
7
6
86
1
15
P.mirabilis
6
5
83
1
15
Strept sp.
5
4
80
1
17
Others species
18
14
78
4
45
Total number
71
58
N/A
13
164
% of total
N/A
81.7%
N/A
18.3%
N/A
51
It can be seen that of the species listed in Table 2, the lowest percentage of culture able
to form coaggregation partnerships was found in the C.striatum culture at 75% (i.e
9/12). The highest was in the P. aeruginosa cultures (89%).
Amongst the bacteria testd in this study, some culture were shown to form
coaggregation partnerships with six others bacteria (Table 3). These included not only
those bacteria such as Staphylococcus and Corynebacterium and Pseudomonas which
are commonly isolated from chronic wounds, but those less commonly reported such as
Gram negative bacteria (Cooper et al , 2009).
Table 3. Coaggregation frequencies of selected bacteria
Species
Number of partnerships
S. aureus (RC03)
5
C. striatum (JT17)
3
P. aeruginosa (JS13)
6
E. faecalis (MS04)
2
Strept. anginosus (JS13)
6
P. mirabilis (RC03)
5
C. amalonaticus (JF06)
5
Porphyromonas spp (SG09)
3
M. morganii (AP12)
4
3.1.4 Matrix obtained from constructed network
In order to investigate the network of coaggregation partnerships, a matrix from five
selected wounds was constructed (Figure8, 9, 10, 11, 12). Selection criteria were based
52
on the number of bacteria per wound (at least 3 bacteria and the number of
coaggregation partnership were at least 4).
Seven bacteria has been isolated from wound one (patient JS13). For the purpose of this
matrix they are labelled as follows: i) Strept. anginosus, j) Enterob. cloacae, k) S.
aureus l) E. faecalis, m) P. aeruginosa, n) K. oxytoca, o) M. luteus.
3.1.4. 1 Matrix from wound 1
Strept. Anginosus and P. aeruginosa formed 6 coaggregation partenerships each. (Fig.
8) The remaining bacteria were able to connect with only two bacteria at a time except
S. aureus that connected with 3 bacteria. From the constructed matrix it is possible to
suggest what would happen if the most connected bacteria were to be removed
sequentially, or if there was a random removal of any given bacterium. In this network
organisms like S. aureus seem to produce no significant impact on the overall physical
net work, but in the biofilm assay the absence of this bacterium showed some changes
in the intensity of biofilm changes that will be discuss later.
Organisms found in wound one (from patient JS13)
i) Strept. anginosus
j) Enterobact. cloacae
k) S. aureus
l) E. faecalis
m) P.aeruginosa
n) K. oxytoca
o) M. luteus
i
o
n
l
j
k
m
Figure 8. Coaggregation network of wound 1
53
3.1.4.2 Matrix from wound 2
The wound of patient two had a less connected system than the wound of patient one
and each of the bacterium was connected with at least three bacteria (Fig. 9). This
matrix suggested that total disruption of the network would not occur if there were to be
sequential or random removal of any member. This is because, unlike in wound one, no
bacterium appeared to be more connected than the rest and the removal of a single
bacterium would have less physical effect to the network as a whole. Here the
community of bacteria proved to be more important than any single bacterium.
i
j
l
k
m
n
Figure. 9 Coaggregation network in wound 2
Wound 2 Organisms (from patient JF06)
i) Strept. agalactiae
j) Citrobact. amalonaticus
k) P. aeruginosa
l) C. striatum
m) S. aureus
n) P. mirabilis
54
3.1.4.3 Matrix from wound 3
The wound of patient 3 had the same characteristic as wound 2 and here again each
bacterium was highly connected so there was no major impact expected on the overall
network if sequential or random node removal were to be carried out (Fig.10). We can
observe that known wound pathogens as well as commensal bacteria showed a high
degree of connectivity. Once again the integrity of the overall matrice was dependent on
the entire bacteria community.
j
l
k
i
n
m
Figure 10. Coaggregation network in wound 3
Wound 3(from patient RC03)
Organisms
i) P. mirabilis
j) S. aureus
k) C. striatum
l) E. faecalis
m) Strept. agalactiae
n) P. aeruginosa
55
3.1.4.4 Matrice from wound 4
Wound 4 showed a different picture from the other matrices. P. aeruginosa and
S.aureus are the most connected bacteria and if one were to be removed an organism
like C. striatum would remain connected to only one bacterium andtherefore have a
weak bond with the overall network (Fig. 11). Integrity of the network in this case
depends on these bacteria ( P. aeruginosa and S.aureus) rather than whole bacteria
community .
i
j
l
k
m
Figure 11. Coaggregation network in wound 4
Wound 4 (from patient JT17)
Organisms
i) P. aeruginosa
j) S. aureus
k) E. faecalis
l) Enterobact. claocae
m) C.striatum
56
3.1.4.5 Matrice from wound 5
Wound five has the characteristics of both wounds one and four (Fig. 12). The high
connectivity of bacteria was seen as in wound four but on removal of Citrobact feundii
for instance the resulting net work is made of weak bonds the removal of B. cepacia
and Citrobact. fundii at the same time leave S.aureus with no connectivity with the
network in this case the ―vulnerability‖ of a potential wound pathogen was recognised
if any commensal bacteria were to be removed from the network.
i
l
k
j
n
m
Figure 12. Coaggregation network in wound 5
Wound 5 (from patient CR01)
Organisms
i) K. pneumoniae
j) P. aeruginosa
k) B. cepacia
l) C. striatum
m) Citrobact. feundii
n) S. aureus
57
3.1.5 Analysis of networks
3.1.5.1 Network properties
The network characteristics of the selected chronic wounds based on coaggregation
partnerships of at least 4 bacteria are summarised in Table 4.
The number of bacteria found in each of the wounds was relatively small and therefore
gave rather small networks. Wound environments can be considered to be somewhat
limited in the area of coaggregation and biofilm studies in relation to the number of
bacteria found. In the two most highly studied coaggregation systems, dental plaque and
freshwater, for instance the number of bacteria can be far greater and this gives bigger
networks. Coaggregation ability of freshwater biofilm was studied and 28 out of 29
biofilm strains coaggregated with one or more planktonic strains given a total of 95 of a
possible 406 coaggregation partnerships (Rickard et al, 2003). Dental plaque represents
the most relatively well-describe system in terms of coaggregation partnerships with
more than 1000 oral bacterial strain shown to coaggregate (review paper Kolenbrander
et al, 2000; 2002). Barabasi (Barabasi et al, 2002) recognised the existence of small
networks, and proposed that there are many nodes linked to the network via just one
connection: fewer have two, some three, and so forth. Unlike an exponential network,
these remain small but with significant number of nodes connections (Ball, 2000).
The network formulae used for the construction of the various matrix in this study are
based on mathematical methods described by Albert and Barabasi (2002). The number
of nodes (bacteria) for all patients was ≥4≤7
The numbers of edges (M) in wound 3, are the highest followed by wound 2, wound 1
and 5. Wound 4 had the smallest number of edges. The average degree of graph was
greater in wound five while the characteristic path length (L) was greater in wound 1.
