Abstract

Transfer of the blaKPC genes encoding the Klebsiella pneumoniae carbapenemase (KPC) are increasingly responsible for emerging carbapenem resistance. The modified Hodge test (MHT) is recommended for the detection of KPC. We compared MHT with a real-time polymerase chain reaction (PCR) assay targeting common subtypes of blaKPC, using previously described forward and reverse primer sequences. The PCR product was detected using SYBR Green (Applied Biosystems, Foster City, CA) and confirmed by melt curve analysis. PCR was positive in 96% (52/54) of isolates that were MHT+, 90% (28/31) of MHT– isolates were PCR–, and the results were strongly correlated (P = .0001; Fisher exact test). The PCR assay is a sensitive, specific, and rapid test for detecting blaKPC genes. It could help optimize patient care by reducing the time taken to institute appropriate antimicrobial therapy and so help improve patient outcomes.

Carbapenems are highly efficacious drugs for treating infections with extended-spectrum β-lactamase–producing gram-negative bacteria.1 Previously, resistance to carbapenems has been rare; however, the emergence of transmissible carbapenem resistance is now a growing concern.1–9 An increasingly common mechanism of carbapenem resistance is the class-A, Klebsiella pneumoniae carbapenemase (KPC).2,8,10–13 KPCs have been reported in K pneumoniae and in Klebsiella oxytoca, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Citrobacter freundii, Enterobacter spp, Serratia spp, and Salmonella spp.2,8,14–21 The blaKPC genes that encode KPCs are present on transferable plasmids and are flanked by transposable elements, thus allowing for the gene to move from plasmid to the bacterial chromosome and back.8,17,22,23 This potential to disseminate resistance to several classes of β-lactam antibiotics has been demonstrated in several reported outbreaks with high mortality rates.9,13,21,24,25 Laboratory detection of KPC in clinical isolates is, therefore, crucial to limit this spread.

Automated susceptibility testing methods do not reliably detect KPC-mediated resistance.7,15 In 2009, the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100) recommended use of the modified Hodge test (MHT) to definitively identify KPC producers.26 Alternatively, the blaKPC genes have been detected by conventional and real-time polymerase chain reaction (PCR) methods.15,20,27–29 However, there are limitations to using PCR: (1) It is more technically challenging and prone to inhibition.30 (2) It is target-specific and may miss new variants of KPC owing to genetic mutation. Carbapenemase-like patterns of nonsusceptibility leading to false-positive MHT results have also been described in association with endemic CTX-M production and AmpC hyperproduction.31,32

In this study, we compared the detection of the blaKPC genes by an in-house real-time PCR assay with the detection of KPC by the current CLSI-recommended standard of practice,26 the MHT. We also tested isolates with discordant MHT and PCR results for the production of AmpC β-lactamase.

Materials and Methods

Selection of Test Isolates

Clinical isolates of Enterobacteriaceae that had been analyzed with the VITEK2 automated system (BioMérieux, Durham, NC) for antimicrobial susceptibility and were intermediate to resistant to imipenem or ertapenem were subsequently tested by using the MHT. Stocks of these isolates were deidentified and stored in tryptic soy broth with 20% glycerol at −20°C. These included isolates that tested MHT+ and MHT–. We selected 61 MHT+ isolates and 18 MHT– isolates at random from among the stored isolates to perform DNA extraction. A reference K pneumoniae strain (American Type Culture Collection, Manassas, VA [ATCC] BAA-1705) was selected as the positive control and a second reference K pneumoniae strain (ATCC 700603) was selected as the negative control for the PCR assay.

