Meropenem Disc Research Articles

Superbugs causing infections at a tertiary care hospital and the return of pre-antibiotic era!

Aims and Objectives: Increase in the incidence of Escherichia coli and Klebsiella pneumoniae carrying New Delhi metallo beta lactamase-1 (NDM-1) gene are called superbugs is of great concern as presence of blaNDM-1 gene makes E.coli and K.pneumoniae highly resistant to most of currently available antibiotics. This study was planned to observe the burden of blaNDM-1 gene carrying E. coli and K.pneumoniae at a tertiary care hospital in northern India. Materials and Methods: A total of 1709 E. coli and 327 K. pneumonia nonrepitive isolates were taken from various clinical samples received in a tertiary care hospital in northern India Lucknow during the period from May 2012 to April 2013. Carbapenemase production was phenotypically detected in all the carbapenem resistant isolates by modified Hodge test. Metallo-β-lactamase production was detected by using meropenem and imipenem discs with and without EDTA and blaNDM-1 gene was detected by polymerase chain reaction. Results: Over all metallo β- lactamase production was found in 82% and 88.89% of carbapenem resistant E.coli and K. pneumonia respectively. Out of 366 carbapenem resistant isolates, 102 were found positive for blaNDM-1 gene out of which 89 were E.coli and 13 were K. pneumoniae. Conclusions: With limited treatment options left for this crisis situation like the pre-antibiotic era; it is an alarm for rational antibiotic therapy usage and intensive education programs.Asian Journal of Medical Sciences Vol.7(2) 2015 76-79

Incidence of carbapenem-resistant pseudomonas aeruginosa in clinical samples

Background: Pseudomonas aeruginosa have been reported to show resistance to carbapenem drugs. Detection of carbapenem resistance pseudomonas is now important to prevent their spread as discriminates of these bacteria could be fetal to the hospitalized patients. The main objective of this study was to detection of carbapenem resistant pseudomonas aeruginosa. We also aimed to search for the treatment option for this multidrug resistant P.aeruginosa. Method: A present study was conducted over a period of 6 months. 239 isolates of Pseudomonas aeruginosa were obtained from the samples from hospitals of Pt.J.N.M.Medical College, Raipur (C.G.) from January 2015 to June 2015. These isolates were tested for susceptibility to antipseudomonal drugs and considered to be resistant to carbapenem .When the zone of inhibition around imipenem and meropenem discs was <13mm. Antibiogram generated by disc diffusion susceptibility testing for clinically relevant antibiotics. Results: Out of total 239 Pseudomonas aeruginosa isolates, 25(10.46%) were found to be carbapenem resistant. All imipenem resistant isolates sensitive to polymyxin B (300g) and colistin (10g). Conclusion: The rapid dissemination of carbapenem resistance is worrisome and calls for the implementation of surveillance studies as well as judicious use of antibiotics. This therapeutic challenge to the clinicians and also need proper selection of antibiotics especially carbapenems.

Is there a relationship between rectal colonization and nosocomial infection of patients in intensive care unit?

Nosocomial infections caused by multidrug-resistant (MDR) microorganisms are a major problem in intensive care units (ICUs) with high mortality and morbidity rates and the prior colonization is an important risk factor for these infections. The aim of this study was to investigate the prevalence of rectal colonization of MDR microorganisms and the association between the microorganisms that caused colonization and infection in the patients with nosocomial infections in ICUs. Rectal swabs were obtained on the day of 0, 3, 7, 14, 21 and weekly thereafter from 80 patients over 18 years of age hospitalized in ICU for more than 48 hours, and cultured for vancomycin-resistant enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)- producing gram-negative bacilli (GNB) and carbapenem-resistant enteric and nonenteric bacilli. Patients whose rectal swabs were not obtained on admission (on the day of 0), were excluded even they were hospitalized more than 48 hours. Bile esculin agar containing 64 μg/mL ceftazidime and 6 μg/mL vancomycin, chromogenic MRSA agar and blood agar media, MacConkey agar containing 1 mg/L ceftazidime and ceftriaxone, and 5 mL tryptic soy broth media containing 10 µg imipenem and meropenem discs were used for identification. Identification of GNB was determined by conventional methods and ESBL production was determined by double-disc synergy test. Patients have been followed up for nosocomial infections. Bacterial identification and antibiotic susceptibility tests were performed with standard microbiological methods. In 37 (46%) of the 80 patients, at least one MDR microorganism was isolated in rectal swab cultures on the day of 0. The most common microorganisms were ESBL-positive E.coli (19%), followed by ESBL-positive K.pneumoniae (13%), carbapenem-resistant P.aeruginosa (10%), ESBL-positive K.oxytoca (3%), MRSA (1%), VRE (1%), carbapenem-resistant Acinetobacter sp. (1%) and carbapenem-resistant K.pneumoniae (1%), respectively. The number of microorganisms isolated from rectal swab cultures on the following days have increased, and on the 7th day, the rate of the patients with rectal colonization ascended to 72%. Out of 80 patients, 52 (65%) had nosocomial infections in the follow-up and the mean duration of infection development was 11.8±9.9 days in these patients. Patients with and without rectal colonization were compared in terms of subsequent nosocomial infection rates. While no statistically significant difference has been detected between two groups on the day of 0, patients with rectal colonization detected on the day of 3 and 7, had a significantly higher incidence of nosocomial infections (p=0.02, p=0.01). Among the patients with ESBL-positive GNB, carbapenem-resistant K.pneumoniae, carbapenem-resistant P.aeruginosa and VRE infections, the same microorganisms have been isolated in the rectal swab cultures taken before the development of infection. This result was statistically significant for each of these microorganisms (p=0.00-0.03). However, such a correlation was not observed for Acinetobacter infections. Since MRSA infections developed in only two patients, no istatistical analysis has been done for this microorganism. In conclusion, our data suggest that MDR microorganisms that cause nosocomial infections, initially colonize the gastrointestinal tract, and early detection of colonized patients in ICUs may help an effective infection control by preventing the spread of these resistant microorganisms.