Wound 3 had the highest connectedness (C). All five wounds had a higher probability
that a node will be connected (γ) and this was due to the small sample sizes of the
58
network constructed in this study. An example of calculation details of each parameter
below can be found in appendix 2.
Table 4. The network characteristic of chronic wound bacteria base on
coaggregation assay
Network
Characteristics
Wound 1
Wound 2
Wound 3
Wound 4
Wound 5
n
7
6
6
5
6
M
11
12
15
7
10
K
3.1
4
5
2.8
3.33
L
1.66
1.2
1
1.25
1.5
γ
0.64
0.67
0.71
0.43
0.51
C
0.224
0.33
0.41
0.28
0.27
M/n
1.57
2
2.5
1.4
1.6
n = number of nodes = number of bacteria
M = number of edges = size
K = the average degree of graph 2M/n
L= characteristic path length
(γ) = clustering co-efficient = average of the number of realized link/average of total
possible link
C = connectedness = M/n2
M/n = links per species
NB. (Definitions of these terms are given in section 1.6.4 and an example of calculation
in appendix 3)
59
3.1.5.2 Effect of node loss
Probability that a node (bacterium) will be connected in this study was high (γ values in
table 4) and results in the survival of a network and leads to the formation of a
secondary network, with at least three nodes remaining connected. This reduces the
network by about 50% in size (figures 9-12). However the removal of the two most
connected nodes in wounds 1 and 4 (figures 8-11) would result in a breakdown of the
main network and the formation of a secondary network of two nodes. This reduces the
network by 40 % in size.
3.1.6 Biofilms formation in microtitre plate
3.1.6.1 Biofilms formation of individual bacteria
The ability of individual isolates to form biofilm was tested by the method of
Christensen el al, (1985) using a 1/100 dilution of overnight nutrient broth culture. 59
clinical isolates derived from chronic wounds and two reference organisms were tested.
Standard microorganisms were P. aeruginosa NCIB 8626 and S. aureus 74022. The
extent of biofilms formed was related to the optical density of crystal violet extracted
from adherent bacteria (Fig 13.). Each culture was replicated six times, and the mean
optical densities reported
Figure 13. Stained biofilms in a microtiter plate (Organism from Patient PP15) Well
2) S. aureus, 3) P. aeruginosa 4) P. mirabilis, 5) Strept. species (group G), 6)
Peptostrept. magnus, 7) E. faecalis, 8) empty well, 9) C. striatum C+) positive control
(P. aeruginosa NCIB8626), B) negative control (blank, TSB alone.
60
The quantitative assay for the biofilm formation (method described in section 2.1.5.1)
was performed according to the method described by Christensen et al. (1985), the 96
well flat bottomed plastic tissue culture microtite plate above was used. It provided a
measure of the rate of adherence and hence biofilm biomass formed by each of the
tested bacteria. The adherence capacity of a bacterium can be appreciated
macroscopically by the deep purple crystal violet sediment. For instance well 2.3,5,7
represented respectively by S. aureus P. aeruginosa, Streptococcus species (group G)
and E. faecalis are more adherent (deep purple) than P. mirabilis in well 4, Peptost.
magnus in well 6 and C. striatum in well 9 (light purple). The positive control
represented by P. aeruginosa NCIB8626 also appears deep purple compare to the
negative control(B) that show no colour as TBS alone was used.
3.1.6.2 Screening for biofilm formation
All 59 (100%) of the clinical bacteria isolated from chronic wound indicated an ability
to form biofilms in vitro with varying degrees of adherence (Table 5). Using
Christensen classification strains were classified into the following categories (Fig. 14):
non adherent (0), weakly (6), moderately (9), or strongly (44) adherent. The cut-off OD
(ODc) for the microtiter plate test was defined as three standard deviation above the
mean OD of the negative control (Calculation in appendix 3).
61
50
No of organisms
45
40
35
30
25
20
15
10
5
0
Non adherent
Weakly adherent Moderate adherent
Strong adherent
Classified groups
Figure.14 Classification of the result of biofilms in microtiter plate into 4
categories.
Bacteria in this study were all able to form biofilms in vitro and those with strong
adherence were greater in number than any other group. Those that showed weak
adherence were the least with moderate adherence occupying the middle position.
Adherence properties of the most common species tested in this study are given in
Table 5.
62
Table 5. Adherence of various single species of bacteria isolated from
chronic wounds in microtiter plate
species
Number
of
bacteria
tested
number of bacteria showing
NA
WA
MA
SA
S.aureus
11
_
1
_
10
P.aeruginosa
11
_
_
_
11
C. striatum
11
_
1
1
9
P. mirabilis
4
_
1
2
1
Strep. spp
4
_
1
2
1
E. faecalis
7
_
_
2
5
Others spp.
11
_
2
2
7
TOTAL
59
0
6
9
44
%
100
0
10.16
15.25
74.57
NA= no adherence, WA= weak adherence, MA= Moderate adherence, SA=Strong
adherence
Statistical analysis, one way ANOVA, of the optical density obtained from the 1/100
inoculum and p value <0.05 were considered significant for ability to form biofilm
(Appendix 4). Given confirmation that a higher proportion of bacteria isolated from
chronic wounds in this study were able to form biofilm in vitro
3.1.6.3 Biofilms formation with mixed cultures of bacterial isolates
Wounds containing at least 4 bacteria were selected for this assay where selected
bacteria were removed. A total of 5 wounds with 31 bacteria (5 patients out of 16) were
studied. The five mixed cultures were strongly adherent however some individual
63
bacteria showed weak and moderate adherence (Table 6). Details of calculation of OD
found in appendix 3.
Table 6. Adherence of mixed cultures of bacteria isolated from chronic wounds in
microtiter plate /coaggregation score
Wound
Biofilm
adherence
highest coaggregation
mixed
node with the
partnership in
highest
each group
coaggregation
included)
2.2
factor
Strongly
0.26
adherent
1.3
Strongly
1.9
Strongly
0.38
1.5
Strongly
0.22
3.4
Strongly
adherent
Moderately
Citro. amalonaticus 5
adherent
P. mirabilis 5
weakly
0.68
adherent
5
Strept. anginosus 6
adherent
adherent
4
adherent
weakly
adherent
3
Bacteria with the
biomass of the
isolate
2
adherence
biomass of
culture (all
1
Biofilm
Strongly
P. aeruginosa 4
adherent
0.10
adherent
K.pneumoniae 4
weakly
Selected wounds 1, 2,3,4,5 from table 6 show that the mixed culture of their various
individual bacteria gave strongly adherent attached cells with high biofilm biomass.
However the biofilm biomass of any bacterium with the highest coaggregation
parternership in each wound gave a reduce OD indicating that the biofilm produce by a
community of bacteria is greater to that of a single bacterium irrespective of its ability
to coaggregate. Biofilm biomass was expressed as OD of crystal violet stained cells at
550 nm. Three out of five bacteria with the highest score of coaggregation showed a
weak adherence, one had a moderate adherence and one showed a strong adherence
(Table 7) indicating that a greater ability to coaggregate do not always imply strong
64
ability to form biofilm. However some individual biofilm biomass show that some
individual bacteria biofilm production account for a higher percentage in the overall out
put ie P. aeruginosa alone gave an OD of 0.68 while the mixture accounted for 1.5.