Detection of KPC by the MHT

The MHT was performed as previously described.26 Briefly, a 0.5 McFarland suspension of E coli, ATCC 25922, was used to prepare a lawn culture on a Mueller Hinton agar plate (Becton Dickinson, Sparks, MD), and a 10-μg ertapenem susceptibility disk (Becton Dickinson) was placed in the center of the test area. Test isolates were subcultured onto sheep blood agar plates (Becton Dickinson) to establish pure cultures. The isolate was then streaked in a straight line from the edge of the disk to the edge of the plate and was incubated overnight at 35°C in ambient air. After 16 to 24 hours of incubation, the plate was examined for a cloverleaf-shaped indentation at the intersection of the test organism and the E coli ATCC 25922 within the zone of inhibition. The presence of a cloverleaf-shaped indentation was considered MHT+.

DNA Extraction From Cultured Bacterial Colonies

Fresh, well-isolated test colonies grown on sheep blood agar plates following overnight incubation were used for DNA extraction using the Qiagen EZ1 Virus Mini Kit, version 2.0, on the Qiagen Biorobot automated DNA extraction platform (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Briefly, a 0.5 McFarland-standard bacterial suspension was prepared in saline. A final elution volume of 90 μL containing bacterial DNA was extracted from 400 μL (2.4 × 108 colony-forming units) of the suspension and stored at −20°C before using them for PCR.

blaKPC Detection by Real-Time PCR

The forward primer sequence (5′-ATG TCA CTG TAT CGC CGT C-3′) and reverse primer sequence (5′-CTC AGT GCT CTA CAG AAA ACC-3′) used in this assay have been described by Tibbetts et al.20

The amplicons were detected using the intercalating nucleic acid dye SYBR Green (Invitrogen, Eugene, OR) and were confirmed by melt curve analysis. The blaKPC primers were diluted to a concentration of 25 pmol/μL. The final 25 μL of the PCR mixture contained 5 μL of the extracted DNA and 20 μL of PCR buffer composed of 2 μL each of the forward and reverse primers and 12 μL of the SYBR Green, nucleotides, and Taq polymerase solution (Invitrogen) and 4 μL of nuclease-free water (Ambion, Austin, TX). The PCR was performed under the following conditions: stage 1, 95°C for 5 minutes (“hot-start” step) with optics off; stage 2, 95°C for 25 seconds with optics off, 60°C for 30 seconds, 72°C for 30 seconds with optics on, for 30 cycles, followed by a melt curve analysis from 65°C to 95°C at 0.2°/second. A sample was considered positive by PCR if it crossed the preset threshold within 30 cycles and the melting temperature was within 0.1°C of the positive control as determined by melt curve analysis.

AmpC Testing

The AmpC disk test was performed in the manner previously described.33 Briefly, AmpC disks containing tris(hydroxymethyl)aminomethane (Tris)-EDTA (Becton Dickinson) were prepared in house by applying 20 μL of a 1:1 mixture of saline and 100 μL Tris-EDTA (Sigma-Aldrich, St Louis, MO) to sterile filter paper disks, allowing the disks to dry, and storing them at 2°C to 8°C. The surface of a Mueller Hinton agar plate (Becton Dickinson) was inoculated with a lawn of cefoxitin-susceptible E coli ATCC 25922 according to the standard disk diffusion method. Immediately before use, AmpC disks were rehydrated with 20 μL of saline. Colonies of K pneumoniae and Enterobacter cloacae that had differing test results by the MHT and PCR were applied to a disk. A 30-μL cefoxitin disk (Becton Dickinson) was placed on the inoculated surface of the Mueller Hinton agar. The inoculated AmpC disk was then placed almost touching the antibiotic disk with the inoculated disk face in contact with the agar surface. The plate was then inverted and incubated overnight at 35°C in ambient air. After incubation, plates were examined for an indentation or a flattening of the zone of inhibition, indicating enzymatic inactivation of cefoxitin (positive result), or the absence of a distortion, indicating no significant inactivation of cefoxitin (negative result).

