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Multidrug-Resistant Organisms:
Where Are We with Detection and
Reporting in 2015?
Audrey N. Schuetz, MD, MPH
1
Faculty Disclosure
The Association of Public Health Laboratories adheres to established standards
regarding industry support of continuing education for healthcare professionals.
The following disclosures of personal financial relationships with commercial
interests within the last 12 months as relative to this presentation have been
made by the speaker(s):
Nothing to disclose.
Analysis.
Answers.
Action.
2
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Objectives
Multidrug-resistant organisms (MDROs)
1. Address the new challenges in detection
of certain MDROs
2. Recognize the importance of accurate
detection and reporting of these
organisms
3
Bruce McCall, The New Yorker, 2008
4
Multidrug-resistant Organisms
Organisms that are resistant to one or more
classes of antimicrobial agents
• Methicillin-resistant Staphylococcus
aureus
• Vancomycin-resistant Enterococcus
• Extended-spectrum β-lactamases (ESBLs)
• Carbapenem-resistant Organisms
(Enterobacteriaceae, Acinetobacter
baumannii, Pseudomonas aeruginosa)
Healthcare Infection Control Practices Advisory Committee (HICPAC) - Management of MultidrugResistant Organisms in Healthcare Settings, 2006
5
Multidrug-resistant Organisms
Organisms that are resistant to one or more
classes of antimicrobial agents
• Methicillin-resistant Staphylococcus
aureus
• Vancomycin-resistant Enterococcus
• Extended-spectrum β-lactamases (ESBLs)
• Carbapenem-resistant Organisms
(Enterobacteriaceae, Acinetobacter
baumannii, Pseudomonas aeruginosa)
Healthcare Infection Control Practices Advisory Committee (HICPAC) - Management of MultidrugResistant Organisms in Healthcare Settings, 2006
6
Why is detection of MDROs
important?
• MDROs have increased in prevalence
over the past three decades in the U.S.
• MDROs are associated with
– Increased mortality
– Increased length of stay and increased costs
• Offer appropriate individualized patient
treatment
• Control the spread of organisms
Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013
Marchaim D et al. Antimicrob Agents Chemother. 2008; 52:1413
7
Carbapenem-resistant Organisms
8
Ambler Classification of β-lactamases
Molecular
Class
Enzymes
A
TEM, SHV, CTX-M, KPC, GES, SME,
IMI, NMC
B
NDM, IMP, VIM, SPM, SIM, GIM
C
AmpC, CMY-10
D
OXA
Red = Carbapenemases
9
Klebsiella pneumoniae Carbapenemases
(KPCs)
• Class A β-lactamases
• Enterobacteriaceae and nonEnterobacteriaceae
• KPC-2 through KPC-23
• blaKPC gene
• Plasmid allows for horizontal gene transfer
• Encodes for resistance to other
antimicrobial classes
10
Metallo-β-lactamases (MBLs)
• Class B β-lactamases
• P. aeruginosa, A. baumannii,
Enterobacteriaceae
• Require zinc to catalyze hydrolysis of
antimicrobials
• New Delhi Metallo-β-lactamases (NDMs)
– Located on very mobile genetic element
– Carry additional resistance genes
– Reservoirs – colonized or infected persons on
Indian subcontinent, also Middle East and Balkan
countries
Yong D et al. Antimicrob Agents Chemother. 2009; 53:5046
11
Oxacillinases (OXAs)
•
•
•
•
Class D β-lactamases
Oxacillin- and cloxacillin-hydrolyzing
Most are plasmid-encoded
Enterobacteriaceae, P. aeruginosa, A.
baumannii
• 488 OXA enzymes, many of which are
carbapenemases
12
AmpC β-lactamases
• Class C β-lactamases
• SPACE/SPICE organisms
• Hydrolyze cephalosporins, cephamycins
(cefoxitin or cefotetan), aztreonam
• Most are not carbapenemases
– AmpC-expressing organisms can be resistant to
carbapenems due to co-existent porin changes
• Very low hydrolysis rates for cefepime,
carbapenems
13
Question 1
Which confirmatory test for suspected carbapenemase
production is now described in the new CLSI M100-S25
document?
