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1.  Multiplex Real-Time PCR Assay for Detection and Classification of Klebsiella pneumoniae Carbapenemase Gene (blaKPC) Variants▿ 
Journal of Clinical Microbiology  2011;49(2):579-585.
Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (blaKPC) have been reported, with KPC-2 (blaKPC-2) and KPC-3 (blaKPC-3) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of blaKPC variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described blaKPC variants (blaKPC-2 to blaKPC-11). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each blaKPC variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with blaKPC-3 and blaKPC-2 identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel blaKPC variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.
doi:10.1128/JCM.01588-10
PMCID: PMC3043520  PMID: 21123529
2.  Rapid and sensitive detection of blaKPC gene in clinical isolates of Klebsiella pneumoniae by a molecular real-time assay 
SpringerPlus  2013;2:31.
Background
The aim of this study was the rapid identification of blaKPC gene in 38 Klebsiella pneumoniae clinical isolates with reduced susceptibility to carbapenems. The modified Hodge Test (MHT) was carried out to phenotypically determine whether resistance to carbapenems was mediated by a carbapenemase. The detection of the blaKPC gene was performed by real-time acid nucleic sequence-based amplification (NASBA™™), specifically designed for the detection of KPC RNA target.
Results
Thirty-two/38 isolates evaluated by MHT showed the production of carbapenemases, while all the strains exhibited the production of KPC by inhibition test with phenylboronic acid (the combined disk test with IPM/IPM plus phenylboronic acid). The detection of blaKPC gene by Nuclisens EasyQ KPC yielded positive results in 38/38 (100%) strains. The presence of blaKPC gene was confirmed in all K. pneumoniae isolates when tested by the gold standard PCR assay.
Conclusions
In consideration of the serious challenge represented by infections due to K. pneumoniae it appears necessary the rapid identification of carbapenemases in clinical settings as it is made possible by the use of NASBA™ assay.
doi:10.1186/2193-1801-2-31
PMCID: PMC3581762  PMID: 23450269
Klebsiella pneumoniae; Carbapenem resistance; blaKPC; NASBA™
3.  Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses 
In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of blaKPC and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various β-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC β-lactamase. The real-time PCR assay was able to detect the presence of blaKPC in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying blaKPC-2 and 12 isolates carrying blaKPC-3. This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes blaKPC-2-like and blaKPC-3-like alleles. Therefore, this assay is a rapid method to identify the presence of blaKPC enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.
doi:10.1128/JCM.02193-12
PMCID: PMC3536237  PMID: 23077125
4.  The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria 
The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.
doi:10.1186/1476-0711-12-30
PMCID: PMC3816589  PMID: 24143953
Duplex; Real-time PCR; Infection control; Carbapenemase
5.  Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes▿  
Journal of Clinical Microbiology  2008;47(2):322-326.
We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of blaKPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the blaKPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the blaKPC PCR assay was used to confirm the presence of blaKPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected blaKPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of blaKPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two blaKPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with blaKPC-2 and blaKPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.
doi:10.1128/JCM.01550-08
PMCID: PMC2643690  PMID: 19036932
6.  blaKPC gene Detection in Clinical Isolates of Carbapenem Resistant Enterobacteriaceae in a Tertiary Care Hospital 
Introduction: Carbapenem resistance among Enterobacteriaceae, especially in Klebsiella pneumoniae and Escherichia coli, is an emerging problem worldwide. A common mechanism of carbapenem resistance is the production of class-A, Klebsiella pneumoniae carbapenemase (KPC).
Aims and Objectives: The present study focused on determining the antibiotic resistance pattern and prevalence of bla KPC gene coding for KPC in carbapenem resistant Enterobacteriaceae.
