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1.  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™
2.  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
3.  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
4.  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
5.  Comparative Evaluation of Multiplex PCR and Routine Laboratory Phenotypic Methods for Detection of Carbapenemases among Gram Negative Bacilli 
Background: Carbapenem resistant pathogens cause infections associated with significant morbidity and mortality.
Objective: This study evaluates the use of Multiplex PCR for rapid detection of carbapenemase genes among carbapenem resistant Gram negative bacteria in comparison with the existing phenotypic methods like modified Hodge test (MHT), combined disc test (CDT) and automated methods.
Material and Methods: A total of 100 Carbapenem resistant clinical isolates, [Escherichia coli (25), Klebsiella pneumoniae (35) P. aeruginosa (18) and Acinetobacter baumannii (22)] were screened for the presence of carbapenemases (blaNDM-1, blaVIM, blaIMP and blaKPC genes) by phenotype methods such as the modified Hodge test (MHT) and combined disc test (CDT) and the molecular methods such as Multiplex PCR.
Results: Seventy of the 100 isolates were MHT positive while, 65 isolates were positive by CDT. All the CDT positive isolates with EDTA and APB were Metallo betalactamase (MBL) and K. pneumoniae carbapenemase (KPC) producers respectively. blaNDM-1 was present as a lone gene in 44 isolates. In 14 isolates blaNDM-1 gene was present with blaKPC gene, and in one isolate blaNDM-1 gene was present with blaVIM, gene. Only one E. coli isolate had a lone blaKPC gene. We didn’t find blaIMP gene in any of the isolates. Neither of the genes could be detected in 35 isolates.
Conclusion: Accurate detection of the genes related with carbapenemase production by Molecular methods like Multiplex PCR overcome the limitations of the phenotypic methods and Automated systems.
doi:10.7860/JCDR/2014/10794.5322
PMCID: PMC4316255  PMID: 25653949
blaNDM-1; blaVIM; blaIMPblaKPCgenes; Combined disc test; Modified hodge test; Multiplex PCR
6.  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
7.  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
8.  “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
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.  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
13.  Rapid and Direct Real-Time Detection of blaKPC and blaNDM from Surveillance Samples 
Journal of Clinical Microbiology  2013;51(11):3609-3615.
We assessed the performance of a duplex real-time PCR assay for blaKPC and blaNDM performed directly (D-PCR) on perianal and perirectal swabs and stool. Spiked specimens and 126 clinical surveillance swabs (comprising a sensitivity panel of 46 perirectal double swabs previously determined to be culture positive for blaKPC-PCR-positive Enterobacteriaceae and a specificity panel of 80 perianal swabs from patients at risk of carbapenemase-producing Enterobacteriaceae [CPE] colonization) were studied. For the surveillance swabs, D-PCR was compared to PCR after broth enrichment (BE-PCR) and two culture-based methods: the HardyCHROM ESBL agar (HC-A) and the CDC screening (CDC-A) methods. PCR was performed on morphologically distinct colonies that were isolated by culture. All of the initial PCR testing was done without extraction using a simple lysis procedure. The analytical sensitivities of D-PCR for blaKPC were 9 CFU/μl (for swabs) and 90 CFU/μl (for stool), and for blaNDM, it was 1.9 CFU/μl (for both swabs and stool). In the clinical sensitivity panel, D-PCR and BE-PCR were initially positive for blaKPC in 41/46 (89.1%) and 43/46 (93.5%) swabs, respectively. The swabs that were initially negative by D-PCR (n = 5) and BE-PCR (n = 3) were visibly stool soiled; all swabs were blaKPC positive upon repeat testing after lysate extraction. The CDC-A and HC-A yielded blaKPC-positive Enterobacteriaceae from 36/46 (78.3%) and 35/46 (76.1%) swabs, respectively (sensitivities of D-PCR/BE-PCR postextraction of soiled specimens versus HC-A, P = 0.0009, and versus CDC-A, P = 0.0016). All swabs in the specificity panel were negative for CPE by all four methods. D-PCR allows for the timely detection of blaKPC and blaNDM carriage with excellent sensitivity when specimens visibly soiled with stool undergo preparatory extraction.
doi:10.1128/JCM.01731-13
PMCID: PMC3889741  PMID: 23966498
14.  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
15.  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
16.  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
17.  The modified Hodge test is a useful tool for ruling out klebsiella pneumoniae carbapenemase 
Clinics  2012;67(12):1427-1431.