99 Active surveillance and containment of carbapenemase producing Enterobacteriaceae (CPE) in cystic fibrosis (CF) patients attending an Italian care centre

Objectives Carbapenemases, that hydrolyse most b-lactams, have emerged and spread worldwide. Currently are not many data of CPE in CF. The accesses to different hospitals, such as transplant centres, make these patients at high risk for transmission of CPE. The study aims to quantify and describe the CPE isolated from CF patients in a year about active surveillance. Methods During 2014, 147 rectal swabs were analyzed. The swabs were inoculated on McConkey agar plate with meropenem disk. The susceptibility testing was performed with automatic method (Phoenix). Carbapenemase were tested by disk diffusion synergy test. For molecular characterization was used GeneXpert. The fingerprinting of isolates were performed by Box-PCR. Results In ten samples we not evaluated the CPE because there was no bacterial growth. Three patients were colonized by CPE identified as E. cloacae. Clonal identity among strains was founded. The strains show a reduced susceptibility to carbapenems and the resistance to other b-lactams and aminoglycosides. The strains were susceptibility to fluoroquinolones, colistin and tigecycline. The molecular assay shows the presence of bla VIM gene. Conclusion The screening using culture methods has unsuitable for some CF patients while molecular method has more effective. Moreover the genotyping suggests a spread among patients. The CPE represents a danger for CF patients: this resistance may be transmitted to the multi-resistant pathogens that support CF pulmonary infection. Then an active surveillance, with an appropriate method of screening for CF patients, is needed to decrease and prevent the transmission of CPE.

Chromogenic agar medium for rapid detection of extended-spectrum β-lactamases and Klebsiella pneumoniae carbapenemases producing bacteria from human immunodeficiency virus patients.

Sir, Bacterial infections in the human immunodeficiency virus (HIV) patients are mostly caused by the pathogenic bacteria with a high level of antibiotic resistance.[1] Extended-spectrum beta(β)-lactamases (ESBLs) are plasmid-encoded enzymes and confer resistance to penicillins, cephalosporins, and aztreonam.[2] Klebsiella pneumoniae carbapenemases (KPC) belong to the family of serine carbapenemases and are usually found in Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E.coli). KPC hydrolyze β-lactam antibiotics and thereby, reducing their activity and misidentification of KPC-producing bacteria is common with standard susceptibility testing.[3] The aim of this study is to identify the ESBLs and KPC-producing gram-negative bacteria from HIV patients using HiCrome agar medium. HiCrome ESBL Agar Base (HiMedia, India) is a chromogenic medium used for the selective isolation of ESBL-producing bacteria on the basis of colour colonies. HiCrome ESBL supplement, containing antibiotics such as ceftazidime, cefotaxime, ceftriaxone, aztreonam, and flucanazole, is used to inhibit other contaminating microorganisms and non-ESBL-producing bacteria. HiCrome KPC Agar (HiMedia, India) is used in the identification of KPC-producing bacteria and the supplement contains antibiotics that inhibit the growth of yeast, gram-positive organisms, and gram-negative organisms that do not produce carbapenemases. The HIV patients were identified by standard combination rapid tests as per National acquired immune deficiency syndrome Control Organisation (NACO) guidelines (NACO, 2007).[4] The median cluster of differentiation 4 (CD4) cell count of the HIV patients was 142 cells/mm3. Among the 173 bacterial isolates from HIV patients, 126 were from urine, 27 from pus, 16 from sputum, 2 from blood, 1 from fine needle aspiration cytology, and 1 from vaginal swab. Using ESBL and HiCrome KPC agar media, a total of 108 (P ≤ 0.001) and 132 (P ≤ 0.001) bacterial isolates were found to be positive for ESBLs and KPC production, respectively. Out of 108 ESBLs-producing strains, 64 were E. coli, 11 K. pneumoniae, 18 Klebsiella oxytoca (K. oxytoca), 10 Pseudomonas aeruginosa (P. aeruginosa), and 5 Proteus mirabilis (P. mirabilis). Of the 132 KPC-producing isolates, 73 were E. coli, 17 K. pneumoniae, 15 K. oxytoca, 18 P. aeruginosa, 5 P. mirabilis, 3 Proteus vulgaris, and 1 Acinetobacter baumannii. A number of 59 isolates showed positive for both ESBLs and KPC production. ESBLs production was compared by the combination disc method (CDM) using cefotaxime and ceftazidime alone and in combination with clavulanic acid and KPC production by the modified Hodge test (MHT) using meropenem disc.[5] It was found that only 88 (P ≤ 0.001) isolates showed positive for ESBLs production using CDM and 110 (P ≤ 0.001) for KPC production using MHT. Comparison of results of the above methods in this study thus revealed that HiCrome agar is more sensitive than CDM in ESBLs production, as well as MHT in KPC production. Ongut et al. (2014)[6] from Turkey reported that among 237 bacterial isolates from various clinical samples from non-HIV patients, 143 showed positive for ESBLs production using Brilliance ESBL agar (Oxoid; Thermo Fisher Scientific, UK) and among 143 ESBL-positive isolates, 76 were E.coli and 67 K. pneumoniae. Samra et al. (2008)[7] reported a new CHROMagar KPC medium for rapid and direct detection of carbapenem-resistant K. pneumoniae from clinical samples. They found that among 122 swab cultures, 43 K. pneumonaiae isolates showed positive for KPC production. This is the first report of the detection of ESBLs and KPC-producing gram-negative bacteria from HIV patients using chromogenic agar medium in India. Identification of ESBLs and KPC production among gram-negative bacteria using HiCrome agar is cost-effective and in addition, has the advantage of rapid identification within 24 h with higher sensitivity. The limitations of this method include the occurrence of false-positive results in case of multiple β-lactamases producing bacteria and that a few other carbapenemases producing bacteria may show positive results as well. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Characterization of KPC, NDM and VIM Type Carbapenem Resistance &amp;lt;i&amp;gt;Enterobacteriaceae&amp;lt;/i&amp;gt; from North Eastern, Nigeria