Methodology: Forty six carbapenem resistant isolates belonging to the family Enterobacteriaceae were tested for antibiotic sensitivity pattern. Modified Hodge Test (MHT) and PCR for bla KPC gene detection were performed on these isolates. Of these, 22 were Klebsiella pneumoniae, 21 were Escherichia coli, 2 were Citrobacter species and 1 was Proteus mirabilis
Results: Forty three (93.4%) out of the 46 isolates were resistant to Meropenem, 34 (73.9%) were resistant to Imipenem and 30 (65.2%) were resistant to both Imipenem and Meropenem. Modified Hodge Test was positive in 38 (82.6%) out of 46 isolates and blaKPC gene was detected in 31 (67.4%) isolates. bla KPC gene was detected in 28 out of the 38 MHT positive isolates.
doi:10.7860/JCDR/2013/7759.3747
PMCID: PMC3919306  PMID: 24551626
Enterbacteriaceae; Carbapenem resistance; Modified Hodge Test; blaKPC gene
7.  First report of Klebsiella pneumonia carbapenemase-producing Pseudomonas aeruginosa isolated from burn patients in Iran: phenotypic and genotypic methods 
Wound infection associated with carbapenem-resistant Pseudomonas aeruginosa in burn patients is a growing problem. One of the main mechanisms of resistance to carbapenem antibiotics is the ability of P. aeruginosa to produce carbapenemase enzymes. Klebsiella pneumonia carbapemenase (KPC) is an important type of carbapenemase which can hydrolyze carbapenem antibiotics. The Modified Hodge Test (MHT) and boronic acid as a KPC inhibitor are two phenotypic methods used for detection of carbapenemase. The sensitivity and specificity of these two phenotypic tests for the identification of KPC can be measured by PCR.
In this study, 241 P. aeruginosa strains were isolated from wounds of hospitalized burn patients. Carbapenem-resistant P. aeruginosa isolates were determined by the disk diffusion method. KPC-producing carbapenem-resistant strains were examined using the Modified Hodge Test, followed by boronic acid. Further, strains with positive responses to MHT and boronic acid tests were analyzed with the PCR molecular method. One hundred eighty-six of 241 isolates were resistant to carbapenems and 75 were positive in the MHT. Three exhibited an at least 5-mm diameter difference when meropenem was combined with boronic acid vs meropenem alone in the boronic acid test. Two strains had a specific band with primer No.1 after gel electrophoresis.
This study showed that MHT, despite excellent sensitivity, has variable specificity independent of bacterial species. Further, the use of KPC inhibitors such as boronic acid did not yield favorable sensitivity and specificity among the specimens from Iranian patients. Thus, it seems that sequencing after PCR should be considered the gold standard for the detection of KPC-producing P. aeruginosa.
doi:10.3205/dgkh000226
PMCID: PMC3960934  PMID: 24653970
P. aeruginosa; KPC; boronic acid; Modified Hodge Test; blaKPC
8.  Evaluation of the New NucliSENS EasyQ KPC Test for Rapid Detection of Klebsiella pneumoniae Carbapenemase Genes (blaKPC) 
Journal of Clinical Microbiology  2012;50(8):2783-2785.
KPC-type carbapenemases are emerging in Klebsiella pneumoniae and other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for blaKPC detection with 300 members of the Enterobacteriaceae, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring blaKPC genes, with no false positives, and results were available within 2 h.
doi:10.1128/JCM.00284-12
PMCID: PMC3421519  PMID: 22622445
9.  Rapid Detection of blaKPC Carbapenemase Genes by Real-Time PCR▿  
Journal of Clinical Microbiology  2008;46(9):2879-2883.