OBJECTIVE:
Enterobacteriaceae bacteria harboring Klebsiella pneumoniae carbapenemase are a serious worldwide threat. The molecular identification of these pathogens is not routine in Brazilian hospitals, and a rapid phenotypic screening test is desirable. This study aims to evaluate the modified Hodge test as a phenotypic screening test for Klebsiella pneumoniae carbapenemase.
METHOD:
From April 2009 to July 2011, all Enterobacteriaceae bacteria that were not susceptible to ertapenem according to Vitek2 analysis were analyzed with the modified Hodge test. All positive isolates and a random subset of negative isolates were also assayed for the presence of blaKPC. Isolates that were positive in modified Hodge tests were sub-classified as true-positives (E. coli touched the ertapenem disk) or inconclusive (distortion of the inhibition zone of E. coli, but growth did not reach the ertapenem disk). Negative results were defined as samples with no distortion of the inhibition zone around the ertapenem disk.
RESULTS:
Among the 1521 isolates of Enterobacteriaceae bacteria that were not susceptible to ertapenem, 30% were positive for blaKPC, and 35% were positive according to the modified Hodge test (81% specificity). Under the proposed sub-classification, true positives showed a 98% agreement with the blaKPC results. The negative predictive value of the modified Hodge test for detection was 100%. KPC producers showed high antimicrobial resistance rates, but 90% and 77% of these isolates were susceptible to aminoglycoside and tigecycline, respectively.
CONCLUSION:
Standardizing the modified Hodge test interpretation may improve the specificity of KPC detection. In this study, negative test results ruled out 100% of the isolates harboring Klebsiella pneumoniae carbapenemase-2. The test may therefore be regarded as a good epidemiological tool.
doi:10.6061/clinics/2012(12)13
PMCID: PMC3521806  PMID: 23295597
Modified Hodge Test; KPC; Carbapenemase; Ertapenem
18.  Molecular Characterization of Klebsiella pneumoniae Carbapenemase (KPC)-Producing Enterobacteriaceae in Ontario, Canada, 2008-2011 
PLoS ONE  2014;9(12):e116421.
Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of blaKPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as blaKPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored blaKPC-2 (n = 23) or blaKPC-3 (n = 7). blaTEM-1 (n = 27) was commonly detected and occasionally blaOXA-1 (n = 3) and blaCTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried blaSHV-11. blaKPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258.
doi:10.1371/journal.pone.0116421
PMCID: PMC4280202  PMID: 25549365
19.  Detection of Colonization by Carbapenemase-Producing Gram-Negative Bacilli in Patients by Use of the Xpert MDRO Assay 
Journal of Clinical Microbiology  2013;51(11):3780-3787.
Detecting colonization of patients with carbapenemase-producing bacteria can be difficult. This study compared the sensitivity and specificity of a PCR-based method (Xpert MDRO) for detecting blaKPC, blaNDM, and blaVIM carbapenem resistance genes using GeneXpert cartridges to the results of culture with and without a broth enrichment step on 328 rectal, perirectal, and stool samples. The culture method included direct inoculation of a MacConkey agar plate on which a 10-μg meropenem disk was placed and plating on MacConkey agar after overnight enrichment of the sample in MacConkey broth containing 1 μg/ml of meropenem. Forty-three (13.1%) samples were positive by PCR for blaKPC and 11 (3.4%) were positive for blaVIM; none were positive for blaNDM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay for blaKPC were 100%, 99.0%, 93.0%, and 100%, respectively, compared to broth enrichment culture and sequencing of target genes. The sensitivity, specificity, PPV, and NPV of the assay for blaVIM were 100%, 99.4%, 81.8%, and 100%, respectively. Since none of the clinical samples contained organisms with blaNDM, 66 contrived stool samples were prepared at various dilutions using three Klebsiella pneumoniae isolates containing blaNDM. The PCR assay showed 100% positivity at dilutions from 300 to 1,800 CFU/ml and 93.3% at 150 CFU/ml. The Xpert MDRO PCR assay required 2 min of hands-on time and 47 min to complete. Rapid identification of patients colonized with carbapenemase-producing organisms using multiplex PCR may help hospitals to improve infection control activities.
doi:10.1128/JCM.01092-13
PMCID: PMC3889767  PMID: 24006011
20.  Phenotypic and Enzymatic Comparative Analysis of the KPC Variants, KPC-2 and Its Recently Discovered Variant KPC-15 
PLoS ONE  2014;9(10):e111491.
