Major Carbapenemase Research Articles

Fecal carriage and molecular epidemiology of carbapenem-resistant Enterobacteriaceae from outpatient children in Shanghai

BackgroundFecal colonization with carbapenem-resistant Enterobacteriaceae (CRE) is a risk factor for bacterial translocation resulting in subsequent endogenous infections. The purpose of this study is to investigate the prevalence of CRE strains colonization in stool samples of outpatient in a tertiary pediatric hospital of Shanghai, China.MethodsIn a retrospective study, fecal samples were consecutively obtained from patients in 2016 and screening test for CRE was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the broth microdilution method and β-lactamases were characterized by polymerase chain reaction (PCR) assays and DNA sequencing. Multilocus sequence typing (MLST) was performed for the genetic relationships of the isolates.ResultsA total of 880 fecal samples were included for this screening test and 32 CRE strains were identified in 32 non-duplicate fecal samples from 32 children (1.3 ± 1.5 years), with a carriage rate of 3.6%. These strains mainly distributed in Klebsiella pnuemoniae (37.5%) and Escherichia coli (37.5%). All CRE strains showed high resistance to most of the routinely used antibiotics (> 90%) except for polymyxin B and tigecycline. The blaNDM gene was the major carbapenemase gene harbored by gastrointestinal CRE strains, followed by blaKPC-2, blaIMP-26, and blaIMP-4. Other β-Lactamase genes including blaCTX-M, blaSHV, blaTEM-1, and blaDHA-1 were also detected. MLST analysis revealed that various sequence types (STs) were detected in these strains, with ST11 and ST37 being more prevalent in K.pneumoniae and ST101 in E.coli.ConclusionsThis study revealed the prevalence of CRE fecal carriage in children from outpatient and urgent implementation of infection control measure should be conducted to limit the spread of CRE strains.

Role of the Hydrophobic Bridge in the Carbapenemase Activity of Class D β-Lactamases.

Class D carbapenemases are enzymes of the utmost clinical importance due to their ability to confer resistance to the last-resort carbapenem antibiotics. We investigated the role of the conserved hydrophobic bridge in the carbapenemase activity of OXA-23, the major carbapenemase of the important pathogen Acinetobacter baumannii We show that substitution of the bridge residue Phe110 affects resistance to meropenem and doripenem and has little effect on MICs of imipenem. The opposite effect was observed upon substitution of the other bridge residue Met221. Complete disruption of the bridge by the F110A/M221A substitution resulted in a significant loss of affinity for doripenem and meropenem and to a lesser extent for imipenem, which is reflected in the reduced MICs of these antibiotics. In the wild-type OXA-23, the pyrrolidine ring of the meropenem tail forms a hydrophobic interaction with Phe110 of the bridge. Similar interactions would ensue with ring-containing doripenem but not with imipenem, which lacks this ring. Our structural studies showed that this interaction with the meropenem tail is missing in the F110A/M221A mutant. These data explain why disruption of the interaction between the enzyme and the carbapenem substrate impacts the affinity and MICs of meropenem and doripenem to a larger degree than those of imipenem. Our structures also show that the bridge directs the acylated carbapenem into a specific tautomeric conformation. However, it is not this conformation but rather the stabilizing interaction between the tail of the antibiotic and the hydrophobic bridge that contributes to the carbapenemase activity of class D β-lactamases.

Molecular and epidemiological analysis of IMP-1 metallo-β-lactamase-producing Klebsiella pneumoniae in a tertiary care hospital in Japan.

This study investigated the molecular and phenotypic characteristics of carbapenemase-producing Klebsiella pneumoniae, and identified the risk factors underlying its acquisition. We evaluated K.pneumoniae isolated in Nagasaki University Hospital between January 2009 and June 2015. The presence of carbapenemase genes and plasmid characteristics were investigated. We performed multilocus sequence typing (MLST), and generated a dendrogram based on the results of pulsed-field gel electrophoresis (PFGE) for carbapenemase-producing strains. We also performed a case-control study of patients. Of the 88 K.pneumoniae strains that showed minimum inhibitory concentration ≥1μg/mL for imipenem and/or meropenem, and that were available from our bacterial collection, 18 had the IMP-type carbapenemase gene, all of which were IMP-1 according to sequencing analysis. Strains included seven different sequence types (STs), of which the most common was ST1471. A dendrogram showed the significant similarity of some strains with relationships in PFGE patterns, STs, and the wards in which they were isolated. Plasmid incompatibility group was similar among the IMP-1 producers. Regarding risk factors, multivariate analysis showed that liver disease and previous uses of carbapenems and anti-fungal drugs were significant factors for the acquisition of IMP-1-producing strains. Our results demonstrate that IMP-1 is a major carbapenemase produced by K.pneumoniae. The PFGE results indicated the possibility of transmission in the hospital. The identified risk factors should be considered for appropriate antibiotic therapy and infection-control measures.