Table 7. Adherence of selected single species of bacteria isolated from chronic
wounds in microtiter plate/adherence of mixed culture
wound
number of bacteria showing adherence
Number of bacteria tested
in the mixure
NO
weak
Moderate
Strong
adherence
adherence
adherence
adherence
1
7
_
2
_
5
2
6
_
1
1
4
3
6
_
1
0
5
4
6
_
2
_
4
5
6
_
1
1
4
_
_
_
5
Mixed
cultured
3.1.6.4 Biofilm of mixed culture without key organisms (bacteria
showing highest score of coaggregation)
There were no differences between mixed cultures of isolates from each of selected five
wounds in their ability to form biofilm and the extent of biofilm produced when the
bacterium with the highest coaggregation score was taken out of the mixure in terms of
the interpretation of biofilm (i.e strongly adherent). However there were some
numerical differences in the culture density. e.g. bacteria in wound one mixed culture
gave an OD of 2.2 and an OD of 1.8 when Strept. anginosus was removed although
both had been rated as strongly adherent (table 8). In wound 5 the optical density of
the mixed culture was 3.5 but following the removal of P. aeruginosa the OD was 1.1
(organism here were also rated strongly adherent). Bacteria in wound 3 gave the same
65
OD of 1.9 of the mixed culture even when P. mirabilis was removed. Mixed culture
from wound 4 gave an increase in OD from 1.7 to 2.4 after the removal of K.
pneumonia (Table 8).
Table 8. Biofilm formation of bacteria isolated from patient 1 the in mixed culture,
each without one respective node.
WOUND 1
OD of mixed
% loss
Adherence
2.2
N/A
SA
Strept. anginosus*
1.8
18.2
SA
Enterobact. cloacae*
1.7
22.8
SA
S. aureus*
0.9
59.1
SA
E. feacalis
1.2
45.5
SA
P.aeruginosa*
0.7
68.2
SA
K. oxytoca*
1.4
36.4
SA
M. luteus*
1.7
22.8
SA
cultures
All organisms of wound1
*= bacteria omitted from the mixed culture
The greatest lost of biofilm biomass in wound 1 was observed with the omission of P.
aeruginosa (68.2%).
Table 9. Biofilm formation of bacteria isolated from patient 2 in the mixed culture,
each without one respective node
WOUND 2
OD of mixed cultures
% loss
Adherence
All organisms of wound 2
1.3
N/A
SA
Strept. agalactiae*
1.1
15.4
SA
Citrobact. amalonaticus*
0.7
46.1
SA
P. aeruginosa*
0.7
46.1
SA
C. striatum*
1.1
15.4
SA
S. aureus *
0.8
38.5
SA
46.1
SA
P. mirabilis*
0.7
*= bacteria omitted from the mixed culture
66
In patient 2 the removal of any one member of the community from the mixture led to a
reduction of OD but the most noticeable was for Citrobact. amalonaticus, P.
aeruginosa, S. aureus, therefore it is on these three bacteria that the greatest loss on
biofilm biomass occured but the adherences remain the same that is strongly adherent.
In wound 3 the greatest lost in biofilm biomass occurred with the removal of S. aureus
as shown below.
Table 10. Biofilm formation of bacteria isolated from patient 3 in the mixed
culture, each without one respective node.
WOUND 3
OD of mixed cultures
% loss
Adherence
All organisms of wound 3
1.9
N/A
SA
P. mirabilis*
1.9
00
SA
S. aureus *
0.1
94.8
WA
C. striatum*
0.7
63.1
SA
E. faeallis*
0.8
57.9
SA
P. aeruginosa*
0.4
78.9
WA
Strept. gr G
0.4
78.9
WA
*= bacteria omitted from the mixed culture
The mixture of bacteria in wound 3 have also shown that a bacterium can be removed
without an effect on the overall OD of the mixture e.g the removal of P. mirabilis did
not affect the OD of the mixture in any way on like the removal of S aureus, P.
aeruginosa, Strept. gr G that resulted in a week adherence (Table 10).
67
Table 11. Biofilm formation of bacteria isolated from patient 1 in the mixed
culture, each without one respective node.
WOUND 4
OD of mixed cultures
% loss
Adherence
All organisms of wound4
1.7
N/A
SA
E. faecallis*
0.7
58.9
SA
S. aureus *
0.9
47.1
SA
P. aeruginosa
0.7
58.9
SA
Strept. anginosus
1.1
35.3
SA
K. pneumoniae*
2.7
58.8 increase
SA
M. luteus*
1.4
17.6
SA
*= bacteria omitted from the mixed culture
The removal of a bacterium in this mixture reveals it possible that the presence of a
given bacterium could supress biofilm formation in vitro e.g the removal of K.
pneumoniae gave an increase in the OD however the greatest lost was observed with the
removal of P. aeruginosa and E. faecallis (Table 11).
Table 12. Biofilm formation of bacteria isolated from patient 5 in the mixed
culture, each without one respective node.
WOUND 5
OD of mixed cultures
% loss
Adherence
All organisms of wound5
3.5
N/A
SA
K. pneumonaie*
3.4
2.8
SA
P. aeruginosa*
1.1
68.6
SA
B.cepacia*
3.0
14.3
SA
C. striatum*
3.1
11.5
SA
Citrobact. freudii*
3.2
8,6
SA
S. aureus*
3.4
2.8
SA
*= bacteria omitted from the mixed culture
The removal of P. aeruginosa had a noticeable impact on biofilm biomass yet the other
bacteria showed little or no real change to the OD mixture after their individual removal
(Table 12)
68
3.1.6.5 Biofilm of mixed culture the issue of node lost (omission of one
bacterium at a time in the mixture)
The removal one bacterium at a time for all combinations in the biofilm assay gave no
difference to the behavior of all mixed cultures according to the Christensen
classification irrespective of which bacterium was removed. However reductions in
optical density were often observed when some preferential bacteria were omitted in the
mixtures (Table 8, 9, 10, 11, 12). To evaluate the effect of these preferential bacteria,
biofilm formation in selected paired cultures were carried out. Results showed that some
bacteria when paired with P. aeruginosa, S. aureus or K. pneumoniae gave an increase
in OD compared to when paired with other bacteria (Table 13).
