Rapid Detection of the Commonly Encountered Carbapenemases (New Delhi Metallo-β-Lactamase,OXA-48/181) Directly from Various Clinical Samples using Multiplex Real-Time Polymerase Chain Reaction Assay

Background: Resistance due to New Delhi metallo-β-lactamase (NDM) and OXA-48/181 continues to emerge as a threat which is associated with nosocomial outbreaks and is a serious healthcare concern. Phenotypic detection being laborious and time-consuming requires rapid detection of NDM and OXA-48/181, which is achieved through real-time polymerase chain reaction (RT-PCR). Materials and Methods: In this study, RT-PCR assay was developed to simultaneously detect NDM and OXA-48/181. The assay was validated on 102 non-duplicate, phenotypically characterised clinical samples. Results: The assay showed a sensitivity and specificity of 97% and 100% for the detection of carbapenemases in comparison to conventional PCR. The in-house developed multiplex RT-PCR would help to rule-in the presence of NDM and OXA-48/181. Conclusions: Rapid detection of these carbapenemases would be assist in better patient management, in terms of accurate antimicrobial treatment, help in cohorting infected from uninfected patient to prevent spread.     

Rapid Detection of KPC,NDM, and OXA-48-Like Carbapenemases by Real-Time PCR from Rectal Swab Surveillance Samples

We describe a multiplex real-time PCR assay for use on the ABI 7500 Fast TaqMan platform to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-β-lactamases (NDM), and the OXA-48-like family of carbapenemases from bacterial culture lysates or sample enrichment broth lysates.     

Intrapatient emergence of OXA-247: a novel carbapenemase found in a patient previously infected with OXA-163-producing Klebsiella pneumoniae

Two genetically related Klebsiella pneumoniae strains carrying OXA-type carbapenemases were isolated from a single patient 1 month apart. Kpn163 harboured OXA-163 and Kpn247 a new variant named OXA-247 that showed susceptibility to carbapenems and expanded-spectrum cephalosporins similar to OXA-48. Our epidemiological, biochemical and molecular results suggest the intrapatient emergence of bla_OXA-247 from bla_OXA-163.     

Diversity in Acinetobacter baumannii isolates from paediatric cancer patients in Egypt

Acinetobacter baumannii is an important nosocomial pathogen, commonly causing infections in immunocompromised patients. It is increasingly reported as a multidrug-resistant organism, which is alarming because of its capability to resist all available classes of antibiotics including carbapenems. The aim of this study was to examine the genetic and epidemiological diversity of A. baumannii isolates from paediatric cancer patients in Egypt, by sequencing the intrinsic blaOXA–51-like gene, genotyping by pulsed-field gel electrophoresis and multi-locus sequence typing in addition to identifying the carbapenem-resistance mechanism. Results showed a large diversity within the isolates, with eight different blaOXA-51-like genes, seven novel sequence types and only 28% similarity by pulsed-field gel electrophoresis. All three acquired class-D carbapenemases (OXA-23, OXA-40 and OXA-58) were also identified among these strains correlating with resistance to carbapenems. In addition, we report the first identification of ISAba2 upstream of blaOXA-51-like contributing to high-level carbapenem resistance. This indicates the presence of several clones of A. baumannii in the hospitals and illustrates the large genetic and epidemiological diversity found in Egyptian strains.     

Multiple carbapenem hydrolyzing genes in clinical isolates of Acinetobacter baumannii