Performance of the EUCAST disc diffusion method and two MIC methods in detection of Enterobacteriaceae with reduced susceptibility to meropenem: the NordicAST CPE study.

Objectives To examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations. Methods Sixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs. Results A triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization. Conclusions The EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.

Molecular characterization of carbapenemases in gram-negative bacilli isolated from clinical samples in an University Hospital from Buenos Aires, Argentina

Background: Carbapenemase-producing organisms (CPO) are formidable hospital pathogens with rising incidence in health care facilities, high mortality rates associated whit infection, and potential to spread antibiotic resistance. The objective of this study was to investigate the presence and to determinate the type of carbapenemases in gram-negative bacteria isolated from clinical samples. Methods & Materials: From January 2015 to October 2017, clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa (only first isolate from a patient) from any site of infection, showing decreased sensitivity to any carbapenem (imipenem, meropenem or ertapenem) were included in the study. The identification was determined by MALDI-TOF and antibiotic susceptibilities by the automated system Phoenix. In isolates with reduced susceptibility to any carbapenem (MIC > = 1 ug/ml) the presence of genes coding for major carbapenemases (I.e. blaVIM, blaNDM, blaIMP, blaKPC and blaOXA-48) was investigated by polymerase chain reaction using specific primers. Results: KPC carbapenemase was the most prevalent being detected in 125 isolates (61 K.pneumoniae, 25 P.aeruginosa,22 E.cloacae, 7 S.marcescens, 7 K.oxytoca,2 E.coli and 1 C.freundii. Metallo-Beta-Lactamases (MBL) were mostly found in 15 P.aeruginosa (14 VIM and 1 IMP) an in only 3 K.pneumoniae (IMP). OXA-48 carbapenemase was present in 10 isolates (8 K.pneumoniae, 1 E.cloacae, 1 K.oxytoca) K. pneumoniae carring blaKPC showed meropenem-MIC values ranging <0,5->32ug/ml; MIC50 = 8 ug/ml and MIC90 > 8 ug/ml). Other Enterobacteriaceae (specially K.oxytoca and E.coli) showed somehow a more susceptible profile (range, <0,5 - 8ug/ml; MIC50 = < 0,5ug/ml and MIC90 = < 0,5ug/ml). Enterobacteriaceae with OXA-48 carbapenemase showed only resistance to ertapenem (MICs > 1 ug/ml), while remained Meropenem- and Imipenem-susceptible (MICs < 1 ug/ml). On the other hand, P. aeruginosa with KPC carbapenemase showed MICs values for both meropenem and imipenem (MICs > 8 ug/ml). MBL-carring isolates showed a wider range of MICs (0,5->8 ug/ml). Conclusion: Carbapenemase-producing organisms are a real serious threat to our patients. We found several kinds of carbapenemases in diverse gram-negative bacilli. Know of resistance mechanisms is important in view of future therapeutics concerned with the treatment of CPO and for aiding control efforts by surveillance and infection control interventions.

Epidemiology, clinical features and outcome of bacteriemia episodes by carbapenemase-producing and non carbapenemase-producing enterobacteriaceae