Table. 13 Biofilm formation of paired cultured/ coaggregation partnership of
bacteria isolated from patient 5
Paired bacteria
P. aeruginosa/ B. cepacia
Biofilm Adherence
biomass
Of paired
(550nm)
bacteria
2.9
SA
Number of Coaggregation
partnership of each
bacterium
4:4
P. aeruginosa/ C.striatum
2.8
SA
4:2
P. aeruginosa/ C. freudii
3.0
SA
4:3
P. aeruginosa/ S. aureus
2.8
SA
4:1
P. aeruginosa /K.pneumoniae
2.9
SA
4:4
S. aureus/ B. cepacia
2.9
SA
2:4
S. aureus / C.striatum
2.4
SA
2:2
S .aureus / Citrobact. freudii
2.3
SA
2:3
S. aureus/ K.pneumoniae
3.1
SA
2:4
B. cepacia /C.striatum
1.3
SA
4:2
B. cepacia /Citrobact. freudii
1.1
SA
4:3
K. pneumoniae/ B. cepacia
2.6
SA
4:2
K. pneumoniae/ C.striatum
2.8
SA
4:2
K. pneumoniae/ Citrobact. freudii
2.6
SA
4:3
SA=strongly adherent
69
In the above table it was possible to observe that organism like B. cepacia gave an
increase in OD When pair with P. aeruginosa (2.9) but a decrease OD of 1.3 when
paired with C.striatum. Similar result can be observed with C. freudii when paired with
P. aeruginosa (3.0) but a decrease in OD when paired with B. cepacia (1.1). The
number of coaggregation partenership of individual bacteria seems not to influence the
OD of the paired mixture as it can be observed that in 4:4 ratio of coaggregation
partnership the biofilm biomass of paired bacteria were 2.9. In a 4:3 ratio the OD
observed are 3.0, 1.1 and 2.6. In a 2:2 ratio OD is 2.4 (table13).
70
Chapter 4
4.1 Discussion
This is the first study to investigate the ability of bacteria isolated from chronic wounds
to coaggregate in vitro and to determine whether cell to cell interactions might influence
biofilm formation in vitro, and possibly also in the chronic wound. It was shown that the
percentage of bacteria able to coaggregate (81.69) was greater than that of noncoaggregating bacteria. Furthermore all the bacteria included in the study had an ability
to form biofilm in vitro, which supports a previous observation (Rickard et al, 2003)
that a significant proportion of biofilm strains can coaggregate. Bacteria known to be
biofilm formers as well as major wound pathogens and inhabitants of chronic wounds
have been shown to form high coaggregation partnerships (Mousa, 1997; Vindenes and
Bjerknes, 1995).
4.1.2 Relationship between coaggregation ability and growth phase
The growth phase of two known wound pathogens was used prior to coaggregation
assay to harvest bacteria samples used in this study. The rationale behind this was from
previous studies on coaggregation that gave evidence that this phenomenon is growth
phase dependent (Rickard et al, 1999 and 2000). The first observation of growth phase
dependency of coaggreagation was with aquatic bacteria. In B. natatoria and in a strain
of M. luteus, for instance, coaggregation is said to be maximum in the stationary phase.
Studies on coaggregation have also shown that it is possible for a bacterium to gain and
lose its coaggregation ability during growth phase (Rickard et al, 2000). An experiment
where three differents pairs species of M. luteus and that of Blastomonas was carried
out showed that the first pair (B.natatoria 2.1 and M. luteus 2.13) developed the ability
to coaggregate during exponential phase, reaching a maximum score of 4 in stationary
phase, the second pair (M.luteus 2.13 and B. natatoria 2.8) coaggregated optimally upon
71
entry into stationary phase reaching a score of 2, the third pairs (B.natatoria 2.1 and
B.natatoria 2.8) coaggregated only in late stationary phase but all pairs lost ability to
coaggragate as they entered the decline phase. In this study bacterial growth patterns
were not characterised individually but all harvested at a selected time based on the
growth curve of two known pathogen (as mentioned earlier). Even though the result
gave a significant amount of coaggregating bacteria, the outcome of what would have
happen if pairs were matched following their genetic closeness remains unknown.
However bacteria in this study were harvested in the stationary phase and results
confirm previous studies that revealed a great proportion of bacteria do coaggregate in
the stationary phase (Rickard et al, 2002). It is possible however that the proportion of
coaggregating bacteria could have increased given the specific harvest time of
individual bacteria. Studies on P. aeruginosa indicate that quorum-sensing or cell to cell
communication is required to make a robust biofilm under some growth conditions
(Devies et al, 1998). A previous study has shown that biofilm accumulation for the wild
type of P. aeruginosa was more rapid than the quorum –sensing deficient mutant with a
rapid accumulation phase at the stationary phase higher than the mutant (Pei-Ching and
Ching-Tsan, 2007). Biofilm formers that are major wound pathogens (like P.
aeruginosa and S. aureus) have been known to quorum sense, and during this
phenomenon a bacterial cell activates specific genes in response to chemical signals
released by the cells themselves into the environment. The conditions that precipitate
this response could be stress due to reduction in nutrient availability or when a threshold
concentration of the signal chemical is achieved, at high population density. It is
possible that in this study P. aeruginosa and Gram negative bacteria had
a better
chance to form biofilm because of the increase in the production of quorum sensing
molecules known to occur at early stationary phase. Studies on S. aureus on the other
hand have showed that active quorum sensing prevents attachment and development of
72
a biofilm (Vuong et al, 2000; Beenken et al, 2003), and that low agr (the genes
produced during active quorum sensing of S. aureus) is important for biofilm
development. To prevent this, S. aureus and all Gram positive bacteria were harvested
at the mid stationary phase where productions of
quorum sensing molecules are
believed to be reduced. It is also possible that harvesting Gram positive bacteria at mid
stationary phase also increased both the ability to coaggregate and to form biofilm in
vitro.
4.1.3 Coaggregation assay
The present findings suggest that bacteria recently isolated from chronic wounds have
the ability to coaggregate. Coaggregation was not the only cell to cell interaction found
in this study as autoaggregation did occur (24 out of 71 strains), but unlike
coaggregation that is said to occur among genetically distinct bacteria (Kolenbrander,
2000), autoaggregation is a cell to cell interaction that increases the integration of
genetically identical strains. It is possible that when bacteria get access to a wound as
single cells in suspension (planktonic cells), they directly adhere as a primary colonizer
to the wound surface or recognize and adhere to each other as pairs and under
favourable conditions they directly adhere to a surface, multiply and begin biofilm
development. These coadhered cells could also directly integrate to an already preexisting biofilm as secondary colonizers. These suggestions agreed with previous
studies on coaggregation between bacteria in human dental plaque (Kolenbrander et al,
1999) and to proposed theory on the development of multispecies biofilms (Rickard et
al, 2003b). Furthermore, it is possible that a strong ability to recognise several bacteria
and coaggregate (ability to have a +3 or +4 score) confers on bacteria the potential to
recruit a variety of bacteria into a biofilm, encouraging by so doing to form a multispecies biofilm like dental plaque (Rickard et al, 2003b)
73
4.1.4 Microtiter plate assay
This assay demonstrated the ability of bacteria isolated from a chronic wound to form
biofilm in vitro and supports the findings of a previous study where the presence of
biofilms in chronic wounds was established (James et al, 2008). All 59 of the isolates
tested in this study were biofilm
formers (100%). The microtiter plate method used
here is an established means to provide a suitable way to determine the quantity of
biofilm produced by bacteria, however this study showed that the Christensen
classification did not take into account differences in absolute numerical values of test
result by putting into the same category two numerically different results so that an OD
0.7 and that of 3.5 are all classified as strongly adherent (Table 5). Readings over 3.5
were not quantified by the microtiter plate but obviously indicated strong adherence.
Therefore a method that will separate more subtle abilities of biofilm formation is
needed.