Introduction and Aim: Carbapenem resistance among species of Enterobacteriaceae has emerged as a global public health problem that adds to the high cost of care, severity and high mortality of otherwise straightforward infections. Governments around the world are devoting efforts to combat this important threat. The present study was undertaken in our setting to detect and characterize carbapenem resistance among Enterobacteriaceae. Methodology: Confirmed species of Enterobacteriaceae isolated from 225 patients that were admitted in various units of University of Maiduguri Teaching Hospital (UMTH) Maiduguri were screened for carbapenem resistance with meropenem and ertapenem disc (10 μg, Oxoid, England) using clinical and laboratory standards institute (CLSI) breakpoints. Suspected carbapenemase producers were subjected to confirmation using Modified Hodge Test method. Detection of the carbapenemase genes was done by multiplex PCR using KPC, NDM-1 and VIM primers. Results: A total of 225 clinical isolates of Enterobacteriaceae comprising 73 (32.4%) of Klebsiella pneumoniae, 61 (27.1%) of Escherichia coli, 21 (9.3%) of Proteus mirabilis, 18 (8.0%) of Klebsiella oxytoca, 13 (5.8%) of Morganella morganii, 12 (5.3%) of Citrobacter freundii, 12 (5.3%) of Serratia marcescens, 7 (3.1) of Enterobacter aerogenes, 3 (1.4%) of Klebsiella ozaenae, 3 (1.4%) of Hafnia alvei and 2 (0.9%) of Citrobcter sedlakii were isolated. A total of 28 (12.4%) of the isolates screened positive as carbapenemase producers. All the 28 screened isolates were further subjected to confirmation using the Modified Hodge Test for which 23 (10.2%) were confirmed resistant. Therefore a prevalence of 10.2% for carbapenem resistance was recorded in this study. Based on multiplex polymerase chain reaction, the various percentage genotypes of the carbapenemase producers were: 11 (47.8%) for KPC, 2 (8.7%) for VIM while 5 (21.7%) isolates have co-existence of the NDM-1 and VIM genes. However, 5 (21.7%) of the isolates have none of the genes screened for in them. The occurrence of carbapenemase genes among species of Enterobacteriaceae was as follows: 6 (33.3%) for Klebsiella pneumoniae, 4 (22.2%) for Escherichia coli, 4 (22.2%) for Proteus mirabilis, 3 (16.7%) for Serratia marcescens and then 1 (5.6%) for Klebsiella oxytoca. Conclusion: Despite the low use of carbapenem agents in the study area, carbapenem resistance was documented. This calls for an ongoing surveillance and infection control practices of this and other emerging resistance threat in all health centers of Nigeria. Irrational use of antibiotics must be discouraged so as to reduce this resistance threat. Antibiotic stewardship program should be established in all tertiary health centers of Nigeria.

Detection of Carbapenemases in Clinical Enterobacteriaceae Isolates Using the VITEK AST-N202 Card.

BackgroundThe rapid and accurate detection of carbapenemase-producing Enterobacteriaceae (CPE) in clinical microbiology laboratories is essential for the treatment and control of infections caused by these microorganisms. This study was performed to evaluate the ability of the VITEK AST-N202 card to detect CPE isolates.Materials and MethodsA total of 43 (Klebsiella pneumoniae, n = 37; Escherichia coli, n = 3; and Enterobacter cloacae, n = 3) CPE isolates and 79 carbapenemase-non-producing Enterobacteriaceae (CNE) isolates were included in this study. The CPE isolates harbored KPC-2 (n = 11), KPC-3 (n = 20), GES-5 (n = 5), VIM-2 (n = 2), IMP-1 (n = 1), NDM-1 (n = 2), or OXA-232 (n = 2). Of the 79 CNE isolates, eight K. pneumoniae isolates were resistant to ertapenem, imipenem, and meropenem, while the remaining 71 isolates were susceptible to the carbapenems. Antimicrobial susceptibilities were tested using the VITEK AST-N202 card, and the results were interpreted as positive when the isolates showed resistant or intermediate results. Modified-Hodge tests (MHTs) were performed using ertapenem or meropenem disks for the screening of carbapenemase production. Polymerase chain reaction (PCR) and direct sequencing were used to identify β-lactamase genes.ResultsSensitivity of MHT with ertapenem and meropenem disks for the detection of carbapenemase was 81.4% (35/43) and 81.4% (35/43), respectively, and a combination with both antibiotic disks increased the sensitivity to 88.4% (38/43). Specificity of the MHT was 100% (79/79) for the CNE isolates. Sensitivity of ertapenem, imipenem, and meropenem as assessed by the VITEK AST-N202 card was 100% (43/43), 93% (40/43), and 95.3% (41/43), respectively. Specificity (89.8%, 71/79) of the test with each carbapenem was improved to 100% (71/71) when eight carbapenem-resistant CNE isolates were excluded from the testing.ConclusionThe VITEK AST-N202 card showed high sensitivity for the detection of carbapenemases in Enterobacteriaceae strains. PCR and sequencing experiments for the detection of carbapenemases are recommended when clinical Enterobacteriaceae isolates show non-susceptibility to carbapenems.

Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates

With the emergence of antibiotic resistance among Bacteroides fragilis group isolates the need of susceptibility testing in routine laboratories is increasing. The aims of the present study were to evaluate the disk diffusion method for susceptibility testing in case of different clinical isolates of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic susceptibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to amoxicillin/clavulanic acid, cefoxitin, clindamycin, imipenem, metronidazole, moxifloxacin, piperacillin/tazobactam, tigecycline by agar dilution method previously. The inhibition zones of the same antibiotics including meropenem disc were determined by the disc diffusion on Brucella blood agar supplemented with haemin and vitamin K1. Plates were incubated at 37 °C in an anaerobic atmosphere for 24 h. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement between the inhibition zone diameters and the MICs for imipenem, metronidazole, moxifloxacin and tigecyclin. The inhibition zone diameters of meropenem also separated clearly the isolates, which can be considered wild-type isolates. In case of amoxicillin/clavulanic acid and piperacillin/tazobactam intermediate and susceptible isolates according to the MIC determination, overlap during the zone diameter determination. Isolates with an inhibition zone <23 mm for amoxicillin/clavulanic acid and <25 mm for piperacillin/tazobactam should be retested by a MIC determination method. The 10 μg clindamycin disc clearly separated the resistant and the susceptible population of B. fragilis group strains. In the case of cefoxitin only resistant population could be separated with an inhibition zone <17 mm, intermediate and susceptible isolates overlap. In conclusion, we suggest that disk diffusion can be an option for susceptibility testing of B. fragilis group isolates for most relevant antibiotics in routine laboratories.