A. A multiplex 17-target molecular assay for resistance
determinants
B. MBL Etest
C. Carba NP test
D. MALDI-TOF MS
CLSI. M100-S25. Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational
Supplement, 2015
14
Question 1
Which confirmatory test for suspected carbapenemase
production is now described in the new CLSI M100-S25
document?
A. A multiplex 17-target molecular assay for resistance
determinants
B. MBL Etest
C. Carba NP test
D. MALDI-TOF MS
CLSI. M100-S25. Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational
Supplement, 2015
15
Detection of CROs
• Detection of carbapenemases
– Modified Hodge test
– Enzymatic carbapenemase detection (i.e.
Carba NP)
– MALDI-TOF MS
• Characterization of type of
carbapenemase
– Inhibition-based phenotypic assays
– Molecular-based assays
16
Modified Hodge Test
Pos Ctl
Enterobacter AmpC
patient-1
patient-2
Neg Ctl
Slide courtesy of Dr. Paul Schreckenberger
• Carbapenem inactivation
test
• Not specific for
carbapenemases
• AmpC plus efflux pump
or porin loss may be
positive
• Sensitivity 95-100%
• Useful for
Enterobacteriaceae
• False negative results
have been demonstrated
with blaNDM-1 strains
Mochon AB et al. J Clin Microbiol. 2011; 49:1667
17
Carba NP Assay
• CLSI M100-S25 includes more options for
identification of carbapenemase-producing
organisms
• Two-hour tube test
• Imipenem is hydrolyzed which leads to a
color change
• Identifies carbapenemase production in
Enterobacteriaceae, P. aeruginosa and
Acinetobacter spp.
CLSI. M100-S25. Performance Standards for Antimicrobial Susceptibility Testing; 25th Informational
Supplement.
Nordmann P et al. Emerg Infect Dis. 2012; 18:1503
18
Carba NP
• Solution A = phenol red, zinc sulfate,
sodium hydroxide
• Solution B = Solution A plus imipenem
• Positive if “B” tube is yellow compared to
“A” tube
19
CLSI Multi-center Carba NP Study
• 80 gram-negative bacteria (66 Enterobacteriaceae)
• 44 positive for carbapenemases and 36 negative for
carbapenemases
Isolates
Results
14 NDM
Most detected; 2 sites missed 2 NDMs
8 VIM
All detected
4 IMP
2 sites missed one IMP
2 SME
All detected
10 KPC
Most detected; Missed 2 KPCs with low carbapenem MICs*
5 OXA-48
Only 2 were detected
Limbago B et al. Multicenter Evaluation of a Rapid Test for Detection of Carbapenemase Production for
Development of a CLSI-Endorsed Method. ICAAC 2014.
20
CLSI M100-S25
• Under Table 2A
– Laboratories using Enterobacteriaceae MIC
interpretive criteria for carbapenems described in
M100-S20 should perform the MHT, the Carba
NP test, and/or a molecular assay when isolates
of Enterobacteriaceae are suspicious for
carbapenemase production based on imipenem
or meropenem MICs of 2-4 µg/mL or ertapenem
MIC of 2 µg/mL.
– After implementation of the current interpretive
criteria, the MHT does not need to be performed
other than for epidemiological or infection control
purposes.