Background: Carbapenemase-producing Enterobacteriaceae (CPE) is a public health problem due to their spectrum of infections and their high mortality. The aim of this study was to describe the epidemiology, clinical features and outcome of bacteremia by CPE compared to non-CPE. Methods & Materials: Retrospective cohort study. Enterobacteriaceae bacteremia episodes from January 2015 to December 2016 were included. The presence of genes coding for major carbapenemases (I.e. blaVIM, blaNDM, blaIMP, blaKPC and blaOXA-48) was investigated by polymerase chain reaction using specific primer. Chi square analysis and Kruskal-Wallis test were used for categorical and for continuous variables, respectively. To identify risk factors for 7-day and 30-day mortality a multivariate stepwise logistic regression analysis was performed. Results: We included 67 episodes (67,2% male): 14 (23,2%) CPE and 53 (76,8%) non-CPE. CPE isolates were blaKPC (78,5%), blaOXA (14,2%) and blaNDM (7,14%). Klebsiella spp (CPE: 85,7% vs non-CPE: 41,5%) was the most frequently isolated pathogen. The mean days of hospitalization until bacteremia episode and ICU admission in CPE vs non-CPE were: 26,6 vs 8,2 (p = 0.016) and 78% vs 45% (p = 0.027) respectively. The most common source of bacteremia was catheter-related in the CPE group (45% vs 18%; p = 0,005) and intra-abdominal in the non-CPE group (52% vs 9,1%; p = 0,009). Adequate empirical antibiotic treatment was similar: 64% vs 77%; p = 0.31. The risk of death at 7-day was 6.8 times higher in the CPE group compared to the non-CPE group (OR 6.8 CI 95% 1.1-40; p = 0,006) and mortality at 30-day was also higher in the CPE group (OR 4.9, 95% CI 1.1-20, p = 0.011). In multivariate analysis, Pitt bacteremia score > 4 (OR 20, 95% CI = 3,7- 40, p = < 0,0001), APACHE II score > 15 (OR 19, 95% CI 2,5-46, p = 0,0004), shock (OR 36, 95% CI 4,5-55, p = < 0,0001), CPE (OR 4,8, 95% CI 1,1-20, p = 0,01) were risk factors for 30-day mortality. Conclusion: Patients with Enterobacteriaceae bacteremia were slightly different regarding epidemiology and clinical presentation. CPE episodes had a much higher early and late mortality than non CPE, and was one of the independent prognostic factors. It is essential to improve early diagnosis and adequate treatment in patients at risk.

Performance of the EUCAST disc diffusion method and two MIC methods in detection of Enterobacteriaceae with reduced susceptibility to meropenem: the NordicAST CPE study.

ObjectivesTo examine performance of EUCAST disc diffusion and supplementary MIC methods for detection of Enterobacteriaceae with reduced susceptibility to meropenem using EUCAST screening recommendations.MethodsSixty-one Nordic laboratories delivered data on EUCAST disc diffusion (n = 61), semi-automated meropenem MIC (n = 23; VITEK2, n = 20 and Phoenix, n = 3) and gradient meropenem MIC (n = 58) methods. The strains (n = 27) included the major carbapenemase classes (A, n = 4; B, n = 9; D, n = 6) involved in the global spread of carbapenemase-producing Enterobacteriaceae (CPE) and non-CPE strains (n = 8) covering a range of broth microdilution (BMD) meropenem MICs.ResultsA triplicate Klebsiella variicola (meropenem MIC 0.5 mg/L) harbouring OXA-48 and Escherichia coli ATCC 25922 showed an overall good precision. Meropenem zone diameters below the EUCAST screening cut-off (<27 mm) were reported for strains with MIC ≥1 mg/L (n = 21), irrespective of resistance mechanism. For three strains (MIC = 0.5 mg/L) with OXA-48/-181, eight laboratories provided meropenem zone diameters above the screening cut-off. Very major errors (VMEs) were not observed. The overall distributions of major errors (MEs) and minor errors (mEs) were 9% and 36% (disc diffusion), 26% and 18% (VITEK2) and 7% and 20% (gradient MIC), respectively. Differences in ME and mE distributions between disc diffusion and MIC gradient tests compared with semi-automated methods were significant (P < 0.0001), using BMD MICs as a reference for categorization.ConclusionsThe EUCAST disc diffusion method is a robust method to screen for CPE but isolates with meropenem MICs <1 mg/L pose challenges. The high ME rate in semi-automated methods might deter appropriate use of carbapenems in CPE infections with limited therapeutic options.

Evaluation of the Amplidiag CarbaR-VRE kit for the accurate detection of carbapenemase-producing bacteria