Sixteen different variants (KPC-2 to KPC-17) in the KPC family have been reported, and most current studies are focusing on KPC-2 and KPC-3. The KPC-15 variant, which isolated from Klebsiella pneumoniae in a Chinese hospital, was a recently discovered KPC enzyme. To compare the characteristics of KPC-15 and KPC-2, the variants were determined by susceptibility testing, PCR amplification and sequencing, and study of kinetic parameters. The strain harboring the KPC-15 showed resistance to 18 conventional antimicrobial agents, especially to cabapenem antibiotics, and the strain involving the KPC-2 also indicated resistance to cabapenem antibiotics, but both strains were susceptible to polymyxin B and colistin. The conjugation experiments showed that the changes of MIC values to the antibiotics were due to the transferred plasmids. The differences of amino acids were characterised at sites of 119 leucine and 146 lysine with KPC-15 and KPC-2. The minimum evolution tree indicated the KPC alleles evolution, and showed that the KPC-15 appeared to be homogenous with KPC-4 closely. Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study. The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2. Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.
doi:10.1371/journal.pone.0111491
PMCID: PMC4216079  PMID: 25360633
21.  Molecular Dissection of an Outbreak of Carbapenem-Resistant Enterobacteriaceae Reveals Intergenus KPC Carbapenemase Transmission through a Promiscuous Plasmid 
mBio  2011;2(6):e00204-11.
ABSTRACT
Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine β-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene blaKPC. The remaining strains harbored distinct blaKPC plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the blaKPC element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak.
IMPORTANCE
The ease of horizontal transmission of carbapenemase resistance plasmids across strains, species, and genera of bacteria observed in this study has several important public health and epidemiological implications. First, it has the potential to promote dissemination of carbapenem resistance to new populations of Enterobacteriaceae, including organisms of low virulence, leading to the establishment of reservoirs of carbapenem resistance genes in patients and/or the environment and of high virulence, raising the specter of untreatable community-associated infections. Second, recognition of plasmid-mediated outbreaks, such as those described here, is problematic because analysis of resistance plasmids from clinical isolates is laborious and technically challenging. Adaptation of nested arbitrary PCR (ARB-PCR) to investigate the plasmid outbreak facilitated our investigation, and the method may be broadly applicable to other outbreaks due to other conserved mobile genetic elements. Whether infection control measures that focus on preventing transmission of drug-resistant clones are effective in controlling dissemination of these elements is unknown.
doi:10.1128/mBio.00204-11
PMCID: PMC3202755  PMID: 22045989
22.  Evaluation of PCR-Based Testing for Surveillance of KPC-Producing Carbapenem-Resistant Members of the Enterobacteriaceae Family▿  
Journal of Clinical Microbiology  2009;47(10):3261-3265.
The spread of carbapenem-resistant members of the Enterobacteriaceae family (CRE) harboring carbapenemases is an emerging public health threat. As KPC-producing Klebsiella species are endemic in our tertiary care hospital, we aimed to evaluate a PCR-based surveillance test for identification of rectal carriage of KPC-producing CRE. We conducted a surveillance study between May and December 2007. Rectal swabs were collected from patients known to harbor CRE and from contacts of newly discovered patients harboring CRE. Specimens were evaluated by culture and by PCR analysis for blaKPC and were defined as positive if CRE was cultured and blaKPC was identified. Discrepant results between the culture and PCR analysis were resolved by subculturing, repeating the PCR, and performing a hydrolysis assay. Positive CRE cultures prior or subsequent to the time of sampling for the study were also taken into consideration. Sensitivity, specificity, and time to result were calculated. A total of 755 swabs were included. Concordant results were documented for 735 specimens; 51 were positive as determined by both PCR and culture. Discrepancies existed for 20 swabs; 9 were blaKPC negative and CRE culture positive, and 11 were blaKPC positive and CRE culture negative. After repeat testing, a total of 64 samples were classified as blaKPC-positive CRE. The sensitivity and specificity of the PCR analysis were 92.2% and 99.6%, respectively, and those of the culture were 87.5% and 99.4%, respectively. Over the last 3 months of the study, the sensitivity of the PCR improved to 96.3%, versus 77.8% for culture. Time to result was 30 h for the PCR and 60 h (negative) and 75 h (positive) for the CRE culture. blaKPC PCR-based testing is a useful method for the surveillance of KPC-producing CRE. Its main advantage over culturing is a shorter time to result, and it may prove to be more sensitive.