DNA Sequencing

Described blaKPC sequences are available online through the GenBank database (Accession No. AF297554; http://www.ncbi.nlm.nih.gov/Genbank/). The ABI Prism sequence detection system (Applied Biosystems, Foster City, CA) was used to amplify and detect the blaKPC amplicons (246 base pairs). We selected 5 positive amplicons and 2 negative samples for direct DNA sequencing using the BigDye Terminator 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer’s protocols. Sequences were detected by the ABI 3100 Capillary Electrophoresis Instrument with Data Collection version 2.1 (Applied Biosystems), and analysis of the sequences was performed with ABI DNA Sequencing Analysis Software, version 5.1.1 (Applied Biosystems).

Results

The specificity of the primers for the detection of blaKPC genes was evaluated by the BLAST program (National Center for Biotechnology Information; available at http://blast.ncbi.nlm.nih.gov/Blast.cgi). No matches to the primers’ sequences were found other than those for the blaKPC genes. Frozen stocks of 76 isolates were available for DNA extraction, including 55 K pneumoniae, 17 E cloacae, 2 E coli, and 2 Serratia marcescens. Of the K pneumoniae isolates selected, 48 were found to be resistant to carbapenems by the VITEK2 automated antibiotic susceptibility testing system, while 7 were found to be susceptible Table 1. In addition, there were 4 E cloacae and 1 S marcescens isolates that were found to be resistant by the VITEK2 system, while 13 E cloacae and 2 E coli isolates were found to be susceptible to carbapenems. In total, there were 61 MHT+ isolates and 15 MHT– isolates.

Table 1

Summary of Isolates and Results of Carbapenem Susceptibility Testing by the VITEK2 System*

Table 2

Preliminary Results of the MHT and PCR Assay

Initial MHT and PCR Results

The PCR assay was positive in 50 of the MHT+ test isolates (50/61 [82%]) and was negative in 13 MHT– isolates (13/15 [87%]). Eleven isolates that had been reported as MHT+ were negative by PCR, including 8 K pneumoniae and 3 E cloacae isolates Table 2. AmpC testing was performed on all 11 of these isolates. One isolate of S marcescens and 1 of E cloacae were negative by MHT and positive by PCR. The results of the MHT and PCR were strongly correlated (P = .0001; Fisher exact test). The threshold temperature for the melt curve analysis for the DNA sequences that were amplified by the PCR was between 91°C and 92°C. We did not encounter a single sample that was reportable as equivocal by PCR.

Repeated MHT, Repeated PCR, and AmpC Results

The MHT and PCR were repeated for the 11 isolates that had discrepant results. Following the repeated MHT, 9 previously MHT+ isolates were reidentified as MHT–, including 6 K pneumoniae and 3 E cloacae isolates. Two K pneumoniae isolates remained MHT+ in repeated testing and were subsequently detected as PCR+ in the repeated test. The AmpC test was positive in both of these K pneumoniae isolates.

AmpC was detected in 4 isolates, including 3 K pneumoniae and 1 E cloacaeTable 3. Of these, the results of 1 K pneumoniae isolate remained MHT+/PCR–. The results of 1 K pneumoniae isolate changed from MHT+/PCR– to MHT–/PCR+, while the results of the third K pneumoniae isolate changed from MHT+/PCR– to MHT+/PCR+. The results for the E cloacae isolate changed from MHT+/PCR– to MHT–/PCR–. AmpC was not detected in 7 organisms for which the results had changed from MHT+/PCR– to MHT–/PCR–, including 5 K pneumoniae and 2 E cloacae isolates.

Following this repeated testing, there were 51 isolates that were positive by the MHT and PCR, while 21 isolates were negative for both Table 4. Based on these results, the sensitivity of the PCR was 98% (51/52), the specificity was 88% (21/24), and the results of the MHT and PCR were strongly correlated (P = .0001; Fisher exact test).