A subclass B3 metallo- -lactamase found in Pseudomonas alcaligenes

Sir, Metallo-b-lactamase (MBL) is an important resistance determinant among Gram-negative bacteria, and its clinical relevance is increasing. Some MBL genes are carried on mobile gene elements that have spread among various clinically important bacterial species. Here we report a case of a novel MBL-positive Pseudomonas alcaligenes strain, MRY13-0052, that caused a bloodstream infection in a medical institution in Japan. P. alcaligenes is a Gram-negative aerobic bacillus belonging to the bacterial family Pseudomonadaceae, the members of which are common inhabitants of soil and water, and it is a rare opportunistic human pathogen. However, little is known about the clinical importance of P. alcaligenes, mainly because of the difficulties in identifying and distinguishing this bacterium from closely related Pseudomonas species, such as Pseudomonas aeruginosa, Pseudomonas mendocina and Pseudomonas pseudoalcaligenes, in medical settings. We report here our investigation of the draft genome sequence of P. alcaligenes strain MRY13-0052 and our finding that this strain contains a subclass B3 MBL, PAM-1 (P. alcaligenes MBL-1), that can hydrolyse cephalosporins and carbapenems. In 2012, Pseudomonas strain MRY13-0052 was recovered from a blood sample of a patient who was receiving therapy for Guillain–Barre syndrome. The patient had no recent history of travel abroad. Although the primary site of infection was unknown, the patient became afebrile soon after combination therapy with ceftazidime and clindamycin. The MRY13-0052 strain was identified as P. mendocina by the VITEK2 system (bioMerieux; 96% probability), but subsequently as P. alcaligenes based on 16S rRNA gene sequence analysis. MRY13-0052 was resistant to penicillins, cephalosporins, aztreonam and fosfomycin, but susceptible to imipenem, meropenem, amikacin, fluoroquinolones, minocycline and trimethoprim/sulfamethoxazole, according to MICs determined using the VITEK2 system and the Etest (bioMerieux), applying the recommended breakpoints described by CLSI (2013). The production of MBL was screened for using a disc containing sodium mercaptoacetic acid (SMA) (Eiken). Apparent expansion of the growth inhibitory zone around the ceftazidime and meropenem discs was observed around the SMA disc following overnight incubation at 378C, strongly suggesting that MRY13-0052 produces MBL. PCR tests to detect the blaNDM, blaIMP, blaVIM and blaTMB genes in MRY13-0052 were all negative; therefore, we analysed the whole-genome shotgun (WGS) sequence of MRY13-0052, obtained using the GS Junior system (Roche), to identify the responsible MBL gene (DDBJ/EMBL/GenBank accession number of the WGS project: BATO01000000). BLAST-based similarity searches revealed that MRY13-0052 carries three class C b-lactamase genes and a novel subclass B3 MBL gene [which we named blaPAM-1 (DDBJ/EMBL/GenBank accession number of the gene: AB858498)] that might confer resistance to b-lactams. The PCR product of the blaPAM-1 gene was ligated into pUCP19 (ATCC), a Pseudomonas–Escherichia shuttle vector, resulting in the PAM-1 expression vector pUCP19-blaPAM-1. P. aeruginosa strain PAO1 and Escherichia coli strain MC1061 were transformed with this vector, and transformants were selected on agar plates containing 20 mg/L piperacillin. Expression of the blaPAM-1 gene was driven by the tac promoter regardless of IPTG induction and confirmed by SMA disc-mediated expansion of the growth inhibitory zone around ceftazidime and meropenem discs. As shown in Table 1, blaPAM-1-producing P. aeruginosa bacteria were more resistant to ceftazidime, imipenem, meropenem and doripenem than control bacteria harbouring the empty vector (MICs increased 32-fold, 2-fold, 6-fold and 21-fold, respectively), but were still as susceptible as control bacteria to aztreonam. Although blaPAM-1producing E. coli bacteria were slightly more resistant to ceftazidime and meropenem than control bacteria (MICs increased 4-fold and 1.4-fold), there was no apparent change in the susceptibility to aztreonam and other carbapenems. The differences in the contribution of the PAM-1 enzyme to cephalosporin and carbapenem resistance among P. aeruginosa and E. coli could reflect differences in expression levels, outer-membrane permeability and/or efflux systems in these hosts. The blaPAM-1 gene in P. alcaligenes strain MRY13-0052 is encoded in contig 73, which is part of the chromosome, and there is no transposable element, such as a transposon or integron, around the gene, suggesting that blaPAM-1 is an intrinsic species-specific MBL gene of P. alcaligenes. Pseudomonas otitidis, a Pseudomonas species that is associated with otic infections in humans, also produces a resident MBL named POM-1 (P. otitidis MBL-1), which is active against carbapenems. The PAM-1 protein exhibits close similarity to POM-1 (72.4% amino acid identity), suggesting that these enzymes have a common ancestor (Figure 1). PAM-1 and POM-1 are homologous with the L1 MBL of Stenotrophomonas maltophilia (63.3% and 62.1% identity,

Detection of NDM-2-producing Acinetobacter baumannii and VIM-producing Pseudomonas aeruginosa in Palestine.