Carbapenem resistance among Enterobacteriaceae is an emerging problem worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) enzymes are among the most common β-lactamases described. In this study, we report the development and validation of a real-time PCR (q-PCR) assay for the detection of blaKPC genes using TaqMan chemistry. The q-PCR amplification of blaKPC DNA was linear over 7 log dilutions (r2 = 0.999; slope, 3.54), and the amplification efficiency was 91.6%. The q-PCR detection limit was 1 CFU, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria. Perianal/rectal swabs (n = 187) collected in duplicate from 128 patients admitted to Sheba Medical Center surgical intensive care units were evaluated for the presence of carbapenem-resistant bacteria by culturing on MacConkey agar-plus-carbapenem disks and for blaKPC genes by q-PCR. Carbapenem-resistant organisms, all K. pneumoniae, were isolated from 47 (25.1%) of the 187 samples collected, while blaKPC genes were detected in 54 (28.9%) of the patient samples extracted by the NucliSENS easyMAG system. Of these, seven samples were positive for blaKPC genes by q-PCR but negative for carbapenem resistance by culture, while all samples in which no carbapenem-resistant bacteria were detected by culture also tested negative by q-PCR. Thus, the sensitivity and specificity of the q-PCR assay after extraction by the NucliSENS easyMAG system were 100% and 95%, respectively. Similar values were obtained after DNA extraction by the Roche MagNA Pure LC instrument: 97.9% sensitivity and 96.4% specificity. Overall, the blaKPC q-PCR assay appears to be highly sensitive and specific. The utilization of q-PCR will shorten the time to blaKPC detection from 24 h to 4 h and will help in rapidly isolating colonized or infected patients and assigning them to cohorts.
doi:10.1128/JCM.00661-08
PMCID: PMC2546719  PMID: 18614657
10.  Phenotypic and Enzymatic Comparative Analysis of the Novel KPC Variant KPC-5 and Its Evolutionary Variants, KPC-2 and KPC-4▿  
A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated blaKPC-5, was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of blaKPC-5 showed significant differences from the flanking regions of other blaKPC variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro103→Arg), while KPC-4 contained Pro103→Arg plus an additional amino acid change (Val239→Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.
doi:10.1128/AAC.00734-08
PMCID: PMC2630594  PMID: 19015357
11.  Rectal Screening for Klebsiella pneumoniae Carbapenemases: Comparison of Real-Time PCR and Culture Using Two Selective Screening Agar Plates 
Journal of Clinical Microbiology  2012;50(8):2596-2600.
Klebsiella pneumoniae carbapenemases (KPCs) have recently been described in Chicago, IL, especially among residents of long-term acute care hospitals (LTACHs). These patients are frequently transferred to local Chicago hospitals for higher acuity of medical care, and rapid detection and isolation of KPC-colonized LTACH residents may interrupt the introduction of KPCs into acute care hospitals. We evaluated the performance of a real-time PCR for blaKPC from enrichment broth versus direct plating of rectal surveillance swabs on two selective culture media, CHROMagar extended-spectrum-β-lactamase (ESBL) and vancomycin, amphotericin B, ceftazidime, and clindamycin (VACC) plates. Rectal surveillance swabs were collected as part of a point prevalence study of KPC carriage rates among 95 residents of two Chicago area LTACHs. Discrepant results between PCR and culture were resolved by subculturing the enrichment broth. Overall, 66 of 95 patients (69.5%) were colonized with KPCs, using the cumulative results of culture as a reference standard. Real-time PCR from enrichment broth was positive in 64 of 66 (97%) colonized patients, including nine surveillance swabs that were missed by both selective culture media. PCR demonstrated higher sensitivity, 97.0%, than culture using either CHROMagar or VACC plates (both with sensitivity of 77.3%). In addition, turnaround time was significantly shorter for the PCR-based method than for culture, with a mean of 24 h versus 64 to 72 h for CHROMagar and VACC plates (P < 0.0001). Overall, PCR for blaKPC represents the best screening test for KPCs with significantly higher sensitivity and with less hands-on time, resulting in a shorter time to results.
doi:10.1128/JCM.00654-12
PMCID: PMC3421488  PMID: 22622443
12.  A Long-Term Low-Frequency Hospital Outbreak of KPC-Producing Klebsiella pneumoniae Involving Intergenus Plasmid Diffusion and a Persisting Environmental Reservoir 
PLoS ONE  2013;8(3):e59015.
Background
To study the molecular characteristics of a long-term, low frequency outbreak of blaKPC-2 in a low prevalence setting involving the hospital environment.