By including only K pneumoniae and E cloacae in the analysis, there were 48 isolates that were positive by the MHT and PCR, while 21 isolates were negative for both. This yielded a sensitivity of 98% (48/49) and specificity of 91% (21/23) for the PCR, with the results of the MHT and PCR being strongly correlated (P = .0001; Fisher exact test). Furthermore, with only isolates of K pneumoniae included in the analysis, 46 (98%) of 47 isolates were positive by the MHT and PCR, and 7 (88%) of 8 isolates were negative by the MHT and PCR, with a strong correlation between the results of the MHT and PCR (P = .0001; Fisher exact test).

Table 3

Results of AmpC Testing Among Discordant Isolates

Table 4

Results After Repeating the MHT and PCR for Discordant Isolates

DNA Sequencing

The DNA sequence of 5 randomly selected amplicons was confirmed to correspond to the blaKPC genes using a BLASTn analysis. The protein sequence encoded by all 5 was also confirmed to correspond to a part of the KPC enzyme structure using a BLASTp analysis. Two nonamplified DNA samples were negative.

Discussion

The class-A carbapenemase enzyme, KPC, is an emerging mechanism of resistance to all classes of β-lactams.2,8,10–13 The laboratory detection of KPC among clinical isolates is crucial to limit its spread.9,13,21,24,25 Automated susceptibility testing methods have not been shown to reliably detect KPC-mediated resistance.7,15

In 2009, the CLSI guidelines (M100) recommended the MHT to definitively identify KPC producers.26 This is a phenotypic test to detect carbapenemase production as the cause of reduced susceptibility to carbapenems. The procedure was first described by Lee et al34 in 2001 and was shown to have 100% sensitivity and specificity for detecting KPC. This complex test requires skilled laboratory personnel to set up and trained operators to interpret the results. Consequently, the test suffers from subjectivity in interpretation of the results and might not be optimally sensitive or specific. Being a phenotypic test, it also has a lengthy turnaround time, with reportable results not available for up to 24 hours. Hence, the test is more useful as a confirmatory test rather than as a screening tool.

In accordance with the current CLSI guidelines, clinical samples of E cloacae, S marcescens, E coli, and P aeruginosa are not routinely tested for the presence of KPC by the MHT in our laboratory. The carbapenem resistance detected in these organisms has been considered to have different causes, such as loss of the porin channel combined with the presence of an AmpC β-lactamase.35,36 Carbapenemase-like patterns of nonsusceptibility leading to false-positive MHT results have also been described in association with endemic CTX-M production and AmpC hyperproduction.31,32

Among the 13 isolates that had initially tested positive by the MHT and negative by PCR in our study, 10 were negative by MHT on repeated testing, including 6 K pneumoniae, 3 E cloacae and 1 P aeruginosa. The 2 K pneumoniae isolates that remained MHT+ and had discrepant PCR results were both positive for AmpC. Two possibilities exist that may explain these 2 MHT+/KPC PCR– isolates. First, as reported by Schechner et al,30 KPC PCR could be falsely negative due to inhibitory substances in the reaction or technical inexperience. However, the second and most probable reason could be the presence of an up-regulated AmpC enzyme, extended-spectrum β-lactamase, primarily CTX-Ms with mutation as described by Carvalhaes et al32 and Pasteran et al.37

The blaKPC genes encoding the KPCs are present on plasmids.8,17,22,23 During the past few years, conventional and real-time PCR methods have been described to detect these genes.15,20,27–29 A PCR assay, such as ours, offers the advantage of faster genotyping and a shorter turnaround time (4 hours for the PCR, compared with >24 hours for the MHT). There is also less subjectivity in interpreting the results. However, the complexity of these tests requires skilled and trained operators as well. Because these assays are specific for the particular target sequence, they cannot be used to monitor the emergence of novel variants. Furthermore, the presence of the target blaKPC genes does not always correlate with the phenotypic expression of KPC, nor is it able to quantify the level of expression. These assays are not able to detect mechanisms of resistance apart from KPC.