4.1.5 Coaggregation, biofilm and the issue of chronic wound
Bacteria used in this study were isolated from patients with chronic wounds (Cooper et
al, 2009). Each wound had a range of different species of bacteria and confirmed the
idea that bacteria colonizing chronic wounds exist in polymicrobial communities
(Bowler et al, 2001). Communities of microorganisms are likely to exist in their own
microenviroment in chronic wounds. Studies on coaggregation and biofilm formation of
bacteria present in dental plaque have shown that there are pioneer bacteria that are
important in the formation of biofilm. This study suggests that the ability of a bacterium
to form a coaggregation partnership with more than one other organism bacterium may
gives it the potential to initiate attachment which creates a microenviroment that
encourages secondary bacteria to colonise and begin to create a complex multi-species
community of microorganism. Most research to date has focused on the role of key
74
pathogens in a wound rather than the effect of all members of the microbial
communities. This study has shown that even though organisms like P. aeruginosa
could be a pioneer for biofilm formation, it could function as a bridging organism in a
similar way to Fusobacterium nucleatum in dental plaque. However other bacteria that
inhabit a chronic wound may also be pioneers since they also have property of cell to
cell interaction. It is possible that where the secondary colonizer also has coaggregating
ability, this increases recruitment and tends to promote the formation of a complex
multi-species community. This scenario has been validated in dental plaque as
mentioned earlier. As proven in dental plaque and fresh water biofilm, it is possible that
coaggregation enhances biofilm formation even in chronic wound. Results from this
study show that a biofilm from a mixed culture is robust compared to a biofilm formed
by a single species (Table 6), suggesting that influence of microbial community is
important as well as the role play by individual bacteria inhabiting chronic wounds.
Knowing which bacteria are likely to adhere with a given bacterium could give an
overview about the expected bacterial community. These suggestions have been
validated in dental plaque and freshwater coaggregation studies and for both areas
phylogenetic trees have been proposed (Rickard et al, 2002: 2003a). In wound
environments some bacteria may be unable to satisfy the requirement to initiate disease
alone but combining forces in a multiple-species to form biofilms may increase ability
to cause disease. This study has, therefore, shown that strong, moderate, weak biofilm
formers as well as non coaggregating, and strong coaggregating bacterial pathogens and
commensals have been found in wound enviroments. This may suggest that some
bacteria thrive in an environment where they are not alone. This senario has been
documented for gum and periodontal disease (Brook et al, 1987) and leg ulcers. It is
possible that communal protection from phagocytocis, production of growth factor,
modification of local environment and protection of sensitive bacteria by production of
75
inactivating enzyme are the ″benefit″ of a life in a multi-species biofilm. Pathogens
found to be sensitive may be rendered resistant by other members of the mixed
infection. Treatment in this case will be focus on community as well as single bacterium
(Brook, 1989; 1996).
4.1.6 Biofilm and wound healing
Biofilm as a barrier to wound healing was not addressed in this study, however in the
light of its findings the debate as to the overall role of the biofilm in delaying healing
remains open. There is an increasing evidence to support the existence of biofilm in
chronic wounds (James et al, 2008). Chronic wounds are known to be resistant to
antimicrobial treatment. In this study all 59 clinical isolates tested were able to form
biofilm in vitro. Knowing that bacteria in biofilm are up to 1000 times more resistant to
antibiotics that their planktonic counterparts (Prosser et al, 1987), it is possible that
biofilm offers an explanation for the pathophysiology of nonhealing wounds as
proposed by some authors (Schultz, et al 2003). A group of scientists also found
similarities between bacterial infections found in cystic fibrosis patients and that of
chronic wounds. They hypothesized that the presence of a biofilm is the main cause of
immune system failure to eradicate chronic infection and also why chronic wounds will
not heal (Bjarnsholt et al, 2008). It is also possible that the polymicrobial nature of
chronic wounds and the resulting biofilms result over time to chronic infection just like
in previous studies (Van Steenbergen et al, 1984) carried on periodontal disease, where
a diverse community of microorganisms acts in consort over time to result in a chronic
infection. The extracellular polysaccharide matrix (EPS) or the glycocalyx prevents the
access of antibiotics to the bacterial cells embedded in the biofilm community. Recent
suggestions (Wolcott and Rhoads, 2008) that treatment of chronic wounds that includes
antibiofilms agents to improve the healing outcome may well be justified. This practice
76
in the United State of America called Biofilm-based wound care (BBWC) management
has been demonstrated to provide a strategy that significantly improves healing
frequency.
4.1.7 Network theory and the issues of node loss
The decrease in characteristic path length with the increasing connectance (Table 4) is
in agreement with other network properties. The characteristic path lengths of the
constructed matrix were short which is consistent with a small world topology and
observations of most real-world networks (Albert and Barabasi, 2002). However the
clustering coefficient was high in constructed network (tendancy several bacteria to be
connected to one same bacterium) this is generally inapplicable to small word topology
(Dunne et al, 2002). This may be due either to the relatively small sample size or that
biofilm networks do not display a typical small word topology.
Some networks showed an increased vulnerability to a node removal when the most
connected bacteria were removed (Fig 8.) as opposed to a random node removal
(Fig10.). This agrees with other real –world networks (Barabasi, 1999). The presence of
key bacteria in the formation of biofilm through coaggreagtion is part of the general
universal biofilm network topology (Austin and Rodgers, 2004). However this work
suggests that all members of the biofilm community are close neighbours and that
treatment that focuses on the whole biofilm system is a better option.
The sample size in chronic wounds was relatively small compared to other network
environments. The resulting networks were also of small size and the connectedness
high (close neighbours). Results showed that the probability of a node (bacterium) to be
connected was high and this could suggest that each bacterium will always be connected
either to a bacterium to which it will have a cell to cell recognition or to an already
connected bacterium. Destruction or removal of one node will therefore affect the next
77
connected node and vice versa the survival or the virulence of one node will definitely
affect the next node and all connected nodes. This study confirms the work of Watts,
(1971) on several networks who came to the conclusion that this world is a ―Small
World‖. The small-world phenomenon formalizes the anecdotal notion that you are only
ever six ―degrees of separation‖ away from anybody else on planet. Even when two
people do not have a friend in common, they are separated by only a short chain of
intermediaries. It is possible that treatment that focuses on breaking the entire
community network will improve healing outcomes.
4.1.8 Clinical implications
Even though the pathogenesis of biofilms in infectious diseases is still a controversial
topic among researchers, this study has confirmed once more that bacteria isolated from
chronic wounds can form biofilms in vitro. According to Daniel Rhoads (South
Regional Wound Care Centre, Lubbock, TX, USA) biofilms are the new threat to health
and he believes:‖the developing world is still struggling with acute infections like
cholera and malaria. The developed world has largely overcome these diseases, and is
now facing a foe that it does not know how to conquer: chronic bacterial infections
associated with biofilms‖. This work has also shown that bacteria inhabiting chronic
wounds have the ability to coaggregate. If more research confirms this and proves
coaggregation actually influences biofilm formation and species diversity even in
chronic wounds then preventing coaggregation could be another away to prevent and
control biofilm formation. Knowing how hard it is to eradicate biofilm, a more gentle
approach in treating and controlling biofilm could be considered through research to
find means to prevent in vivo cell to cell recognition. In fact it has been reported that
honey interferes with adherence ability of P.aeruginosa to surfaces (Lerrer et al, 2007).