Introduction Carbapenemase-producing enterobacteriaceae (CPE) and non fermenters (CPNF; pseudomonadaceae and Acinetobacter sp.) have been increasingly reported worldwide. The most clinically relevant carbapenemases belong either to Ambler class A (KPC-type), Ambler class B, i-e metallo-b-lactamases (MBLs such as IMP-, VIM- and NDM-types) or Ambler Class D (OXA-48-like in enterobacteriaceae, Acinetobacter OXA group: OXA-23, OXA-40, OXA-58, OXA-143 and the intrinsic OXA-51-like enzymes, OXA-198 in P. aeruginosa ). Materials and method The Amplidiag ® Carba-R + VRE, a qualitative multiplex nucleic acid-based in-vitro diagnostic test intended for the detection of carbapenemase-producing bacteria and vancomycin resistant enterococci, has been tested on a collection of 100 characterized Gram-negative isolates with reduced susceptibility to carbapenems, and on 200 isolates collected at the National Reference Center for Antibiotic Resistance from 20st January to 10th February 2016. The markers detected by this assay are bla KPC-lik e, bla NDM-like , bla VIM-like , bla IMP-like , bla OXA-48-like , Acinetobacter OXA genes including bla OXA-23 , bla OXA-24/-40 , bla OXA-58 , and bla OXA-51 including upstream promoter ISAba1 , and vanA and vanB . DNA was extracted using QiaAmp DNA extraction kits or by boiling extract. Results The Amplidiag ® CarbaR + VRE was able to detect all KPC, NDM, VIM, IMP, OXA-48 and variants including OXA−162, −181, −204, −232, −244. Similarly, all A. baumannii producing OXA−23, OXA−24/−40, OXA−58 as well as over expression of the chromosomally-encoded OXA−51-like b-lactamase, due to the presence of ISAba1 upstream of the blaOXA-51 gene were detected. The most prevalent carbapenemases encountered in P. aeruginosa have also been detected. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES−5 in P. aeruginosa , GES−14 in A. baumannii ), GIM−1, AIM−1, SPM−1, DIM−1 or OXA−198 in P. aeruginosa , or OXA−143-like in A. baumannii were not detected by the Amplidiag® CarbaR + VRE assay. The PCR worked equally well on purified DNA and on boiling extracted DNA from a colony. The biological performances of the Amplidiag® CarbaR + VRE on the prospective study of 200 CREs isolates collected at the NRC were of 100% and 96% sensitivity and specificity, respectively. Conclusion Amplidiag® Carba-R's performances were high as it was able to detect the five major carbapenemases: NDM, VIM, IMP, KPC, and OXA−48, as well as OXA-type carbapenemases from Acinetobacter sp. It can provide a result directly on colonies growing on selective screening media within two hours. This test needs now to be evaluated on rectal swabs, since it is able to detect the most worrisome resistant traits in Gram negative bacteria.

First Description of KPC-2-Producing Escherichia coli and ST15 OXA-48-Positive Klebsiella pneumoniae in Tunisia.

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011-2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The blaOXA-48 and blaKPC carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had blaOXA-48 gene, and 14/19 strains also harbored the blaCTX-M-15 gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. Two out of three R/IR-ERT E. coli isolates carried blaOXA-48 and one coproduced the blaCTX-M-15 gene. One E. coli strain, ascribed to the new sequence type ST5700, harbored the blaKPC-2 gene. E. coli isolates were not clonally related and belonged to different sequence types (ST5700, ST227, and ST58). To our knowledge, this is the first report in Tunisia of either KPC-2 carbapenemase in E. coli or OXA-48 carbapenemase in K. pneumoniae of lineage ST15.

Development and evaluation of a multiplex real-time PCR for the detection of IMP, VIM, and OXA-23 carbapenemase gene families on the BD MAX open system

A multiplex real-time polymerase chain reaction (PCR) assay was developed for the detection of clinically prevalent IMP, VIM, and OXA-23 gene families. The assay was designed to work on the BD MAX open platform which is a fully automated system for all PCR processes including sample extraction to PCR resulting. A total of 107 well-characterized carbapenem resistant Enterobacteriaceae were evaluated and the results were 100% concordant with the reference test isolates. This assay will serve to complement PCR screens that detect the major carbapenemase families of NDM, KPC, and OXA-48-like.

Improvement of the Xpert Carba-R Kit for the Detection of Carbapenemase-Producing Enterobacteriaceae.

The Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of blaOXA-181 and blaOXA-232 genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceae epidemiology of many countries worldwide.

Clearance Rate of Carbapenemase-Producing Enterobacteriaceae Carriage Among Hospitalized Patients.

During the past decade, carbapenemase-producing Enterobacteriaceae (CPE) has emerged and spread across the world. 1 The major carbapenemase enzymes currently being reported are KPC, NDM-1, VIM, IMP, and OXA. 2 Because carbapenemase can be effectively transmitted via mobile genetic elements, and current therapeutic options for CPE infections are extremely limited, CPE may be one of the most serious contemporary threats to public health. However, very little is known about the characteristics of CPE carriage during hospitalization. The aims of this study were to investigate the clearance rate of CPE carriage and determine the number of consecutive negative cultures required to confirm CPE clearance. We also examined CPE transmission among hospitalized patients.