During the preparation of this manuscript, new CLSI guidelines were published (CLSI, M100-S20U, 2010) with 2-fold lower minimum inhibitory concentrations (MICs) for each category (susceptible, intermediate, resistant) for the Enterobacteriaceae for meropenem, imipenem, and ertapenem.38 Furthermore, it is suggested that the use of these breakpoints will obviate the need for confirmatory testing on specimens if only for infection control purposes. However, Daikos et al39 have shown that K pneumoniae isolates with MICs to imipenem as low as 0.12 μg/mL still possess carbapenemases. Although these were metallo-β-lactamases rather than KPCs, they would have been detected using MHT. In addition, some of the current automated system platform configurations will make it difficult if not impossible to implement these new guidelines in the next year or two. In our laboratory, we have detected MHT+ isolates with MICs to imipenem as low as 1 to 2 μg/mL and confirmed as KPC with PCR. Despite these new recommendations, we will continue to confirm carbapenemase activity using MHT or PCR.

The PCR assay shows strong correlation with the results of the MHT while offering comparable sensitivity and specificity. These findings, combined with the more rapid turnaround time of PCR assays, suggest this assay might be more suitable as an initial screening test for detecting KPC-mediated carbapenem resistance. This rapid identification of KPC would help optimize selection and commencement of appropriate antibiotic therapy in a timely manner and so might be of value in improving clinical outcomes.

CME/SAM

Upon completion of this activity you will be able to:

  • recognize emerging mechanisms for resistance to β-lactam antibiotics.

  • cite the current Clinical and Laboratory Standards Institute standard for laboratory detection of Klebsiella pneumoniae carbapenemase–mediated carbapenem resistance.

  • discuss the advantages and disadvantages of the currently recommended method for detection of carbapenem resistance.

The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this educational activity for a maximum of 1 AMA PRA Category 1 Credit ™ per article. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module.

The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Questions appear on p 643. Exam is located at www.ascp.org/ajcpcme.

Funded by Residents Research Grant No. 9006 from the Department of Medical Education, Henry Ford Health System, Detroit, MI.