Purpose: Carbapenem resistance in Acinetobacter baumannii has become highly rampant, which has been ascribed to the presence of multiple carbapenemases. The objective of the present study was to prospectively investigate the presence of multiple carbapenemase encoding genes in clinical isolates of A. baumannii. Materials and Methods: A total of 30 imipenem resistant, consecutive non-repeat clinical isolates A. baumannii from a Tertiary Care Centre of Delhi were subjected to antimicrobial susceptibility testing (AST), screening for carbapenemase production by modified Hodge test (MHT) and determination of minimum inhibitory concentration for imipenem by E-Test®. These were subjected to Real time PCR for bla_{IMP-1 and 2}, bla_{VIM-1 and 2}, bla_{OXA23, 24, 51 and 58} using SYBR green-I. These were grouped together on the basis of their genotype as each isolate harboured multiple carbapenemases and correlated with their AST profile. Detection of the novel carbapenemase bla_NDM-1 was performed by real time PCR using TaqMan probes on 14 isolates. Results: Colistin appeared to be the most effective drug in vitro, followed by tetracycline and beta lactam/beta lactamase inhibitor combinations. All, but one isolate were positive for the MHT. All 30 isolates were positive for bla_OXA-51 like gene as well as bla_IMP-1 and bla_VIM-1 genes. bla_{OXA 24 and 58} were not detected in any of the isolates. bla_IMP-2, bla_VIM-2, bla_OXA-23 were present in 15, 6 and 14 isolates respectively. Grouping based on the genotypic profile did not correlate with susceptibility pattern. Nine among the 14 isolates also harboured the novel bla_NDM-1 gene. Conclusions: This is the first study from North India, which comprehensively detected the presence of multiple carbapenemases as well the bla_NDM-1 gene. The presence of the novel gene bla_NDM-1indicated ability of A. baumannii to acquire new carbapenemase genes despite the existence of multiple carbapenemase genes. The present study confirmed the presence of multiple genetic mechanisms for carbapenemases production among the clinical isolates of A. baumannii in north India.     

Genotypic characterization of carbapenem resistant Enterobacterales in clinical isolates from western Maharashtra

PurposeTo study distribution of carbapenemase genes namely; New Delhi metallo-beta-lactamase (blaNDM), Oxacillinase-48 (blaOXA48), Verona Integron-Encoded Metallo-beta-lactamase (blaVIM) and Imipenemase (blaIMP) in carbapenem resistant Enterobacterales (CRE), isolated from clinical samples.MethodThis cross-sectional study was conducted at a tertiary care hospital of western Maharashtra over six months period. CREs were identified by conventional disc diffusion and modified carbapenem inactivation method (mCIM). A total of 50 consecutive CRE isolates from clinical samples were subjected to home brewed polymerase chain reaction (PCR) for detection of carbapenemases.ResultsOut of the 50 CRE isolates, at least one of the four carbapenemase genes was detected in 49 (98%) isolates. The frequency of distribution of these genes were NDM 90% (n = 45), OXA48 60% (n = 30) and VIM 12% (n = 6). Dual combination of blaNDM and blaOXA48 (50%) was the commonest pattern observed, which was frequently associated with Klebsiella pneumoniae.ConclusionsThe study indicate high prevalence of NDM warranting strict anti-microbial stewardship practices. Surveillance of CRE and resistance mechanism is essential to monitor the trend and take informed decision for appropriate anti-microbial therapy.     

OXA- and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla_OXA-51-like, bla_OXA.-_23-like, bla_OXA-40-like and bla_OXA-58-like genes. ISAba1, bla_IMP-like, bla_VIM-like, bla_GES, bla_VEB, bla_PER-2, aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla_OXA-51, 79 strains carried bla_OXA-23 and one strain carried bla_OXA-40. bla_OXA-51 and bla_OXA-23 were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of bla_OXA-51. GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla_PER-2, bla_VEB-1, bla_NDM-1, bla_IMP- and bla_VIM-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey.     

The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide

The spread of carbapenemase producers in Enterobacteriaceae has now been identified worldwide. Three main carbapenemases have been reported; they belong to three classes of β-lactamases, which are KPC, NDM, and OXA-48. The main reservoirs of KPC are Klebsiella pneumoniae in the USA, Israel, Greece, and Italy, those of NDM are K. pneumoniae and Escherichia coli in the Indian subcontinent, and those of OXA-48 are K. pneumoniae and Escherichia coli in North Africa and Turkey. KPC producers have been mostly identified among nosocomial isolates, whereas NDM and OXA-48 producers are both nosocomial and community-acquired pathogens. Control of their spread is still possible in hospital settings, and relies on the use of rapid diagnostic techniques and the strict implemention of hygiene measures.     