Carbapenemase Genes among Multidrug Resistant Gram Negative Clinical Isolates from a Tertiary Hospital in Mwanza, Tanzania

The burden of antimicrobial resistance (AMR) is rapidly growing across antibiotic classes, with increased detection of isolates resistant to carbapenems. Data on the prevalence of carbapenem resistance in developing countries is limited; therefore, in this study, we determined the prevalence of carbapenemase genes among multidrug resistant gram negative bacteria (MDR-GNB) isolated from clinical specimens in a tertiary hospital in Mwanza, Tanzania. A total of 227 MDR-GNB isolates were analyzed for carbapenem resistance genes. For each isolate, five different PCR assays were performed, allowing for the detection of the major carbapenemase genes, including those encoding the VIM-, IMP-, and NDM-type metallo-beta-lactamases, the class A KPC-type carbapenemases, and the class D OXA-48 enzyme. Of 227 isolates, 80 (35%) were positive for one or more carbapenemase gene. IMP-types were the most predominant gene followed by VIM, in 49 (21.59%) and 28 (12%) isolates, respectively. Carbapenemase genes were most detected in K. pneumoniae 24 (11%), followed by P. aeruginosa 23 (10%), and E. coli with 19 isolates (8%). We have demonstrated for the first time a high prevalence of MDR-GNB clinical isolates having carbapenem resistance genes in Tanzania. We recommend routine testing for carbapenem resistance among the MDR-GNB particularly in systemic infections.

Structural Basis for Carbapenemase Activity of the OXA-23 β-Lactamase from Acinetobacter baumannii

Dissemination of Acinetobacter baumannii strains harboring class D β-lactamases producing resistance to carbapenem antibiotics severely limits our ability to treat deadly Acinetobacter infections. Susceptibility determination in the A.baumannii background and kinetic studies with a homogeneous preparation of OXA-23 β-lactamase, the major carbapenemase present in A.baumannii, document the ability of this enzyme to manifest resistance to last-resort carbapenem antibiotics. We also report three X-ray structures of OXA-23: apo OXA-23 at two different pH values, and wild-type OXA-23 in complex with meropenem, a carbapenem substrate. The structures and dynamics simulations reveal an important role for Leu166, whose motion regulates the access of a hydrolytic water molecule to the acyl-enzyme species in imparting carbapenemase activity.

Activity of biapenem (RPX2003) combined with the boronate -lactamase inhibitor RPX7009 against carbapenem-resistant Enterobacteriaceae

The proliferation of carbapenemases in Enterobacteriaceae demands new therapies, with current interest centred on β-lactamase inhibitor combinations. RPX7009 is a new boron-based inhibitor of several class A and C β-lactamases and is being developed in combination with biapenem (RPX2003). We investigated the in vitro activity of the combination. Three hundred Enterobacteriaceae isolates, representing major carbapenemase types, were tested. MICs were determined by CLSI agar dilution with RPX7009 at 2, 4 and 8 mg/L or in a chequerboard format with RPX7009 in doubling dilutions from 0.25 to 32 mg/L. RPX7009 lacked direct antibacterial activity but achieved a dose-dependent potentiation of biapenem against Enterobacteriaceae possessing KPC, SME or IMI/NMC-A carbapenemases: concentrations as low as 2 mg/L reducedthe MICs of biapenem to ≤1 mg/L for over 90% of isolates. RPX7009 also gave a weak potentiation of biapenem against Enterobacteriaceae with combinations of AmpC or extended-spectrum β-lactamase activity and impermeability, although any practical gain against such strains will depend on the breakpoints assigned. RPX7009 had no effect on the MICs of biapenem for isolates with metallo- (IMP, NDM or VIM) or OXA-48 β-lactamases; however, most isolates with these enzymes were less resistant to biapenem than to imipenem or, especially, ertapenem. Biapenem/RPX7009 (Carbavance) overcame most resistance due to KPC and other class A carbapenemases. Class B and D carbapenemases were not inhibited but conferred less consistent resistance to biapenem than to other carbapenems.