References

1.
Zanetti
G
Bally
F
Greub
G
et al
Cefepime versus imipenemcilastatin for treatment of nosocomial pneumonia in intensive care unit patients: a multicenter, evaluator-blind, prospective, randomized study
.
Antimicrob Agents Chemother
 .
2003
;
47
:
3442
3447
.
2.
Deshpande
LM
Rhomberg
PR
Sader
HS
et al
Emergence of serine carbapenemases (KPC and SME) among clinical strains of Enterobacteriaceae isolated in the United States medical centers: report from the MYSTIC Program (1999–2005)
.
Diagn Microbiol Infect Dis
 .
2006
;
56
:
367
372
.
3.
Deshpande
LM
Jones
RN
Fritsche
TR
et al
Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000–2004)
.
Microb Drug Resist
 .
2006
;
12
:
223
230
.
4.
Leavitt
A
Navon-Venezia
S
Chmelnitsky
I
et al
Emergence of KPC-2 and KPC-3 in carbapenem-resistant Klebsiella pneumoniae in an Israeli hospital
.
Antimicrob Agents Chemother
 .
2007
;
51
:
3026
3029
.
5.
Naas
T
Nordmann
P
Vedel
G
et al
Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France [letter]
.
Antimicrob Agents Chemother
 .
2005
;
49
:
4423
4424
.
6.
Samra
Z
Ofir
O
Lishtzinsky
Y
et al
Outbreak of carbapenem-resistant Klebsiella pneumoniae producing KPC-3 in a tertiary medical centre in Israel
.
Int J Antimicrob Agents
 .
2007
;
30
:
525
529
.
7.
Tenover
FC
Kalsi
RK
Williams
PP
et al
Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing
.
Emerg Infect Dis
 .
2006
;
12
:
1209
1213
.
8.
Villegas
MV
Lolans
K
Correa
A
et al
First detection of the plasmid-mediated class A carbapenemase KPC-2 in clinical isolates of Klebsiella pneumoniae from South America
.
Antimicrob Agents Chemother
 .
2006
;
50
:
2880
2882
.
9.
Yigit
H
Queenan
AM
Rasheed
JK
et al
Carbapenem-resistant strain of Klebsiella oxytoca harboring carbapenem-hydrolyzing beta-lactamase KPC-2
.
Antimicrob Agents Chemother
 .
2003
;
47
:
3881
3889
.
10.
Bradford
PA
Bratu
S
Urban
C
et al
Emergence of carbapenem-resistant Klebsiella species possessing the class A carbapenem hydrolyzing KPC-2 and inhibitor-resistant TEM-30 β-lactamases in New York City
.
Clin Infect Dis
 .
2004
;
39
:
55
60
.
11.
Lomaestro
BM
Tobin
EH
Shang
W
.
The spread of Klebsiella pneumoniae carbapenemase-producing K pneumoniae to upstate New York
.
Clin Infect Dis
 .
2006
;
43
:
e26
e28
. doi:.
12.
Nordmann
P
Poirel
L
.
Emerging carbapenemases in gram negative aerobes
.
Clin Microbiol Infect
 .
2002
;
8
:
321
331
.
13.
Woodford
N
Tierno
PM
Jr
Young
K
et al
Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A β-lactamase, KPC-3, in a New York Medical Center
.
Antimicrob Agents Chemother
 .
2004
;
48
:
4793
4799
.
14.
Bonnet
R
Marchandin
H
Chanal
C
et al
Chromosome-encoded class D β-lactamase OXA-23 in Proteus mirabilis
.
Antimicrob Agents Chemother
 .
2002
;
46
:
2004
2006
.
15.
Bratu
S
Landman
D
Alam
M
et al
Detection of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp from Brooklyn, New York
.
Antimicrob Agents Chemother
 .
2005
;
49
:
776
778
.
16.
Hossain
A
Ferraro
MJ
Pino
RM
et al
Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in an Enterobacter sp
.
Antimicrob Agents Chemother
 .
2004
;
48
:
4438
4440
.
17.
Miriagou
V
Tzouvelekis
LS
Rossiter
S
et al
Imipenem resistance in a Salmonella clinical strain due to plasmid-mediated class A carbapenemase KPC-2
.
Antimicrob Agents Chemother
 .
2003
;
47
:
1297
1300
.
18.
Navon-Venezia
S
Chmelnitsky
I
Leavitt
A
et al
Plasmid-mediated imipenem-hydrolyzing enzyme KPC-2 among multiple carbapenem-resistant Escherichia coli clones in Israel
.
Antimicrob Agents Chemother
 .
2006
;
50
:
3098
3101
.
19.
Samra
Z
Bahar
J
Madar-Shapiro
L
et al
Evaluation of CHROMagar KPC for rapid detection of carbapenem-resistant Enterobacteriaceae
.
J Clin Microbiol
 .
2008
;
46
:
3110
3111
.
20.
Tibbetts
R
Frye
JG
Marschall
J
et al
Detection of KPC-2 in a clinical isolate of Proteus mirabilis and first reported description of carbapenemase resistance caused by a KPC β-lactamase in P mirabilis