doi:10.1128/JCM.02368-08
PMCID: PMC2756929  PMID: 19675211
23.  Molecular Survey of the Dissemination of Two blaKPC-Harboring IncFIA Plasmids in New Jersey and New York Hospitals 
Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a major threat in health care facilities. Transmission of blaKPC, the plasmid-borne KPC gene, can be mediated by clonal spread and horizontal transfer. Here, we report the complete nucleotide sequences of two novel blaKPC-3-harboring IncFIA plasmids, pBK30661 and pBK30683. pBK30661 is 74 kb in length, with a mosaic plasmid structure; it exhibits homologies to several other plasmids but lacks the plasmid transfer operon (tra) and the origin of transfer (oriT) that are required for plasmid transfer. pBK30683 is a conjugative plasmid with a cointegrated plasmid structure, comprising a 72-kb element that highly resembles pBK30661 (>99.9% nucleotide identities) and an extra 68-kb element that harbors tra and oriT. A PCR scheme was designed to detect the distribution of blaKPC-harboring IncFIA (pBK30661-like and pBK30683-like) plasmids in a collection of clinical Enterobacteriaceae isolates from 10 hospitals in New Jersey and New York. KPC-harboring IncFIA plasmids were found in 20% of 491 K. pneumoniae isolates, and all carried blaKPC-3. pBK30661-like plasmids were identified mainly in the epidemic sequence type 258 (ST258) K. pneumoniae clone, while pBK30683-like plasmids were widely distributed in ST258 and other K. pneumoniae sequence types and among non-K. pneumoniae Enterobacteriaceae species. This suggests that both clonal spread and horizontal plasmid transfer contributed to the dissemination of blaKPC-harboring IncFIA plasmids in our area. Further studies are needed to understand the distribution of this plasmid group in other health care regions and to decipher the origins of pBK30661-like and pBK30683-like plasmids.
doi:10.1128/AAC.02749-13
PMCID: PMC4023724  PMID: 24492370
24.  Complete Nucleotide Sequence of a blaKPC-Harboring IncI2 Plasmid and Its Dissemination in New Jersey and New York Hospitals 
Antimicrobial Agents and Chemotherapy  2013;57(10):5019-5025.
Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a significant public health threat. blaKPC, the plasmid-borne KPC gene, was frequently identified on numerous transferable plasmids in different incompatibility replicon groups. Here we report the complete nucleotide sequence of a novel blaKPC-3-harboring IncI2 plasmid, pBK15692, isolated from a multidrug-resistant K. pneumoniae ST258 strain isolated from a New Jersey hospital in 2005. pBK15692 is 78 kb in length and carries a backbone that is similar to those of other IncI2 plasmids (pR721, pChi7122-3, pHN1122-1, and pSH146-65), including the genes encoding type IV pili and shufflon regions. Comparative genomics analysis of IncI2 plasmids reveals that they possess a conserved plasmid backbone but are divergent with respect to the integration sites of resistance genes. In pBK15692, the blaKPC-3-harboring Tn4401 was inserted into a Tn1331 element and formed a nested transposon. A PCR scheme was designed to detect the prevalence of IncI2 and pBK15692-like plasmids from a collection of clinical strains from six New Jersey and New York hospitals isolated between 2007 and 2011. IncI2 plasmids were found in 46.2% isolates from 318 clinical K. pneumoniae strains. Notably, 59 pBK15692-like plasmids (23%) have been identified in 256 KPC-bearing K. pneumoniae strains, and all carried KPC-3 and belong to the epidemic ST258 clone. Our study revealed that the prevalence of IncI2 plasmids has been considerably underestimated. Further studies are needed to understand the distribution of this plasmid group in other health care regions and decipher the association between IncI2 plasmids and blaKPC-3-bearing ST258 strains.
doi:10.1128/AAC.01397-13
PMCID: PMC3811408  PMID: 23896467
25.  KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units 
Gut Pathogens  2012;4:7.
Background
Klebsiella pneumoniae carbapenemases (KPCs) producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection.
Methods
KPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing.
Results
KPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs) indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids.
Conclusions
The acquired molecular epidemiology demonstrated that 1) both acquisitions from outward sources and patient relocation within the hospitals were responsible for the observed spreading; 2) KPC-3-encoding Klebsiella pneumoniae ST258 prevailed over other strains. In addition, the described massive transfer of KPC-mediated resistance to non-intensive care units may anticipate spreading of resistance to the non-hospitalized population. Therefore, genotypic analysis alongside phenotypic identification of carbapenemase producers, also at the carriage state, is advisable to prevent and contain further carbapenemase resistance dissemination.
doi:10.1186/1757-4749-4-7
PMCID: PMC3411510  PMID: 22800501
KPC; Carbapenemase; Klebsiella pneumoniae; Plasmid-mediated antimicrobial resistance; Gram-negative; Nosocomial infections

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