Validation of carbapenemase and extended-spectrum -lactamase multiplex endpoint PCR assays according to ISO 15189

To validate and accredit a set of three multiplex endpoint PCR assays, targeting the most important carbapenemase and minor extended-spectrum β-lactamase (ESBL) resistance genes, according to the international ISO 15189 particular requirements for the quality and competence of medical laboratories. Specific primers targeting ESBLs and carbapenemases were collected from the literature or designed internally. The multiplex PCRs were validated for sensitivity, specificity, intra- and inter-run reproducibility and accuracy by means of external quality control (EQC) using a collection of 137 characterized and referenced isolates. For each multiplex PCR assay, the presence of an extraction control ruled out false-negative results due to PCR inhibition or extraction faults. Amplicons were separated by capillary electrophoresis (QIAxcel system, Qiagen). The protocols and validation files were reviewed in the setting of an external audit conducted by the Belgian organization for accreditation (BELAC). Sensitivity, specificity and reproducibility for each targeted gene were 100%. All isolates from the three EQC panels were correctly identified by each PCR assay (accuracy 100%). The validation files were controlled by BELAC, and the PCR protocols were accepted as accredited according to ISO 15189. Three home-made multiplex PCRs targeting the major carbapenemases and four minor class A ESBL genes encountered in Gram-negative bacteria were accredited according to the ISO 15189 standards. This validation scheme could provide a useful model for laboratories aiming to accredit their own protocols.

Complete Sequence of an IncN Plasmid, pIMP-HZ1, Carrying bla IMP-4 in a Klebsiella pneumoniae Strain Associated with Medical Travel to China

In China, IMP-4 is a major plasmid-mediated carbapenemase among multidrug-resistant Enterobacteriaceae, but little is known about the plasmid scaffolds involved in the dissemination of bla IMP-4 ([1][1], [2][2]). In 2010, we identified a Klebsiella pneumoniae CRE1 strain carrying bla IMP-4 from the

Detection systems for carbapenemase gene identification should include the SME serine carbapenemase

Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens.

First Clinical Cases of OXA-48-Producing Carbapenem-Resistant Klebsiella pneumoniae in the United States: the “Menace” Arrives in the New World

OXA-48 has emerged as a major carbapenemase associated with the Enterobacteriaceae in Europe, North Africa, and Asia. We report the first two clinical cases of OXA-48-type carbapenemase-producing Enterobacteriaceae in the United States from patients recently hospitalized in Saudi Arabia and India. Each is more carbapenem resistant than nearly all previously reported OXA-48-type-producing Enterobacteriaceae.

Molecular epidemiological analysis of KPC-2 and IMP-4 carbapenemase possessing Klebsiella pneumonia isolated from children

Objective To investigate the distribution of acquired carbapenemases in carbapenemresistant strains of Klebsiella pneumoniae, and explore its role in epidemiology of nosocomial infection. Methods From November 2008 to March 2009, twenty clinical isolates of carbapenem-resistant Klebsiella pneumoniae were collected from children hospitalized in Wuhan children's hospital. MICs of antibiotics were tested by DNA of Klebsiella pneumoniae. Modified Hodge test was used to screen strains producing carbapenemases,combined imipenem(IPM)-EDTA , meropenem(MEM)- EDTA and ceftazidime(CAZ) - EDTA double-disk synergy test (DDST) were used to detect metallo-β-lactamase-producing. PCR amplification of the carbapenemase and integrase genes, and sequencing were performed. Plasmid conjugation transfer experiments and Southern hybridization were applied to study the mode of drug resistance transmission. Results Four types of Klebsiella pneumoniae were detected by PFGE, type A consisted of 5 strains, including 3 strains of type Al and 2 strains of type A2), type B (2 strains), type C (12strains) and type D (1 strain). Type A and C were the main drug resistant clones. Eight strains of Klebsiella pneumoniae carried both KPC-2 and IMP-4 genes, 10 strains carried IMP-4 gene, 2 strains carried KPC-2 gene. None of NDM-1 ,GIM, SPM, SIM, OXA-23, and VIM carbapenemase genes was detected in 20 isolates. All of 20 isolates carried lntl which were found to be located on bacterial chromosome by Southern blot. Conclusions KPC-2and IMP-4 genes are the major carbapenemase genes in Klebsiella pneumoniae isolated in Wuhan.Transmission of drug resistance is mainly through vertical transmission of type C resistant clone and horizontal transmission of Intl on bacteria chromosome. Key words: Klebsiella pneumoniae; Carbapenems; Beta-Lactamases; Integrons