Multisite Evaluation of Cepheid Xpert Carba-R Assay for Detection of Carbapenemase-Producing Organisms in Rectal Swabs

Rapid identification of patients who are colonized with carbapenemase-producing organisms (CPO) is included in multiple national guidelines for containment of these organisms. In a multisite study, we evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative diagnostic test that was designed for the rapid detection and differentiation of the bla_KPC, bla_NDM, bla_VIM, bla_OXA-48, and bla_IMP-1 genes from rectal swab specimens. A double rectal swab set was collected from 383 patients admitted at four institutions (2 in the United States, 1 in the United Kingdom, 1 in Spain). One swab was used for reference culture (MacConkey broth containing 1 mg/liter of meropenem and subcultured to a MacConkey agar plate with a 10-μg meropenem disk) and for sequencing of DNA obtained from carbapenem-nonsusceptible isolates for carbapenemase identification. The other swab was used for the Xpert Carba-R assay. In addition to the clinical rectal swabs, 250 contrived specimens (108 well-characterized CPO and 142 negative controls spiked onto negative rectal swabs) were tested. Overall, 149/633 (23.5%) samples were positive by the Xpert Carba-R assay. In 6 samples, multiple targets were detected (4 VIM/OXA-48, 1 IMP-1/NDM, and 1 NDM/KPC). The Xpert Carba-R assay detected 155 targets (26 IMP-1, 30 VIM, 27 NDM, 33 KPC, 39 OXA-48) within a time range of 32 to 48 min. The sensitivity, specificity, and positive and negative predictive values of the Xpert Carba-R assay compared to those of the reference culture and sequencing results were 96.6% (95% confidence interval [CI], 92.2% to 98.9%), 98.6% (95% CI, 97.1% to 99.4%), 95.3%, and 99.0%, respectively. The Cepheid Xpert Carba-R assay is an accurate and rapid test to identify rectal colonization with CPO, which can guide infection control programs to limit the spread of these organisms.     

A disc diffusion assay for detection of class A,B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid,dipicolinic acid and temocillin

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 μg), temocillin (30 μg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96–100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae.     

Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India

Background: Surveillance of antimicrobial resistance (AMR) is of great importance. Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens and emergence of resistance in these have increased the morbidity and mortality rates. This surveillance study was initiated by the Government of India - Indian Council of Medical Research. The aim of this study is to determine the antimicrobial susceptibility profile and to characterise the enzyme mediated antimicrobial resistance such as extended spectrum beta-lactamases (ESBLs) and carbapenemases among multidrug-resistant (MDR) P. aeruginosa and A. baumannii. Materials and Methods: A multi-centric study was conducted from January 2014 to December 2015 with a total number of 240 MDR P. aeruginosa and 312 MDR A. baumannii isolated from blood, cerebrospinal fluid, respiratory, pus, urine and intra-abdominal infections. Kirby–Bauer disc diffusion was done to determine the antimicrobial susceptibility profile. Further, MDR isolates were characterised by multiplex polymerase chain reaction to determine the resistance genes for ESBLs and carbapenemases. Results: Among the ESBLs, bla_VEB (23%), bla_TEM (5%) and bla_SHV (0.4%) in P. aeruginosa and bla_PER (54%), bla_TEM (16%) and bla_SHV (1%) in A. baumannii were the most prevalent. Likewise, bla_VIM (37%), bla_NDM (14%), bla_GES (8%) and bla_IMP (2%) in P. aeruginosa and bla_OXA-23like (98%), bla_OXA-58like (2%), bla_NDM (22%) and bla_VIM (3%) in A. baumannii were found to be the most prevalent carbapenemases. bla_OXA-51like gene, intrinsic to A. baumannii was present in all the isolates tested. Conclusion: The data shown highlight the wide difference in the molecular mechanisms of AMR profile between P. aeruginosa and A. baumannii. In P. aeruginosa, plasmid-mediated mechanisms are much lesser than the chromosomal mediated mechanisms. In A. baumannii, class D oxacillinases are more common than other mechanisms. Continuous surveillance to monitor the trends in AMR among MDR pathogens is important for implementation of infection control and to guide appropriate empirical antimicrobial therapy.     

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm

ObjectivesThe aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories.MethodsThe immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric β-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2).ResultsThe overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6–89.2%)/100% (CI 91.8–100%) for RESIST-4, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for CARBA-5, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for Xpert Carba-R, 73.7% (CI 66.2–80.0%)/100% (CI 93.4–99.0%) for β-CARBA, and 97.4% (CI 87.9–99.6%)/97.7% (CI 87.9–99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for β-CARBA (76.6% (CI 68.4–83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3–98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed.ConclusionsExcept for β-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.     