The aim of this study was to screen for carbapenem-resistant Gram-negative bacteria in Palestine and subsequently to identify and investigate the mechanisms of resistance. For a period of 6 weeks, all Gram-negative isolates were collected from six Palestinian hospital laboratories and were tested for susceptibility using 10μg meropenem disks. Isolates showing resistance to meropenem were further investigated. The presence of carbapenemases was assessed by PCR. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing carbapenemases were further investigated by multilocus sequence typing (MLST). In total, 248 Gram-negative isolates were collected from the six laboratories. Among the 248 tested isolates, 15 Acinetobacter baumannii and 6 Pseudomonas aeruginosa were resistant to meropenem. One A. baumannii from Gaza produced NDM-2 and belonged to ST103. Thirteen of the carbapenem-resistant A. baumannii isolates possessed the intrinsic upregulated blaOXA-66 gene and one isolate carried blaOXA-51. All but one of the OXA-66-producing A. baumannii belonged to ST2; the remaining isolate belonged to ST183. One of the carbapenem-resistant P. aeruginosa was classified as VIM-4-producing and three were VIM-2-producing isolates. The three VIM-2-producing isolates belonged to three new sequences types (ST1562, ST1563 and ST1564). All of the carbapenemase-producing isolates were multiresistant non-fermenters. To the best of our knowledge, this is the first report on NDM-producing A. baumannii and VIM-producing P. aeruginosa from Palestine.

Detection of Colonization by Carbapenemase-Producing Gram-Negative Bacilli in Patients by Use of the Xpert MDRO Assay

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.

A Combined Disk Test for Direct Differentiation of Carbapenemase-Producing Enterobacteriaceae in Surveillance Rectal Swabs

Carbapenemase-producing Enterobacteriaceae (CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rapidly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a MacConkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of ≤ 25 mm around the MER disk alone indicated carriage of carbapenem-resistant organisms. Furthermore, ≥ 5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniae carbapenemase (KPC), metallo-β-lactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. Moreover, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differentiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures.

Nosocomial transmission of NDM-1-producing Escherichia coli ST101 in a Korean hospital

Sir, Clonally diverse NDM-1-producing Escherichia coli (ST405, ST131, ST156 and ST101) have been reported globally, including on the Indian subcontinent. Indeed, there may be an association between isolates of NDM-1-producing E. coli ST101, phylogroup B1, that have been identified in Australia, Canada, Germany, the UK and Pakistan. In December 2011, NDM-1-producing E. coli (CREC-36) was identified from a patient who was hospitalized in a 260 bed tertiary care hospital in Korea. Medical records were reviewed and 114 stool or rectal swab samples were taken from 67 patients on Ward 1 (19 rooms), 11 patients on the intensive care unit (ICU) and 25 healthcare workers (doctors, nurses and caregivers) working on Ward 1. We screened patients in the ICU because the first patient had been treated in the ICU for 1 month before she was transferred to room 112. In addition, 23 environmental swabs were collected from washbasins, medicine trays and linen. All samples were cultured in 5 mL of tryptic soy broth containing 10 mg meropenem discs and processed using the CDC protocol. The modified Hodge test and the double-disc synergy test were performed on representative colonies grown on MacConkey agar. The blaNDM-1 gene was identified by PCR and sequencing using the primers NDM-1 F (5′-CAATATTATGC ACCCGGTCG-3′) and NDM-1 R (5′-ATCATGCTGGCCTTGGGGAA-3′). Three more patients who had shared a room (no. 112) with the first patient were also colonized by NDM-1-producing E. coli (CREC-45, CREC-47 and CREC-49). All four patients were elderly (.75 years) and had been in hospital for between 1 and 7 months before the NDM-1-producing E. coli were isolated. No NDM-1-producing E. coli were detected in samples from other patients, healthcare workers or the environment. NDM-1-positive individuals were isolated until decolonization had been confirmed by three consecutive negative stool cultures collected 1 week apart. None of the patients received treatment to eradicate the NDM-1 E. coli. Two patients died from their underlying disease. The remaining two were decolonized 21 and 53 days after the first isolation. No further cases of NDM-1 were detected. All the isolates were genetically related (PFGE identifying one pulsotype with .90% similarity), and the clone was identified by multilocus sequence typing (MLST; http://mlst.ucc.ie/mlst/dbs/ Ecoli) as ST101, phylogenetic group B1. E. coli ST101 is an international clone, frequently associated with the Indian subcontinent. However, the four patients had no epidemiological link to the Indian subcontinent as they had been hospitalized in Korea for a long time due to other underlying diseases. All patients shared an epidemiological link in terms of transmission. Although none of the healthcare workers harboured NDM-1, personal contact between the caregivers and the patients resident in the same room is the most likely transmission route; the NDM-1-colonized patients were bedridden, the beds were very close to each other and no NDM-1 cases were detected in any other rooms on Ward 1. Transmission was probably caused by poor standards of hygiene in the hospital; there were no sinks or alcohol-based hand cleansers in the room. Neither did the hospital monitor the hygiene practices of the healthcare workers. The MICs of antimicrobial agents for the NDM-1-producing isolates were determined using the agar dilution method and Etest. The susceptibility test results were interpreted according to CLSI or EUCAST (www.eucast.org; for colistin and tigecycline) guidelines. All NDM-1-producing isolates except CREC-49 showed high MICs of carbapenems (≥32 mg/L) and multidrug resistance; however, they were susceptible to tigecycline and colistin (Table 1). Using a blaNDM-1 probe, we identified the blaNDM-1 gene by Southern blotting of S1 nuclease-digested total DNA. The blaNDM-1 gene was identified in the 170, 180 and 120 kb plasmids. The discrepancy in blaNDM-1-bearing plasmid size may be because the plasmids are quite unstable and prone to rearrangement, as previously reported. Although the conjugal transfer of plasmids carried by the NDM-1-producing isolates failed, the blaNDM-1-harbouring plasmids from the four isolates were successfully transformed into electrocompetent E. coli HB101. PCR-based replicon typing of the major plasmid incompatibility groups showed that the plasmids belonged to the IncA/C incompatibility group. All transformants co-harboured blaCTX-M-15, blaCMY-4, blaDDHA-1 and armA. The genetic elements surrounding blaNDM-1 were examined by overlapping PCR mapping. The overall structure was ISAba125–blaNDM-1–bleMBL–trpF–blaDDHA-1–ampR–hypA– Dqac–sul1–ISCR1–tnpU–armA. The sequence spanning from ISAba125 to armA was identical to that in the E. coli DVR22 plasmid (IncHI1), and the sequence from blaNDM-1 to armA was identical to that in pNDM-HK (IncL/M). The sequence sul1–ISCR1–tnpU–armA was also identified in the IncA/C