21
Other Enzymatic Carbapenemase Tests
• Blue-Carba
– Bromothymol blue indicator
includes the optimal pH
range for most βlactamases
– Rosco Diagnostica
(Denmark) Carba Blue
• RAPIDEC® CARBA NP
(bioMérieux)
• Rapid Carb Screen
(Rosco) with diatabs
– Imipenem plus indicator in
a tablet form
Pires J et al. J Clin Microbiol. 2013; 51:4281
22
Other Enzymatic Carbapenemase Test
•
•
•
•
Rosco Neo-Rapid CARB kit (Key Scientific Products)
FDA-approved*
Enterobacteriaceae and P. aeruginosa
Uninterpretability issues
*Note correction: During this APHL talk on multidrug‐resistant organisms, I relayed some incorrect information regarding the Neo Rapid CARBA‐NP kit from Rosco. The information I relayed was based on the announcement made by Key Scientific Company about FDA clearance/approval. I have since learned that the kit is NOT FDA‐cleared or FDA‐approved. I apologize for this misrepresentation of the information. ‐ Audrey N. Schuetz, MD, MPH, D(ABMM), FCAP
Time Read
Carba NP
Neo-Rapid
CARB
Sensitivity
Specificity
60 minutes
100
100
100 minutes
100
100
30 minutes
94
100
60 minutes
78
100
120 minutes
67
50
Denis CJ et al. Preliminary comparison of Carba NP test and ROSCO Neo-Rapid CARB Screen kit.
ECCMID 2015.
23
Advantages of
Enzymatic Tests
Disadvantages of
Enzymatic Tests
• Carba NP
• Rapid
– Positive results in 10
minutes to 2 hours
– Negative results in 2
hours
– Special reagents
required
– In-house preparation
– Short shelf life of
Solution B
• Useful for a variety of
GNRs but depends on
kit
• Sensitivity and specificity
approximately 90%
• Subjective color
determination of some
assays
• Limited ability to detect
OXA enzymes
• Few FDA-approved
tests
24
MALDI-TOF MS Carbapenem
Hydrolysis
• Incubate a fresh
bacterial culture
with carbapenem
solution
• Measure the degradation products
compared to carbapenem alone
• Automated software are not currently
available for analysis of spectra
Chong PM et al. J Microbiol Methods. 2015; 111:21
Hrabák J et al. J Clin Microbiol. 2012; 50:2441
25
Characterization of the Type of βlactamases
26
KPC Detection Using Boronic Acidbased Tests
•
•
•
•
KPCs are inhibited by boronic acid
Meropenem is tested with and without boronic acid
High sensitivity for KPC
Isolates with AmpCs plus porin losses may also
test positive
Rosco Diagnostica KPC/MPL disk kit
Doi Y et al. J Clin Microbiol. 2008; 46:4083
27
MBL Detection Using Chelating
Agents
• MBLs are inactivated by chelating
compounds that deprive the organism of zinc
• Chelating compounds include
mercaptopropionic acid, EDTA, dipicolinic
acid
MPI
MP
28
Positive MBL
Mastdiscs™ Group
Mercaptopropionic Acid
TE
2-MPA
IP
IP = Imipenem
TE = EDTA
29
EDTA as Indicator of MBL
• EDTA can show moderate to poor specificity
as an indicator of MBL
• Other β-lactamases can be positive
– Permeability action on outer membrane
• Study of EDTA and other phenotypic methods
– Isolates: 34 KPC, 21 VIM, 4 IMP, 9 OXA, 9
AmpC, 9 ESBL
– Positive by EDTA test: 4/34 KPC, 1/9 ESBL, 4/9
OXA-48 K. pneumoniae
Giske CG et al. Clin Microbiol Infect. 2011; 17:552
30
MAST AmpC Kit
• Detects plasmid and chromosomally
mediated
• E. coli and Klebsiella spp.