Evaluation of a New Phenotypic OXA-48 Disk Test for Differentiation of OXA-48 Carbapenemase-Producing Enterobacteriaceae Clinical Isolates

The current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae (CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceae isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceae isolates (57 Klebsiella pneumoniae carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceae species and provide important infection control information.     

Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis

Background: Acinetobacter baumannii is usually multi-drug resistant (MDR), including third generation cephalosporins, amino glycosides and fluoroquinolone. Resistance to these antibiotics is mediated by multiple factors such as: lactamases, efflux pumps and other mechanisms of resistance. Pulsed-field gel electrophoresis (PFGE) was then used to investigate the genetic relationships among the MDR isolates. Aim: The aim of this study was to determine MDR isolates and the existence of OXAs genes among MDR isolates of A. baumannii collected from Kermanshah hospitals in west of Iran. Materials and Methods: Forty-two MDR A. baumannii were collected from patients at Kermanshah hospitals. The isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics by disk diffusion method was determined. Polymerase chain reaction (PCR) was performed for detection of bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like and bla_OXA-58-like betalactamase genes in isolates and clonal relatedness was done by PFGE (with the restriction enzyme ApaI) and patterns analyzed by Bionumeric software. Results: This study showed high resistant to ciprofloxacin, piperacillin, ceftazidime and also resistant to other anti-microbial agents and more spread blaOXA-23-like gene (93%) in MDR isolate. The PFGE method obtained six clones: A (10), B (9), C (5), D (4), E (11) and F (3) that clone E was outbreak and dominant in different wards of hospitals studied. Conclusion: An isolate from the emergency ward of these hospitals had indistinguishable isolates PFGE profile and similar resistance profile to isolates from intensive care unit (ICU), suggesting likely transmission from ICU to emergency via patient or hospital staff contact.     

Multidrug-Resistant Enterobacteriaceae Colonising the Gut of Adult Rural Population in South India

Background: Multidrug-resistant (MDR) colonisers act as a reservoir for transmission of antibiotic resistance and are a source of infection. Exposure to antibiotics by the commensal flora renders them resistant. Antibiotic consumption and hospitalisation are two major factors influencing this. We studied, antibiotic-resistant bacteria colonising rural adult population who had restricted access to health care and presumably had low consumption of antibiotics. Aim: Detection of multidrug resistance genes of extended spectrum β-lactamase (ESBL-CTX-M), AmpC β-Lactamase (CIT), Klebsiella pneumoniae carbapenemase (KPC) and New Delhi Metallo β-lactamase (NDM) in Enterobacteriaceae colonising the gut of adult population in a South Indian rural community. Methodology: Faecal samples of 154 healthy volunteers were screened for Enterobacteriaceae resistant to commonly used antibiotics by standard methods, followed by phenotypic detection of ESBL by double disk synergy method, AmpC by spot inoculation and carbapenemases by imipenem and ethylenediaminetetraacetic acid + imipenem combined E-test strips and modified Hodge test. Polymerase chain reaction was done to detect bla_CTX-M, bla_CIT, bla_KPC-1 and bla_NDM-1 genes coding for ESBL, AmpC, KPC and NDM, respectively. Results: Colonisation rate of enteric bacteria with MDR genes in the community was 30.1%. However, phenotypically, only ESBL (3.2%) and NDM (0.65%) were detected. While the genes coding for ESBL, AmpC and NDM were detected in 35.6%, 17.8% and 4.4% of the MDR isolates, respectively. Conclusions: Carriage of MDR strains with a potential to express multidrug resistance poses a threat of dissemination in the community. Awareness for restricted use of antibiotics and proper sanitation can contain the spread of resistant bacteria.     