Methodology/Principal Findings
KPC-producing bacteria were screened by selective chromogenic agar and Real-Time PCR. The presence of antibiotic resistance genes was ascribed by PCRs and subsequent sequencing, and the KPC-producing isolates were phylogenetically typed using PFGE and multi-locus sequence typing. BlaKPC-2-plasmids were identified and analysed by S1-nuclease-PFGE hybridization and PCR based replicon typing. A ∼97 kb IncFII plasmid was seen to carry blaKPC-2 in all of the clinical isolates, in one of the isolates recovered from screened patients (1/136), and in the Klebsiella pneumoniae and Enterobacter asburiae isolates recovered from the environment (sinks) in one intensive care unit. The K. pneumoniae strain ST258 was identified in 6 out of 7 patients. An intergenus spread to E. asburiae and an interspecies spread to two different K. pneumoniae clones (ST27 and ST461) of the blaKPC-2 plasmid was discovered. K. pneumoniae ST258 and genetically related E. asburiae strains were found in isolates of both human and environmental origins.
Conclusions/Significance
We document a clonal transmission of the K. pneumoniae ST258 strain, and an intergenus plasmid diffusion of the IncFII plasmid carrying blaKPC-2 in this outbreak. A major reservoir in the patient population could not be unveiled. However, the identification of a persisting environmental reservoir of strains with molecular determinants linked to human isolates, suggests a possible role of the environment in the maintenance of this long-term outbreak.
doi:10.1371/journal.pone.0059015
PMCID: PMC3594221  PMID: 23536849
13.  Multiplex Real-Time PCR for Detection of an Epidemic KPC-Producing Klebsiella pneumoniae ST258 Clone 
We describe a multiplex real-time PCR assay capable of identifying both the epidemic Klebsiella pneumoniae ST258 clone and blaKPC carbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone and blaKPC in a collection of 75 K. pneumoniae isolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistant K. pneumoniae clinical isolates.
doi:10.1128/AAC.00316-12
PMCID: PMC3370784  PMID: 22450983
14.  Direct Detection and Genotyping of Klebsiella pneumoniae Carbapenemases from Urine by Use of a New DNA Microarray Test 
Journal of Clinical Microbiology  2012;50(12):3990-3998.
Klebsiella pneumoniae carbapenemases (KPCs) are considered a serious threat to antibiotic therapy, as they confer resistance to carbapenems, which are used to treat extended-spectrum beta-lactamase (ESBL)-producing bacteria. Here, we describe the development and evaluation of a DNA microarray for the detection and genotyping of KPC genes (blaKPC) within a 5-h period. To test the whole assay procedure (DNA extraction plus a DNA microarray assay) directly from clinical specimens, we compared two commercial DNA extraction kits (the QIAprep Spin miniprep kit [Qiagen] and the urine bacterial DNA isolation kit [Norgen]) for the direct DNA extraction from urine samples (dilution series spiked in human urine). Reliable single nucleotide polymorphism (SNP) typing was demonstrated using 1 × 105 CFU/ml urine for Escherichia coli (Qiagen and Norgen) and 80 CFU/ml urine, on average, for K. pneumoniae (Norgen). This study presents, for the first time, the combination of a new KPC microarray with commercial sample preparation for detecting and genotyping microbial pathogens directly from clinical specimens; this paves the way toward tests providing epidemiological and diagnostic data, enabling better antimicrobial stewardship.
doi:10.1128/JCM.00990-12
PMCID: PMC3502996  PMID: 23035190
15.  Rectal Swabs Are Suitable for Quantifying the Carriage Load of KPC-Producing Carbapenem-Resistant Enterobacteriaceae 
It is more convenient and practical to collect rectal swabs than stool specimens to study carriage of colon pathogens. In this study, we examined the ability to use rectal swabs rather than stool specimens to quantify Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae (CRE). We used a quantitative real-time PCR (qPCR) assay to determine the concentration of the blaKPC gene relative to the concentration of 16S rRNA genes and a quantitative culture-based method to quantify CRE relative to total aerobic bacteria. Our results demonstrated that rectal swabs are suitable for quantifying the concentration of KPC-producing CRE and that qPCR showed higher correlation between rectal swabs and stool specimens than the culture-based method.
doi:10.1128/AAC.01275-12
PMCID: PMC3591867  PMID: 23295937
16.  Real-Time Detection of blaKPC in Clinical Samples and Surveillance Specimens ▿  
Journal of Clinical Microbiology  2011;49(9):3338-3339.