Further research on how to prevent coaggregation interaction at the cell surface receptor
78
where cell to cell recognition is believed to occur could be a new target for
antimicrobial agents. Furthermore mature biofilms are known to be more recalcitrant to
therapy (Costerton et al, 1987; Amorena et al, 1999; Stewart, 2001) than young
community of cells, therefore treatment focus in preventing biofilm maturation may
improve healing outcome better than facing the mature complex multispecies biofilm.
Scientists have provided some answers as to how, why, biofilms have an increased
resistance to antibiotic (Stewart 1996; Fux et al, 2005), biocides (Stewart et al, 1994;
Stewart et al 2000) and host defences (Lam et al, 1987; Leid et al, 2005). Having also
confirmed the presence of biofilm in chronic wounds it is suggested that biofilm
community bacteria should not be overlooked even though disupting the biofilm does
not directly kill bacteria (Wolcott, 2007), as perturbations have proved to help other
alternative therapies and natural host mechanisms to work more effectively to promote
healing (Chaignon et al, 2007). Signal molecules could also constitute a target for
antimicrobial agent as already proposed by others researchers (Bjarnsholt et al, 2005).
Networks obtained from constructed matrices in this study have confirmed that bacteria
inhabiting chronic wounds live in a close polymicrobial community and have thrown
more light on the important role played by this community. The community as a whole
should therefore be considered when deciding on treatment options, bearing in mind
that the species diversity present in wounds is often three-fold greater than what
standard culture results demonstrate (James et al, 2008). Currently, there are no clear
methods for the diagnosis of a biofilm infection in chronic wound, except clinical
suspicion and clinical presentation. A sound diagnostic technique is therefore crucial.
Care of a chronic wound poses enormous material and patient costs. If an effective anti–
biofilm treatment were to be developed that would accelerate wound healing, the impact
will be very significant indeed.
79
4.1.9 Conclusion and limitations
This study has presented evidence that bacteria with the ability to form biofilms are
present in chronic wounds. Biofilm comprised of mixed species are likely to be more
adherent than that of a single species suggesting that they form a more robust biofilm.
Even though certain key bacteria are important in how biofilms are established, the
polymicrobial communities as a whole must be taken into account when treatment of a
chronic wound is concerned. Biofilm
forming bacterial phenotype is an excellent model to explain what is observed in
chronic wounds and their responses to antibiotics, biocides and other wound care
treatments. This knowledge could help the management of wound biofilm and the
improvement of healing outcomes. The ability of bacteria to perform cell to cell
recognition (coaggregation) may influence biofilm formation and biofilm diversity
(Lemont and Jenkinson, 2000). Further studies are needed to understand how closely
related are these organisms. Like in dental plaque and fresh water biofim, phylogenic
trees can then be constructed. Knowing the structure of phylogenic trees in a chronic
wound can help forecast and predict bacteria communities when pioneer bacteria are
known. This situation can assist in the issue of viable but non culturable bacteria by
predicting which bacteria can be present in a given wound environment. These
observations are in line with previous research (James et al, 2008) that indicated the
species diversity present in wound was often three –fold greater than standard culture
results demonstrate. Although this work is not a comprehensive study, it stands as a
pilot study that opens the door to further research. The following are limitations to this
research.
The sample size: unlike in other coaggregation and biofilm environment where usually
research deals with large sample size, for this study the sample size was relatively small.
To obtain a more scientific meaningful picture a larger sample size is needed.
80
Growth curve assay: in this study a generalised harvest time was used for several
bacteria but ideally a specific harvest time for every bacterium is important as different
bacteria coaggregate at a different period of their growth phase. It is possible, therefore,
that some bacteria did not coaggregate or coaggregated weakly because of this factor.
Microtiter plate experiment: as discussed earlier the Christensen classification did not
give a definitive categorization by putting into the same category two numerically
distant results, sensitivity may have been lost. A more specific test may have shown a
different interpretation.
Coaggregation assay: the method use in this study was a visual assessment of the
turbidity of the test result and this always give room to human error. A
spectrophotometric reading will surely give a more reliable result. To carry out the
coaggregation assay, organisms in this study were grown in liquid media (from a solid
agar plate to TSB) and results obtained may be limited by this medium. Coaggregation
however been has proved to be influenced by growth environment. A previous study
(Rickard et al, 2004) looked at coaggregation of bacteria in liquid and solid media and
variation in media showed that maximum coaggregation ability lasted longer after
growth in liquid medium (48 h) but beyond this time coaggregate was best after growth
on solid agar. If organisms from this study were grown on various media the
interpretation of the result might have given different results. Specificity of
coaggregation reactions was not studied in this research but appeared to be of interest
when considering what could prevent coaggregation reaction occurring. However
coaggregation among bacteria has been proven to be mediated by specific adhesion
between different bacterial species that contribute to dental plaque (Gibbons et al, 1973;
Jenkinson and Lamont, 1977). Other studies on the nature of these interactions have
revealed the presence of cell surface interaction; an example is lectin-saccharide
interaction that occurs in aquatic bacteria (Rickard et al, 2000). In dental plaque it has
81
been shown that Candida albicans bind to several species of oral streptococci, in
particular Streptococcus gondonii through recognition of a Streptococcus cell wall
polysaccharide receptor (Holmes et al, 1996). Receptors on the surface of bacteria cell
could constitute part of the interaction, where research has been done it appears that
diversity among the receptors molecules is limited (Rickard et al, 2003a).
Understanding what happen at the surface of coaggregating cells will throw more light
in how coaggregation could be enhanced or preveneted.
Network properties theory:
To obtain a more comprehensive analysis of the result using the network properties
theory a larger sample size combined with genetic analysis (gene sequencing) which
will give a better understanding of organisms and community behaviour under study.
4.1.10 Further work
Traditional approaches to wound microbiology utilize cultures to recover bacteria. The
limitation here is that not all species are recovered therefore molecular methods are
necessary to increase the recovery of a wide range of microorganisms, especially
anaerobes. PCR (polymerase chain reaction) that can target 16S ribosomal DNA
(rDNA) or RNA (rRNA) can be used to achieve this objective.
Genetic analyses have revealed a diversity of genetic factors participating in biofilm
formation and multiple pathways have been discovered (O'Toole, 2003). Analysis of
isogenic mutants of streptococci, for instance has confirmed the essential role of a
specific surface polypeptide (Jenkinson, 1995). Finding gene- expression patterns in
bacterial biofilm in general and in a case- by- case will improve our knowledge of
biofilm.
82
Research into the coaggregation ability of a large number of bacteria inhabiting wounds
as well as genetic characterization is needed to construct a phylogenetic tree and to find
out how closely related are these bacteria.
The region of genes encoding for the adherence of some oral bacteria such as
Streptococcus gordonii
have been indentified (Jenkinson, 1994) but how cell to cell
recognition occurs remains poorly understood, therefore surface identification, location
and composition of adhesion and receptor molecules needs to be carried out in order to
fully understand coaggregation interactions.