Emergence of Klebsiella pneumoniae producing NDM-1 carbapenemase in Saint Petersburg, Russia

Sir, Gram-negative bacilli producing NDM-type metallo-carbapenemases present a recognized threat to the healthcare system. Most NDM-positive isolates are multidrug resistant and remain susceptible only to colistin and tigecycline. Although first and most often detected in Klebsiella pneumoniae and Escherichia coli, the NDM-1 gene can spread rapidly and has been found in diverse bacterial strains and species, in healthcare settings and in the environment. NDM producers have been reported from many countries and often, though not always, from patients with travel or healthcare links in other countries. We report here an NDM-1-producing strain of K. pneumoniae that was recovered in a Russian patient with no history of overseas travel. In April 2012, a patient with heart failure (class III, New York Heart Association functional classification) was admitted to Almazov Centre, where the patient received standard treatment for this condition. The patient did not have a urinary catheter during this hospitalization and no antibiotics were administered; the patient was discharged 31 days after admission when the symptoms of heart failure were stabilized. The patient has resided solely in Saint Petersburg throughout their life. At the end of May 2012, the patient complained of frequent urination. An outpatient urine sample yielded 1.5×10 leucocytes/L and a multidrug-resistant K. pneumoniae (10 cfu/mL) was cultured (isolate 1), which was susceptible in vitro to aztreonam, colistin, chloramphenicol, tetracyclines and tigecycline. The MIC of fosfomycin, determined by the agar dilution method, was 16 mg/L and the patient received this preparation (daily dose 3 g) for 2 days with a positive clinical effect. Microbiological reinvestigation was not performed. Multidrugresistant K. pneumoniae (10 cfu/mL) was again isolated from a follow-up outpatient urine sample taken 5 weeks later (isolate 2). CT revealed urolithiasis and chronic pyelonephritis. By this time, both isolates 1 and 2 were known to carry the blaNDM-1 gene (see below) and aztreonam was initiated at a daily dose of 3.0 g after readmission of the patient to Almazov Centre. The treatment was terminated after 4 days due to adverse effects (diarrhoea and hypotension) and urine cultures yielded no bacteria up to the fifth day after cessation of aztreonam treatment. However, 2 weeks later, multidrug-resistant K. pneumoniae (10 cfu/mL) was again recovered from a urine sample (isolate 3). No antibiotics were administered at this time. The patient’s stools did not grow Gram-negative bacilli on selective media for the detection of carbapenem-resistant Enterobacteriaceae: Brilliance CRE agar, MacConkey agar with meropenem and ertapenem discs as well as agar plates supplemented with 0.5 mg/L meropenem. E. coli and Acinetobacter johnsonii isolated on non-selective agar plates were susceptible to carbapenems and did not carry the blaNDM-1 gene. The renal stones were recognized as uric acid deposits. A dietary change as well as medication was prescribed to diminish the size of the stones. The screening of contact patients and careful microbiological investigation of the hospital environment did not reveal carbapenem-resistant Enterobacteriaceae. The patient was discharged home, to the community. The patient was scheduled for hospitalization to the specialized urological department. The three urinary isolates were identified as K. pneumoniae subsp. pneumoniae by sequencing of the first 500 bp of the 16S RNA gene (MicroSeq ID 16S rDNA 500 Library V. 2.1). Multilocus sequence typing was performed as described on the web site of the Pasteur Institute, France (http://www.pasteur.fr/ recherche/genopole/PF8/mlst/Kpneumoniae.html); all three isolates belonged to sequence type ST340, which has been associated with NDM-1-positive isolates from Oman and South Korea. The isolates were tested by PFGE of XbaI-digested DNA (PulseNet standard operating procedure for E. coli). The PFGE patterns of the NDM-1-producing K. pneumoniae isolates are shown in Figure 1. All isolates from the patient’s urine belonged to the same PFGE type. The PFGE profile of a reference strain of K. pneumoniae producing NDM-1 carbapenemase from Finland (K. pneumoniae, NDM-1, ESBL no. 2050, National Institute for Health and Welfare, Turku, Finland) was completely different (Figure 1). Antibiotic susceptibilities were determined on Mueller–Hinton agar by disc diffusion (Oxoid, UK) and by a microdilution method (Sensititre, Trek Diagnostic Systems, USA). The results were interpreted, where possible, using CLSI guidelines or, in the absence of a CLSI breakpoint (e.g. colistin), using EUCAST guidelines. The isolates were resistant to all b-lactams except aztreonam (Table S1, available as Supplementary data at JAC Online); Research letters

First Detection of VIM-4 Metallo-β-Lactamase-Producing Citrobacter freundii in China