A real-time PCR assay was developed targeting the blaKPC responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.
doi:10.1128/JCM.00268-11
PMCID: PMC3165576  PMID: 21734030
17.  Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases 
Annals of Laboratory Medicine  2012;32(5):359-361.
In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.
doi:10.3343/alm.2012.32.5.359
PMCID: PMC3427824  PMID: 22950072
Carbapenemase; Multiplex PCR; KPC; GES
18.  Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae 
Journal of Medical Microbiology  2009;58(Pt 10):1303-1308.
Isolates of Klebsiella pneumoniae harbouring the carbapenemase KPC may have carbapenem MICs that remain in the susceptible range, and may therefore go unrecognized. To understand the mechanisms contributing to the variability in carbapenem MICs, 20 clinical isolates, all belonging to either of two clonal groups of KPC-possessing K. pneumoniae endemic to New York City, were examined. Expression of genes encoding KPC, the porins OmpK35 and OmpK36, and the efflux pump AcrAB was examined by real-time RT-PCR. Outer-membrane profiles of selected KPC-producing isolates were examined by SDS-PAGE, and proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry. The identification of SHV and TEM β-lactamases and the genomic sequences of ompK35 and ompK36 were determined by PCR and DNA sequencing, respectively. For one clonal group, carbapenem MICs increased with decreasing expression of ompK36. A second clonal group also had carbapenem MICs that correlated with ompK36 expression. However, all of the isolates in this latter group continued to produce OmpK36, suggesting that porin configuration may affect entry of carbapenems. For isolates that had the greatest expression of ompK36, carbapenem MICs tended to be lower when determined by the broth microdilution technique, and scattered colonies were seen around the Etest zones of inhibition. All of the KPC-producing isolates were highly resistant to ertapenem, regardless of ompK36 expression. In conclusion, isolates of KPC-possessing K. pneumoniae that express ompK36 tend to have lower MICs to carbapenems and therefore may be more difficult to detect by clinical laboratories. Regardless of ompK36 expression, all of the KPC producers were consistently resistant to ertapenem.
doi:10.1099/jmm.0.012575-0
PMCID: PMC2887543  PMID: 19556371
19.  Laboratory Detection of Enterobacteriaceae That Produce Carbapenemases 
Journal of Clinical Microbiology  2012;50(12):3877-3880.
A study was designed to evaluate the modified Hodge test (MHT), Mastdiscs ID inhibitor combination disks (MDI), Rosco Diagnostica Neo-Sensitabs (RDS), metallo-β-lactamase (MBL) Etest, and in-house multiplex PCR for the detection of well-characterized carbapenemase-producing Enterobacteriaceae. One hundred forty-two nonrepeat clinical isolates of carbapenemase-producing Enterobacteriaceae (including Klebsiella spp., Escherichia coli, Citrobacter freundii, and Enterobacter spp.) obtained from the SMART worldwide surveillance program during 2008 to 2009 were included. These included 49 KPC-, 27 NDM-, 19 VIM-, 14 OXA-48-like enzyme-, and 5 IMP-producing isolates and 28 carbapenem-resistant, carbapenemase-negative isolates. The manufacturer's instructions were followed for MDI, RDS, and MBL Etest and CLSI guidelines for MHT. A multiplex PCR was designed to detect KPC, NDM, VIM, IMP, and OXA-48-like carbapenemases. Overall, the sensitivity and specificity were 78% and 93% for MDI, 80% and 93% for RDS, 58% and 93% for MHT, and 55% and 100% for MBL Etest, respectively. The PCR had 100% sensitivity and specificity. MDI and RDS performed well for the detection of KPCs and NDMs but poorly for VIMs, IMPs, and OXA-48-like enzymes. MHT performed well for KPCs and OXA-48-like enzymes but poorly for NDMs, VIMs, and IMPs. MDI and RDS were easy to perform and interpret but lacked sensitivity for OXA-48-like enzymes, VIMs, and IMPs. MHT and MBL Etest were often difficult to interpret. We recommend using molecular tests for the optimal detection of carbapenemase-producing Enterobacteriaceae.