• Good performance for detection of AmpCs
in Enterobacteriaceae
Halstead FD et al. J Antimicrob Chemother. 2012; 67:2303
31
mastdiscs™
• Carbapenemase detection (MBL and KPC)
• Enterobacteriaecae
• Some limitations in detection of OXAs
– Add temocillin 30 µg disc
– If resistant to temocillin, probably OXA-48
• Recent study demonstrated 91%
sensitivity for detection of KPC and MBL
• Different sensitivity based on different
enzymes
Saito R et al. J Microbiol Methods. 2015; 108:45
32
Limitations of Inhibition-based Disk
Assays
• Interpretation can be subjective and
requires some experience
• Most methods require overnight incubation
• Multiple methods should be used if more
than one resistance mechanism is
suspected
– For instance, if KPC and AmpC is suspected,
cannot rely upon boronic acid alone
33
Genotypic Testing
• Relatively rapid
• Target-driven
• Panels directly from positive blood cultures can detect a
variety of targets
• Commercial products are available for detection directly from
colonies
– Labor-intensive for the routine clinical microbiology laboratory
(RUO)
– BD Max™ CRE assay
• KPC, NDM, OXA-48
– Check-Points (Netherlands) rapid molecular detection within 2
hours
• KPC, VIM, NDM, OXA-48, OXA-181
– 22-minute multiplex assay by Streck (Omaha, NE)
• 9 targets (AmpC, MBL, KPC, ESBL, OXA-48)
• Philisa® thermocycler
34
Question 2
A 68 year old patient from India has a blood culture positive
for carbapenem-resistant Klebsiella pneumoniae. In which
situation below might it be appropriate to assess whether
this isolate possesses an NDM-1?
A. The physician wishes to treat with ceftazidimeavibactam.
B. The physician wishes to treat with polymyxin B.
C. Infection control wishes to place this patient on contact
precautions.
D. There is never a need to differentiate among different
carbapenemase mechanisms.
35
Question 2
A 68 year old patient from India has a blood culture positive
for carbapenem-resistant Klebsiella pneumoniae. In which
situation below might it be appropriate to assess whether
this isolate possesses an NDM-1?
A. The physician wishes to treat with ceftazidimeavibactam.
B. The physician wishes to treat with polymyxin B.
C. Infection control wishes to place this patient on contact
precautions.
D. There is never a need to differentiate among different
carbapenemase mechanisms.
36
Should We Differentiate Among
Carbapenem-resistant Gramnegative β-lactamases?
37
Ceftolozane-tazobactam (Cubist)
• Received FDA approval for complicated
UTI and complicated intra-abdominal
infections
• Ceftolozane is more active than
ceftazidime against P. aeruginosa
• Doesn’t cover KPC or MBL
• Upcoming ventilator-associated
pneumonia trial
38
Ceftazidime-avibactam (Actavis)
• For treatment of complicated
intraabdominal infections (with
metronidazole) and complicated UTIs
• Enterobacteriaceae and P. aeruginosa
• Active against ESBLs and KPCs and
OXA-48
• Not active against MBLs or strains with
both a KPC and AmpC
Vazquez JA et al. Curr Med Res Opin. 2012; 28:1921
Lucasti C et al. J Antimicrob Chemother. 2013; 68:1183
39
Plazomicin (Achaogen)
• Novel, semi-synthetic aminoglycoside
(neoglycoside, formerly ACHN-490)
• Active against aminoglycoside-modifying
enzymes
• Undergoing phase III trials for CRE
bacteremia and pneumonia
• Does not cover NDM
• Low to no nephrotoxicity or ototoxicity
Zhanel GG et al. Expert Rev Anti Infect Ther. 2012; 10:459
40
Aztreonam-avibactam (AstraZeneca)
• Potentially adds MBLs to the spectrum
• Avibactam is active against other
hydrolyzing enzymes (such as AmpCs and
ESBLs) which are carried by organisms
that also carry MBLs
• Protective against KPCs, some OXAs
• Just completed Phase I trial
41
Carbavance™ (Rempex, Medicines Co.)
• Novel β-lactamase inhibitor (RPX7009) with a
carbapenem
– RPX2009 novel boronate
– Combined with biapenem
• Undergoing Phase III clinical trials for a
variety of infections due to KPC-producing
bacteria
• Most OXA-positive organisms will be affected
by Carbavance
• MBLs are unaffected
42
Activity Against Various β-lactamases
KPC
AmpC
OXA MBL
Ceftolozane-tazobactam
N
+/-
Y
N
Ceftazidime-avibactam
Y
+/-
+/-
N
Aztreonam-avibactam
Y
Y
Y
Plazomicin
Y
Carbavance
Y
*Not active against NDM but retains activity against some VIMs
43
N*
Y
N
MRSA
44
Question 3
55 year old female is admitted to a German hospital with fever and
vomiting.