Dear Sir, Carbapenems are one of the most critically important antimicrobials and are considered as last-choice drugs in clinical settings. However, the emergence and dissemination of Enterobacteriaceae carrying acquired metallo-β-lactamases (MBLs) is being increasingly reported throughout the world, presenting a serious threat to the effectiveness of carbapenem treatment [1]. Acquired MBLs, such as VIM, IMP, and NDM, are associated with different mobile elements, and the genes for VIM-type enzymes are carried as mobile gene cassettes inserted in class 1 integrons. In Citrobacter freundii, MBL-mediated resistance is rare and only scattered studies have reported the isolation of a few MBL-producing strains, particularly in the Far East [2]. To our knowledge, there is no report of VIM-producing C. freundii in China. In this study, we describe the first identification of the blaVIM-4 gene in a clinical C. freundii strain isolated from a patient in a hospital in Wuhu, China. The multidrug-resistant C. freundii CF0638 isolate was recovered from the sputum of a 66-year-old man presenting with diabetic foot to a hospital in Wuhu, China, in September 2011. During the period of hospitalization, symptoms of lower respiratory tract infection emerged and this patient received treatment with cefotaxime and ciprofloxacin for 7 days. The effect of the treatment was not satisfactory. The patient continued to be febrile until he was treated with imipenem-cilastatin (3 g/day). Imipenem-cilastatin treatment was continued for a 2-week course, and the symptoms gradually disappeared during the patient's hospital stay. Susceptibility testing was performed by an agar dilution method according to the CLSI guidelines (2012) [3]. C. freundii CF0638 exhibited high-level resistance to most β-lactam antibiotics tested. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were 4 mg/L and 1 mg/L, respectively (Table 1). Positive results of the modified Hodge test with meropenem disc and double-disc synergy test between carbapenems and EDTA indicated the production of an MBL. CF0638 was subjected to PCR analysis using the respective primer pairs and amplifying conditions for blaIMP and blaVIM, as described previously [4], and followed by DNA sequencing. Nucleotide sequence analysis and homology searches were carried out using the BLAST database (http://www.ncbi.nlm.nih.gov). To determine whether the imipenem resistance was transferable, conjugation experiments were carried out in Luria-Bertani (LB) broth with sodium azide-resistant Escherichia coli J53 as the recipient. Transconjugants were selected on LB agar plates supplemented with sodium azide (100 mg/L) and cefotaxime (4 mg/L). Table 1 MIC for VIM-4 metallo-β-lactamase-producing Citrobacter freundii CF0638 and its transconjugant PCR amplification and nucleotide sequence analysis showed that CF0638 harbored blaVIM-4. Conjugation experiments showed that imipenem resistance was successfully transferred to the recipient E. coli J53. PCR analysis of the transconjugant strain was positive for the blaVIM-4 gene. Susceptibility test results of C. freundii CF0638 and its transconjugant are presented in Table 1. C. freundii has been recognized as an opportunistic pathogen that is rarely involved in nosocomial infections. In neonates and immunocompromised patients, however, invasive infections such as bacteremia, meningitis, brain abscesses, pneumonia, endocarditis, and intra-abdominal sepsis have also been reported [5]. VIM-4 is a single amino-acid variant of VIM-1 β-lactamase. VIM-4 was first described in Pseudomonas aeruginosa in Greece [6], but was subsequently identified as the most common integron-encoded MBL in Enterobacteriaceae species in several European countries. C. freundii isolates are usually susceptible to carbapenems and only a limited number of carbapenem-resistant strains have been identified. Prompt and accurate detection of MBL-producing C. freundii strains is necessary to prevent the dissemination of resistance vectors to more virulent pathogens.

Evaluation of Double-Disk Potentiation and Disk Potentiation Tests Using Dipicolinic Acid for Detection of Metallo-β-Lactamase-Producing Pseudomonas spp. and Acinetobacter spp

Accurate detection of metallo-β-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs) and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-μg method showed the highest sensitivity (97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-μg disk method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-μg disks carried bla(IMP-6,) and the high level resistance to MEM (MIC ≥ 512 μg/ml) was reduced by the presence of phenylalanine arginine β-naphtylamide. Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates are highly resistant to MEM due to the overexpression of efflux pumps.

Rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC carbapenemase during an outbreak

Two different approaches are described for rapid detection of intestinal carriage of Klebsiella pneumoniae producing KPC-type carbapenemase (KPC-KP), based on PCR amplification of DNA extracts from rectal swabs (K-PCR), and on direct plating of rectal swabs on to MacConkey agar with a meropenem disc and a meropenem plus 3-aminophenylboronic acid disc (direct KPC screening test, DKST). K-PCR and DKST were tested with a total of 101 samples from 65 patients, during an outbreak. Although less sensitive, DKST could detect high-level carriage, which appears to be common among infected and colonised patients, while being very cheap and easy to perform, and requiring only basic facilities.

Occurrence and phenotypic detection of class A carbapenemases among Escherichia coli and Klebsiella pneumoniae blood isolates at a tertiary care center

Resistance to carbapenems is a significant therapeutic threat. The increasing frequency of car bapenemase enzymes among Gram-negative bacilli makes their early detection and differentiation urgent. Carbapenemases belonging to Class A are most commonly produced by members of family Enterobacteriaceae and are inhibited to various degrees by clavulanic acid. The present study is aimed to determine the occurrence and phenotypic detection of Class A carbapenemases in Escherichia coli and Klebsiella pneumoniae blood isolates from septicemic patients. A total of 75 isolates of K. pneumoniae and 25 E. coli were screened for resistance to carbapenems by using meropenem and imipenem discs and meropenem E-test. Positive strains were then subjected to a modified Hodge test combined with carbapenemase inhibition tests to phenotypically detect and differentiate Class A serine carbapenemases from other classes of carbapenem hydrolyzing enzymes. The screening test showing the number of isolates resistant to meropenem and imipenem were 41 and 35 for K. pneumoniae and nine and four for E. coli, respectively. A total of 25 (33.3%) K. pneumoniae isolates and two (8.0%) E. coli isolates were classified as Class A carbapenemase producers. Multidrug resistance with coexistence of extended spectrum-beta-lactamases occurred in 44.4% isolates. However, all of the isolates were susceptible to colistin, polymyxin B, and tigecycline by disc diffusion test. We conclude from the present study that Class A carbapenemases appear to be the predominant cause of resistance to carbapenems in Enterobacteriaceae at our center and, thus, phenotypic detection based on simple methods should be employed routinely in clinical microbiology laboratories.