doi:10.1128/JCM.02117-12
PMCID: PMC3503014  PMID: 22993175
20.  Evaluation of a DNA Microarray, the Check-Points ESBL/KPC Array, for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum β-Lactamases and KPC Carbapenemases▿  
Extended-spectrum ß-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPC carbepenemases) have rapidly emerged worldwide and require rapid identification. The Check-Points ESBL/KPC array, a new commercial system based on genetic profiling for the direct identification of ESBL producers (SHV, TEM, and CTX-M) and of KPC producers, was evaluated. Well-characterized Gram-negative rods (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii) expressing various ß-lactamases (KPC-2, SHV, TEM, and CTX-M types) were used as well as wild-type reference strains and isolates harboring ß-lactamase genes not detected by the assay. In addition, phenotypically confirmed ESBL producers isolated in clinical samples over a 3-month period at the Bicetre hospital were analyzed using the Check-Points ESBL/KPC array and by standard PCR. The Check-Points ESBL/KPC array allowed fast detection of all TEM, SHV, and CTX-M ESBL genes and of the KPC-2 gene. The assay allowed easy differentiation between non-ESBL TEM and SHV and their ESBL derivatives. None of the other tested ß-lactamase genes were detected, underlining its high specificity. The technique is suited for Enterobacteriaceae but also for P. aeruginosa and A. baumannii. However, for nonfermenters, especially P. aeruginosa, a 1:10 dilution of the total DNA was necessary to detect KPC-2 and SHV-2a genes reliably. The Check-Points ESBL/KPC array is a powerful high-throughput tool for rapid identification of ESBLs and KPC producers in cultures. It provided definitive results within the same working day, allowing rapid implementation of isolation measures and appropriate antibiotic treatment. It showed an interesting potential for routine laboratory testing.
doi:10.1128/AAC.01298-09
PMCID: PMC2916358  PMID: 20547813
21.  Emergence of blaKPC-containing Klebsiella pneumoniae in a long-term acute care hospital: a new challenge to our healthcare system 
Objectives
To characterize isolates of Klebsiella pneumoniae producing KPC carbapenemase (KPC-Kp) associated with an outbreak in a long-term acute care hospital (LTACH) in South Florida.
Methods
During 21 March to 20 April 2008, 241 K. pneumoniae isolates detected at Integrated Regional Laboratories (Ft. Lauderdale, FL) for which the ertapenem MICs were ≥4 mg/L were studied. PCR, cloning and sequence analysis were used to detect blaKPC and to characterize the β-lactamase and outer membrane proteins (Omps). The expression level of KPC enzymes was studied by immunoblotting. Genetic relatedness of isolates was investigated with rep-PCR and PFGE. Clinical records of patients were investigated.
Results
Seven KPC-Kp strains were isolated from different patients located at a single LTACH, with a further three isolates being recovered from patients at different hospitals. All KPC-Kp isolates in patients from the LTACH and from one hospital patient were genetically related and shared PFGE patterns that clustered with known sequence type (ST) 258 strains. These strains were highly resistant to carbapenems (MICs ≥ 32 mg/L) due to an increased level of KPC expression and loss of Omps. Rectal colonization was documented in all LTACH patients with KPC-Kp isolates. Treatment failures were common (crude mortality rate of 69%). Active surveillance and enhanced infection control practices terminated the KPC-Kp outbreak.