83
APPENDIX 1
1.Coaggregation partenership between bacteria isolated from chronic wounds
ID
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
organism
E.faecalis
S. aureus
P. aeruginosa
P. aeruginosa
K. pneumoniae
B.cepacia
C.striatum
S. aureus
Citrobact.freudii
S. aureus
C.striatum
P.mirabilis
E. faecalis
LE08
LE08
LE08
CR01
CR01
CR01
CR01
CR01
CR01
MC04
MC04
MC04
MC04
Autoagg
r score
1
1
1
1
0
1
2
0
0
0
4
0
0
patient
A14
S.aureus
RC03
0
A15
C.striatum
RC03
1
A16
P.mirabilis
RC03
0
A17
A18
E. faecalis
Strept.gr G
RC03
RC03
0
1
A19
P. aeruginosa
RC03
0
A20
A21
K.pneumoniae
C.striatum
NS07
NS07
0
0
A22
Strept. agalactiae
JF06
0
JF06
1
JF06
JF06
0
2
Autoaggr
score
1
A24
A25
Citrobact
amalonaticus
P. aeruginosa
C.striatum
ID
organisms
patient
A26
A27
A28
S. aureus
P. mirabilis
Strept. agalactiae
Porphyromonas
spp
S. aureus
C.striatum
S. epiderrmidis
M.morganii
C.striatum
S. aureus
C. accolens
C.striatum
E. faecalis
S. aureus
P. mirabilis
Strept.gr G
P. aeruginosa
C.striatum
S. aureus
JF06
JF06
SG09
A23
A29
A31
A32
A33
A34
A35
A36
A37
A38
A39
A40
A41
A42
A43
A44
A45
Coaggregation partenership
A1-A3(1)
A2-A3(1)
A3-A1(1), A3-A2(1)
A4-A5(3), A4-A6(2),A4-A7(1)
A5-A4 (3), A5-A6(2),A5-A7(2),A5-A9
A6-A4 (2), A6-A5(2),A6-A8(2),A6-A9(1)
A7-A4(1), A7-A5(2)
A8-A6(1),A8-A9(1)
A9—A5(1),A9-A6(1), A9-A8(1)
A10-A13(4)
0
A12-A13(4)
A13-10(4),A13-A12(4)
A14-A15(3),A14-A16(4),A14-A17(3),A14A18(3),A14-A19(2)
A15-A14(3),A15-A16(4),A15-A17(3),A15A18(3),A19-A19(2)
A16-A14(4),A16-A15(3),A16-A17(3),A16A18(1),A16-A19(3)
A17-A14(3),A17-A16(3),A17-A18(1),A17-A19(2)
A18--A14(3),A18-A16(1),A18-A17(1),A18-A19(2)
A19-A14(3), A19-A15(3),A19-A16(2),A19A17(3),A19-A18(3)
0
0
A22-A23(3), A22-A24(4),A22-A25(2),22A26(3),A22-A27(3)
A23-A22(3), A23-A25(4),A23-A25(2),23A27(3),A22-A27(3)
A24-A22(3), A24-A23(2),A22-A25(2),
A25-A22(2), A25-A23(1),A22-A25(2),25-A26(2)
Total
coaggr
1
1
2
3
5
4
2
2
3
1
0
1
2
5
5
5
4
4
5
0
0
5
5
0
A26-A23(3), A26-A23(1)
A27-A23(3), A27-A24(4), A27-A25(1)
A28-A29(1), A28-A30(2),A 28-A3(1)
3
4
Total
coaggr
2
3
3
SG09
0
A29-A28(1), A29-A30(1),A29-A31(2)
3
SG09
MW10
MW10
AP12
AP12
AP12
AP12
PP15
PP15
PP15
PP15
PP15
PP15
JR14
JR14
1
2
0
0
0
0
0
1
0
0
0
0
0
0
0
A31-28(3),A31-29(2),A31-30(2)
A32-A33(1)
A33-A32(1)
A34-36(2),A34-37(2)
0
A36-A34(2)
A37-34(2)
A38-A39(2)
A39-38(3)
0
0
0
0
0
0
3
1
1
2
0
1
1
1
1
0
0
0
0
0
0
Coaggregation partenership
84
A47
Strept. anginosus
JS13
1
A47-A48(1), A47-A49(3),A47-A50(1),A47A51(2),A47-A52(1),A46-A53(1)
6
JS13
0A
A48-A47(4), A48-A51(1), A48-A53(2)
3
JS13
JS13
0
0
2
2
A49
A50
Enterobact.
cloacae
S. aureus
E. faecalis
A51
P. aeruginosa
JS13
0
A52
A53
A54
A55
A56
A57
A58
K. oxytoca
M.luteus
P. aeruginosa
K.pneumoniae
P. aeruginosa
S. aureus
E. faecalis
Enterobact.
cloacae
C.striatum
P. aeruginosa
C.striatum
P.mirabilis
A. haemolyticum
F. oryzihabitans
Clost. ramosum
M.morganii
S.aureus
C. striatum
C. accolens
Stenotro.
maltophilia
JS13
JS13
GW16
GW16
JT17
JT17
JT17
1
2
0
0
0
2
0
A49-A47(2), A49-53(1)
A50-A47(2), A51-A51(1)
A51-A47(1), A51-A48(2),A51-A49(2),A51A50(1),A51-A52(2),A51-A53(4)
A52-A47(1), A52-A51(1)
A53-47(1), A53-A49(2), A53-A50(1), A53-A52(2)
A54-A55(2)
A55-A54(2)
A56-A57(3).A56-A58(2),A56-A59(4), A56-A60
A57-56(1), A57-A58(2), A57-A59(1), A57-A60
A58-56(3), A58-A57(3), A58-A59(4)
JT17
1
A59-A56(1), A59-A57(2)
2
JT17
PB18
PB18
PB18
PB18
PB18
PB18
AP12
AP12
AP12
AP12
0
0
1
1
4
0
0
1
2
0
2
A60-A57(1), A60-A57(1), A60-A58(1)
A61-A62(1), A61-A64(1)
A62-A61(1), A62-64(2)
A63-A61(1), A63-A66(1)
0
0
0
A67-A69(2), A67-A68(1), A67-A70(1), A67-A71
A68-A67(1), A68-A70(1), A68-A69(1)
A69-A67(2), A69-A68(1), A69-A70(1), A69-A71(2)
A70-A71(1), A70-A67(1), A70-A69(2)
3
2
2
2
0
0
0
4
3
4
3
AP12
1
A71-A68(1), A71-A69(2), A71-A70(1)
3
A48
A59
A60
A61
A62
A63
A64
A65
A66
A67
A68
A69
A70
A71
6
2
4
1
1
4
4
3
85
APPENDIX 2
1.