Blood cultures are positive for gram-positive cocci in clusters. Rapid
blood culture PCR was positive for S. aureus and negative for mecA.
The isolate was resistant to oxacillin (MIC 8 mcg/mL) and to cefoxitin
(MIC 16 mcg/mL). A PBP2a test was negative. What is explanation?
A.
B.
C.
D.
The PCR was incorrect.
This is a heterogeneous population of MRSA and MSSA.
The organism possesses the mecC gene.
The organism is actually coagulase-negative Staphylococcus which
relies on a mechanism other than mecA for resistance.
Adapted from http:// pathquestions.com
45
Question 3
55 year old female is admitted to a German hospital with fever and
vomiting.
Blood cultures are positive for gram-positive cocci in clusters. Rapid
blood culture PCR was positive for S. aureus and negative for mecA.
The isolate was resistant to oxacillin (MIC 8 µg/mL) and to cefoxitin
(MIC 16 µg/mL). A PBP2a test was negative. What is explanation?
A.
B.
C.
D.
The PCR was incorrect.
This is a heterogeneous population of MRSA and MSSA.
The organism possesses the mecC gene.
The organism is actually coagulase-negative Staphylococcus which
relies on a mechanism other than mecA for resistance.
Adapted from http:// pathquestions.com
46
New Challenges in MRSA Detection
• mecC
• mecA homologue (mecALGA251) with 69%
identity
• Phenotypically methicillin resistant but
negative by PCR for mecA
• Encodes the PBP2c protein and confers
resistance to all β-lactams except ceftaroline
Saeed K et al. Curr Opin Infect Dis. 2014; 27:130
47
mecC – “Cryptic Resistance”
• Negative for mecA by PCR
• Negative for PBP2a
• May be oxacillin susceptible (MIC ≤2
µg/mL)
• May or may not grow on chromogenic
MRSA selective media
Cuny et al. PLoS ONE. 2011; 6:e24360
48
mecC
• Cefoxitin resistant
– Vitek 2 study: 55/62 strains with mecC were
oxacillin susceptible and cefoxitin resistant
• Usually susceptible to non β-lactam
antibiotics
• Newer platform can detect mecC
– BD MAX™ MRSA XT Assay with eXTended
Detection Technology (FDA-cleared)
Cartwright EJ et al. J Clin Microbiol. 2013; 51:2732
49
mecC
• Reported in 2007 from milk tanks of a UK dairy
herd
• Primarily in Europe, usually in animals
– Ruminants, pigs, veal calves, poultry
• Colonizes humans but can also cause disease
– Primarily skin and soft tissue infections but also
bacteremia, osteomyelitis
– Does not carry many toxins
• Denmark – 2% of human MRSA cases are mecC
Petersen A et al. Clin Microbiol Infect. 2013; 19:E16
Concepciόn Porrero M et al. Environ Microbiol Rep. 2014; 6:705
50
Conclusions
• Organisms are evolving genetic machinery to
avoid antibiotics
– But, we are developing new antimicrobials
• Rapidity of results and accuracy of testing is
important for both patient care and public
health reasons
• Choice of testing methods varies based upon
laboratory expertise, geographic area and
relative prevalence of various β-lactamases
• Reporting the type of carbapenemase or βlactamase may be important for treatment
purposes
51
References
1. CLSI. M100-S25. Performance Standards
for Antimicrobial Susceptibility Testing; 25th
Informational Supplement, 2015
2. Hrabák J, Chudáčková E, Papagiannitsis
CC. Detection of carbapenemases in
Enterobacteriaceae: a challenge for
diagnostic microbiological laboratories. Clin
Microbiol Infect. 2014; 20:839-853
3. Saeed K, Marsh P, Ahmad N. Cryptic
resistance in Staphylococcus aureus: a risk
for the treatment of skin infection? Curr Opin
Infect Dis. 2014; 27:130-136
52
Questions ?
Analysis.
Answers.
Action.
53
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