Conclusions
The detection of KPC-Kp in an LTACH represents a serious infection control and therapeutic challenge in a new clinical setting. The speed at which the epidemic of KPC-Kp is spreading in our healthcare system mandates urgent action.
doi:10.1093/jac/dkp327
PMCID: PMC2760463  PMID: 19740911
LTCF; porins; carbapenemases; Enterobacteriaceae; outbreak
22.  Characteristics of Meropenem Heteroresistance in Klebsiella pneumoniae Carbapenemase (KPC)-Producing Clinical Isolates of K. pneumoniae▿  
Journal of Clinical Microbiology  2010;48(7):2601-2604.
Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 μg/ml. Heteroresistant colonies had significantly elevated expression of the blaKPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.
doi:10.1128/JCM.02134-09
PMCID: PMC2897536  PMID: 20504985
23.  Rapid and Simultaneous Detection of blaKPC and blaNDM by Use of Multiplex Real-Time PCR 
Journal of Clinical Microbiology  2013;51(4):1247-1249.
The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting blaKPC and blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.
doi:10.1128/JCM.03316-12
PMCID: PMC3666810  PMID: 23325823
24.  Ultrarapid Detection of blaKPC1/2-12 from Perirectal and Nasal Swabs by Use of Real-Time PCR 
Journal of Clinical Microbiology  2012;50(5):1718-1720.
The novel real-time PCR assay developed as described here was able to detect blaKPC1/2-12 (blaKPC-1/2 to blaKPC-12) from easily available clinical specimens in less than 2 h. The genotypic assay was highly sensitive (100%) and specific (98%). In some cases, it was able to detect blaKPC 48 h before positive detection by standard phenotypic assay on patients who were monitored daily. The high sensitivity and rapidity of the molecular method make it the method of choice for KPC surveillance and, thus, containment purposes.
doi:10.1128/JCM.00195-12
PMCID: PMC3347151  PMID: 22378915
25.  “Silent” Dissemination of Klebsiella pneumoniae Isolates Bearing K. pneumoniae Carbapenemase in a Long-term Care Facility for Children and Young Adults in Northeast Ohio 
Our findings reveal that in 2004 the blaKPC-3 gene was present in a children’s long-term healthcare facility. This suggests pockets of Klebsiella pneumoniae carbapenemase–producing K. pneumoniae independent from sequence type 258 existing in locations not identified at that time.
Background. Klebsiella pneumoniae isolates harboring the K. pneumoniae carbapenemase gene (blaKPC) are creating a significant healthcare threat in both acute and long-term care facilities (LTCFs). As part of a study conducted in 2004 to determine the risk of stool colonization with extended-spectrum cephalosporin-resistant gram-negative bacteria, 12 isolates of K. pneumoniae that exhibited nonsusceptibility to extended-spectrum cephalosporins were detected. All were gastrointestinal carriage isolates that were not associated with infection.
Methods. Reassessment of the carbapenem minimum inhibitory concentrations using revised 2011 Clinical Laboratory Standards Institute breakpoints uncovered carbapenem resistance. To further investigate, a DNA microarray assay, PCR-sequencing of bla genes, immunoblotting, repetitive-sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST) were performed.
Results. The DNA microarray detected blaKPC in all 12 isolates, and blaKPC-3 was identified by PCR amplification and sequencing of the amplicon. In addition, a blaSHV-11 gene was detected in all isolates. Immunoblotting revealed “low-level” production of the K. pneumoniae carbapenemase, and rep-PCR indicated that all blaKPC-3-positive K. pneumoniae strains were genetically related (≥98% similar). According to MLST, all isolates belonged to sequence type 36. This sequence type has not been previously linked with blaKPC carriage. Plasmids from 3 representative isolates readily transferred the blaKPC-3 to Escherichia coli J-53 recipients.
Conclusions. Our findings reveal the “silent” dissemination of blaKPC-3 as part of Tn4401b on a mobile plasmid in Northeast Ohio nearly a decade ago and establish the first report, to our knowledge, of K. pneumoniae containing blaKPC-3 in an LTCF caring for neurologically impaired children and young adults.
doi:10.1093/cid/cis036
PMCID: PMC3404693  PMID: 22492318

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