Phenotypic Screening of Carbapenemases and Associated β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

Dissemination of carbapenem resistance among Enterobacteriaceae poses a considerable threat to public health. Carbapenemase gene detection by molecular methods is the gold standard but is available in only a few laboratories. The aim of this study was to test phenotypic methods for the detection of metallo-β-lactamase (MBL)- or Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and associated mechanisms of β-lactam resistance against a panel of 30 genotypically characterized carbapenem-resistant Enterobacteriaceae : 9 MBL, 7 KPC, 6 OXA-48, and 8 extended-spectrum β-lactamase (ESBL) or AmpC β-lactamases associated with decreased permeability. We used carbapenemase inhibitor-impregnated agar to test for carbapenem-resistant strains. Differences in the inhibition zone sizes of the meropenem, imipenem, ertapenem, and doripenem disks were measured between control and inhibitor (EDTA or phenylboronic acid [PBA] with or without cloxacillin)-impregnated Mueller-Hinton agar with a cutoff of 10 mm. All 9 MBL- and 7 KPC-producing Enterobacteriaceae were identified from the differences in zone size in the presence and absence of specific inhibitors, regardless of the carbapenem MICs and including isolates with low-level resistance to carbapenems. We also detected their associated β-lactam resistance mechanisms (11 ESBL-type and 5 class A β-lactamase 2b). No differences in zone size were observed for OXA-48-producing strains or other carbapenem resistance mechanisms such as ESBL and decreased permeability. We propose a new strategy to detect carbapenemases (MBL- and KPC-type) and associated mechanisms of β-lactam resistance (ESBL or class A β-lactamase 2b) by the use of inhibitor-impregnated agar. A rapid phenotypic detection of resistance mechanisms is important for epidemiological purposes and for limiting the spread of resistant strains by implementing specific infection control measures.

Emergence of Klebsiella pneumoniae isolate co-producing NDM-1 with KPC-2 from India

Sir, The emergence of NDM-1-producing isolates and their sources have been clearly identified in several countries worldwide. In particular, the blaNDM-1 gene was identified in various genera of Enterobacteriaceae and in non-fermenting Gram-negative bacilli from environmental samples in India. Furthermore, the increasing co-production of NDM-1 with other carbapenemases has been detected amongst isolates of Enterobacteriaceae and Acinetobacter sp. in many parts of India. – 7 We report here the isolation of a strain of Klebsiella pneumoniae (designated IR98) from a urine sample from a middle-aged patient admitted to the intensive care unit of a tertiary care hospital in Chennai, India in July 2010. Species identification and antibiotic susceptibility, determined using an automated system (VITEK-2, bioMerieux Inc.), showed wide-spectrum resistance to b-lactams, aminoglycoside, fluoroquinolones, co-trimoxazole, nitrofurantoin and tigecycline, but susceptibility to colistin and fosfomycin, according to CLSI guidelines. MICs of various antibiotics were determined using the agar dilution method, while the tigecycline MIC was determined using the broth microdilution method. European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied to interpret the susceptibilities. The isolate was highly resistant to imipenem (256 mg/L), meropenem (128 mg/L), ceftazidime (.256 mg/L), cefotaxime (.256 mg/L), amikacin (.512 mg/L), gentamicin (.512 mg/L), tobramycin (.512 mg/L), netilmicin (.512 mg/L), co-trimoxazole (.32 mg/L), ciprofloxacin (.32 mg/L) and tigecycline (4 mg/L), but remained susceptible to colistin (0.5 mg/L). The double-disc synergy test (DDST), modified Hodge test (MHT) and combined-disc synergy test (CDST) were used for the detection of metallo-b-lactamases (MBLs), other carbapenemases and KPC, or KPC with MBLs. The simultaneous production of MBL and KPC-like carbapenemases was confirmed by positive DDST, MHT and CDST with meropenem discs containing both EDTA and phenylboronic acid (PBA) or EDTA, while meropenem discs supplemented with PBA were negative. PCR assays for genes encoding b-lactamases and 16S rRNA methylases revealed the presence of blaNDM-1, blaKPC-2, blaCTX-M-15, blaSHV-12, blaTEM-1, blaOXA-1 and rmtB genes. Plasmid analysis using the Kieser technique revealed that K. pneumoniae IR98 harboured four plasmids, with sizes of 160, 120, 70 and 40 kb, using Escherichia coli NCTC 50192 as a reference. To study the transferability of these plasmids (encoding the resistance determinants), transconjugation and transformation experiments were performed using E. coli J53 (Azide-R) and E. coli TOP10 as recipient strains. Transconjugants were selected on MacConkey agar plates using sodium azide (200 mg/L) with ceftazidime (2 mg/L), meropenem (0.5 mg/L) or amikacin (20 mg/L). The plasmid extract of K. pneumoniae IR98 was transformed into E. coli TOP10, and transformants were selected on MacConkey agar plates containing 2 mg/L ceftazidime. Selected colonies were replica-plated onto MacConkey agar plates with or without meropenem (0.5 mg/L) or amikacin (20 mg/L). The genes encoding NDM-1, CTX-M15 and 16S rRNA methylase were transferred in conjugation experiments, whereas transfer of KPC-2 was successful only by transformation. The plasmids purified from the clinical isolate, transconjugants and transformants were typed by PCR-based replicon typing (PBRT). The E. coli J53-p98A transconjugant obtained from the meropenem plate showed an MBL phenotype and elevated MICs of all the b-lactams (except aztreonam) and susceptibility to non-b-lactam antibiotics. Plasmid analysis revealed that E. coli J53-p98A harboured a 160 kb plasmid that belonged to the Inc A/C type. Subsequently, this plasmid was found by PCR to carry the blaNDM-1 gene. In addition, the E. coli J53-p98B transconjugant grown on both ceftazidime and amikacin plates showed decreased susceptibility to aminoglycosides and cephalosporins except cefoxitin, and was positive for extendedspectrum b-lactamase (ESBL) production on DDST. Both blaCTX-M15 and rmtB genes were carried on a 120 kb IncF plasmid that was identified in E. coli J53-p98B. In contrast, the ESBL-negative transformant (E. coli TOP10-p98C) from both ceftazidime and meropenem plates was resistant to all the b-lactams except carbapenems (MICs 2 mg/L). The blaKPC-2 gene together with blaTEM-1 was carried on a 70 kb non-typeable plasmid that was identified in E. coli TOP10-p98C. Although the co-existence of blaNDM-1 with different carbapenemase genes has been reported in India, we believe this to be the first report of the co-occurrence of blaNDM-1 with blaKPC-2 and rmtB in a clinical isolate of K. pneumoniae from India. This co-production of NDM-1 with an unrelated carbapenemase and 16S RNA methylase results in very broad-spectrum antibiotic resistance profiles. The growing emergence of these powerful resistance mechanisms in India is cause for great concern as treatment options are virtually exhausted.