.
J Clin Microbiol
 .
2008
;
46
:
3080
3083
.
21.
Yigit
H
Queenan
AM
Anderson
GJ
et al
Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae
.
Antimicrob Agents Chemother
 .
2001
;
45
:
1151
1161
.
22.
Bratu
S
Brooks
S
Burney
S
et al
Detection and spread of Escherichia coli possessing the plasmid-borne carbapenemase KPC-2 in Brooklyn, New York
.
Clin Infect Dis
 .
2007
;
44
:
972
975
.
23.
Wei
ZQ
Du
XX
Yu
YS
et al
Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China
.
Antimicrob Agents Chemother
 .
2007
;
51
:
763
765
.
24.
Alba
J
Ishii
Y
Thomson
EK
et al
Kinetics study of KPC-3, a plasmid-encoded class A carbapenem-hydrolyzing beta-lactamase
.
Antimicrob Agents Chemother
 .
2005
;
49
:
4760
4762
.
25.
Bratu
S
Landman
D
Haag
R
et al
Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: a new threat to our antibiotic armamentarium
.
Arch Intern Med
 .
2005
;
165
:
1430
1435
.
26.
Clinical and Laboratory Standards Institute
.
Performance standards for antimicrobial susceptibility testing; M100-S19 checklist
.
Wayne, PA
:
Clinical and Laboratory Standards Institute
;
2009
.
27.
Bratu
S
Tolaney
P
Karumudi
U
et al
Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents
.
J Antimicrob Chemother
 .
2005
;
56
:
128
132
.
28.
Cole
JM
Schuetz
AN
Hill
CE
et al
Development and evaluation of a real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase genes
.
J Clin Microbiol
 .
2009
;
47
:
322
326
.
29.
Hindiyeh
M
Smollen
G
Grossman
Z
et al
Rapid detection of blaKPC carbapenemase genes by real-time PCR
.
J Clin Microbiol
 .
2008
;
46
:
2879
2883
.
30.
Schechner
V
Straus-Robinson
K
Schwartz
D
et al
Evaluation of PCR-based testing for surveillance of KPC-producing carbapenem-resistant members of the Enterobacteriaceae family
.
J Clin Microbiol
 .
2009
;
47
:
3261
3265
.
31.
Pasteran
F
Mendez
T
Guerriero
L
et al
Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae
.
J Clin Microbiol
 .
2009
;
47
:
1631
1639
.
32.
Carvalhaes
CG
Picão
RC
Nicoletti
AG
et al
Cloverleaf test (modified Hodge test) for detecting carbapenemase production in Klebsiella pneumoniae: be aware of false positive results
.
J Antimicrob Chemother
 .
2009
;
65
:
249
251
.
33.
Black
JA
Moland
ES
Thomson
KS
.
AmpC disk test for detection of plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking chromosomal AmpC β-lactamases
.
J Clin Microbiol
 .
2005
;
43
:
3110
3113
.
34.
Lee
K
Chong
Y
Shin
HB
et al
Modified Hodge and EDTA-disk synergy tests to screen metallo-β-lactamase–producing strains of Pseudomonas and Acinetobacter species
.
Clin Microbiol Infect
 .
2001
;
7
:
88
91
.
35.
Cao
VT
Arlet
G
Ericsson
BM
et al
Emergence of imipenem resistance in Klebsiella pneumoniae owing to combination of plasmid-mediated CMY-4 and permeability alteration
.
J Antimicrob Chemother
 .
2000
;
46
:
895
900
.
36.
Crowley
B
Benedí
VJ
Doménech-Sánchez
A
.
Expression of SHV-2 β-lactamase and of reduced amounts of OmpK36 porin in Klebsiella pneumoniae results in increased resistance to cephalosporins and carbapenems
.
Antimicrob Agents Chemother
 .
2002
;
46
:
3679
3682
.
37.
Pasteran
F
Mendez
T
Rapoport
M
et al
Controlling false-positive results obtained with the Hodge and Masuda assays for detection of class A carbapenemase in species of Enterobacteriaceae by incorporating boronic acid
.
J Clin Microbiol
 .
2010
;
48
:
1323
1332
.
38.
Clinical and Laboratory Standards Institute
.
M100-S20: update performance standards for antimicrobial susceptibility testing, M100-S20U
.
Wayne, PA
:
Clinical and Laboratory Standards Institute
;
2010
.
39.
Daikos
G
Petrikkos
P
Psichogiou
M
et al
Prospective observational study of the impact of VIM-1 metallo-β-lactamase on the outcome of patients with Klebsiella pneumoniae bloodstream infections
.
Antimicrob Agents Chemother
 .
2009
;
53
:
1868
1873
.