Investigation of the presence of carbapenemases in carbapenem-resistant Klebsiella pneumoniae strains by MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) and comparison with real-time PCR method

PurposeWe aimed to develop a new procedure for rapid detection of the carbapenemase activity using MALDI-TOF MS, and to determine the sensitivity and specificity of the method. Also, we aimed to determine the distribution of carbapenemase genes among the K.pneumoniae strains isolated in our hospital using real-time PCR.MethodBetween January 2017–February 2019; K. pneumoniae strains(n = 74) isolated from blood culture samples were included. Klebsiella pneumoniae NCTC 13438 was used as a positive control and Escherichia coli ATCC 25922 as a negative control. First, Imipenem, meropenem, and ertapenem MIC values of strains were determined. Then bla_KPC, bla_OXA-48, and/or bla_NDM genes were investigated with PCR. Carbapenemase activity was investigated in strains with the newly developed method using MALDI-TOF MS. The performance of the new method was evaluated for both the second and fourth hours of the incubation period.ResultsWhile 65 strains were found resistant to tested carbapenems, nine of them were susceptible. Of the 65 resistant strains, 57 had bla_OXA-48, 15 had bla_NDM, and four had bla_KPC genes. Bla_OXA-48 and bla_NDM genes were detected together in 11 strains. Bla_OXA-48, bla_NDM, and bla_KPC genes were not detected in any of the susceptible strains. The sensitivity and specificity of MALDI-TOF MS at the second hour were 83.1% and 100%, respectively. At the fourth hour, the sensitivity and specificity of MALDI-TOF MS were 100%. No false-positive results were observed.ConclusionThe sensitivity of the method at the fourth hour was better than the second hour. The false-negative results observed in the second hour disappeared when the incubation period was extended to 4 h. MALDI-TOF MS which is still under development is a fast, cost-effective, promising method for the detection of carbapenemase activity.     

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1

PurposeCarbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India.MethodsFifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis.ResultsIsolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 ​% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 ​h detected 92.3 ​% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 ​h at cut off 17 ​mm while for NDM-1 CIM was the test of choice at 16 ​mm.ConclusionCIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 ​h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 ​mm was considered adequate for detection of OXA-48-like and 16 ​mm of NDM-1 by mCIM at 4 ​h and CIM respectively.     

Prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China

In order to assess the prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China, we conducted a polymerase chain reaction (PCR)-based surveillance of OXA-type β-lactamase gene clusters for a total of 2,880 Acinetobacter spp. isolates collected from 23 Chinese provinces. All isolates were tested for susceptibility to 12 antimicrobial agents and showed high rates of resistance to all these agents except minocycline. We also found that the vast majority of carbapenem-resistant Acinetobacter spp. were OXA-23-like-producing isolates, predominantly Acinetobacter baumannii isolates. Besides, bla _OXA-58-like and bla _OXA-24-like genes were detected in 32 and 11 isolates, respectively, involving many provinces throughout China. Furthermore, these two carbapenem-resistance determinants were located on transferable plasmids in most cases, indicating an emerging threat for both OXA-58-like- and OXA-24-like-producing Acinetobacter spp. isolates in China. Interestingly, a novel homologue of the bla _OXA-143 gene was identified in a susceptible Acinetobacter pittii isolate. Overall, these observations suggest that the bla _OXA-23-harboring A. baumannii isolates are the most frequent carbapenem-resistant Acinetobacter spp. in China, and the bla _OXA-24-like and bla _OXA-58-like genes have emerged as potential threats of hospital outbreaks of multidrug-resistant Acinetobacter spp.     

Characterization of genetic diversity of carbapenem-resistant Acinetobacter baumannii clinical strains collected from 2004 to 2007

Genotypes of carbapenem-resistant Acinetobacter baumannii collected by the clinical microbiology laboratory of a university hospital in Chicago, IL, between 2004 and 2007 were analyzed by pulsed-field gel electrophoresis. A single genotype established predominance after being introduced in 2005. Analysis of carbapenemases by PCR revealed that imipenem resistance but not meropenem resistance was associated with the presence of bla_OXA-23 and bla_OXA-40 genes.     

Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K-SeT) for Rapid Detection of OXA-48-Like Carbapenemases in Enterobacteriaceae

We evaluated an immunochromatographic lateral flow assay to detect OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n = 82). One hundred percent sensitivity and specificity were observed using bacteria recovered from both solid medium and spiked blood culture bottles, and the results were obtained in <10 min.