Network mathematic calculation e.g wound one (patient JS13)
Node(bacteria)
i
j
k
l
m
n
o
i
N/A
1
1
1
1
1
1
j
1
N/A
3
2
1
2
2
k
1
2
N/A
2
2
2
1
l
1
2
3
N/A
1
2
2
m
1
1
2
1
N/A
1
1
n
1
2
3
2
1
N/A
2
o
2
2
1
2
1
2
N/A
Total link
7
10
13
10
7
10
9
Mean
1
1.42
1.85
1.42
1
1.42
1.28
The characteristic path length of the whole graph is 1.42 (the median of the mean)
n= 7 (number of bacteria or node)
M= 11
K=2M/n =2x11/7= 3.1
C= M/n2=11/49=0.224
L= 1.42
Y= 11/6+6+6+6+6+6=0.40
M/n= 1.57
86
2. .Network mathematic calculation e.g wound two (patient JF06)
Node(bacteria)
i
j
k
l
m
n
i
N/A
1
1
1
1
2
j
1
N/A
1
1
1
1
k
1
1
N/A
1
1
1
l
1
1
1
N/A
2
1
m
1
1
1
2
N/A
2
n
2
1
1
1
2
N/A
Total link
6
5
5
6
7
7
Mean
1
0.83
0.83
1
1.16
1.16
The characteristic pathlength of the whole graph is 1 (the median of the mean)
n= 6 (number of bacteria or node)
M= 12
K=2M/n =2x12/6= 4
C= M/n2=12/36=0.33
L= 1
Y=11/5+5+5+5+5+5=0.36
M/n=2
87
APPENDIX 3
Biofilm OD/ Biofilm Index/ Coaggregation partnership
1
E.faecalis
LE08
SA
3.1
3
No of
Coaggr
parternersh
ip
1
2
S. aureus
LE08
SA
3.3
3
1
3
P. aeruginosa
LE08
SA
3.3
3
2
4
P. aeruginosa
CR01
SA
3.1
3
3
5
K. pneumoniae
CR01
WA
0.1
1
5
6
B.cepacia
CR01
SA
2.4
3
4
7
C.striatum
CR01
MA
0.4
2
2
S. aureus
CR01
WA
0.1
1
2
9
Citrobact.freudii
CR01
SA
0.8
3
3
10
S. aureus
MC04
SA
0.5
3
1
11
C.striatum
MC04
SA
1.2
3
1
12
P.mirabilis
MC04
MA
0.3
2
0
13
E. faecalis
MC04
MA
0.3
2
1
14
S.aureus
RC03
SA
1
3
2
15
C.striatum
RC03
MA
0.4
3
5
16
P.mirabilis
RC03
SA
0.8
1
5
17
E. faecalis
RC03
SA
1.5
3
5
18
Strept.gr G
RC03
SA
1.8
3
4
19
P. aeruginosa
RC03
SA
1,4
3
4
20
K.pneumoniae
NS07
WA
0.2
1
5
21
C.striatum
NS07
SA
3.1
3
0
22
Strept. agalactiae
JF06
SA
0.8
3
0
23
Citrobact amalonaticus
JF06
MA
0.4
2
5
24
P. aeruginosa
JF06
SA
3.5
3
5
25
C.striatum
JF06
MA
0.4
3
3
26
S. aureus
JF06
SA
3.1
3
4
27
P. mirabilis
JF06
SA
2.8
3
2
28
Strept. agalactiae
SG09
SA
3.5
3
3
29
Peptostreptococcus spp
SG09
MA
3.5
3
3
Biofilm index
No of
Coaggr
parternership
Organism
8
Organism
Patient
Patient
Biofilm
adherec
e
Biofilm
adherece
Biofilm actual
OD
Biofilm actual
OD
Biofilm index
88
S. aureus
SG09
SA
3.1
3
3
30
31
S. epiderrmidis
MW10
SA
3.5
3
1
32
C.striatum
AP12
WA
0.1
3
2
33
S. aureus
AP12
SA
3.5
3
1
34
C.striatum
PP15
SA
0.9
3
1
35
E. faecalis
PP15
SA
3.5
3
1
36
S. aureus
PP15
SA
3.5
3
0
37
P. mirabilis
PP15
SA
0.8
3
0
38
Strept.gr G
PP15
WA
0.1
1
0
39
P. aeruginosa
PP15
SA
3.5
3
0
40
C.striatum
JR14
WA
0.1
1
0
41
S. aureus
JR14
SA
2.6
3
0
42
43
Strept. anginosus
Enterobact. cloacae
JS13
JS13
SA
MA
3.5
0.2
3
3
6
3
44
S. aureus
JS13
SA
3.1
3
2
45
E. faecalis
JS13
SA
3.2
3
2
46
P. aeruginosa
JS13
SA
3.5
3
6
47
K. oxytoca
JS13
MA
0.3
2
2
48
M.luteus
JS13
SA
2.8
3
4
49
P. aeruginosa
GW16
SA
2.1
3
1
50
K.pneumoniae
GW16
SA
1.3
3
1
51
P. aeruginosa
JT17
SA
3.5
3
4
52
S. aureus
JT17
SA
3.1
3
4
53
E. faecalis
JT17
SA
1.5
3
3
54
Enterobact. cloacae
JT17
MA
0.2
2
2
55
C.striatum
JT17
SA
0.8
3
3
56
P. aeruginosa
PB18
SA
3.2
3
2
57
P.mirabilis
PB18
SA
0.9
3
2
58
S.aureus
AP12
SA
2.8
3
3
59
C. striatum
AP12
SA
0.8
4
4
Scale use for biofilm index
Strong adherence (SA) = 3
Moderate aherence (MA) = 2
Weakly (WA) =1
Non adherent=0
89
2. An example of Christensen et al, 1985 analysis of biofilm formation. A case of a
mixed culture of patient 1
Well s 1
0.295
0.986
1.335
0.597
1.709
2
0.269
1.046
1.118
0.578
1.421
3
0.393
0.614
0.886
0.512
1.013
4
0.568
1.094
0.882
0.833
1.264
5
0.401
0.514
0.621
0.498
1.101
Threshold value=
0.074667 +3SD=
ODcx2=
0.204
ODcx4=
0.408
Blank
0.089
0.075
0.067
0.079
0.102
0.063
6 mean
OD
0.647 0.428833 0.354167
0.625 0.813167
0.7385
0.957
0.9665 0.891833
0.795
0.6355 0.560833
1.365 1.312167
1.2375
0.831233
0.075
SD=
MA
SA
SA
SA
SA
0.074667
0.009158
90
APPENDIX 4
Statistical analysis
1. One-way ANOVA: Biofilm OD versus Biofilm Index
Source
DF SS
MS
F
P
Biofilm Index 2 24.819 12.410 12.87 0.000
Error
61 58.804 0.964
Total
63 83.623
S = 0.9818 R-Sq = 29.68% R-Sq(adj) = 27.37%
One-way ANOVA: Biofilm Index, coaggregation patnership index
Source DF
SS
MS
F
P
Factor 1 58.369 58.369 124.04 0.000
Error 116 54.587 0.471
Total 117 112.956
S = 0.6860 R-Sq = 51.67% R-Sq(adj) = 51.26%
2. Correlations: Biofims index and Organisms that coaggregated
Pearson correlation of biofilm index and Organisms that coaggregated =
0.467
P=Value = 0.000
91
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