OXA-48 Producers Research Articles

Efficacy of Humanized Carbapenem and Ceftazidime Regimens against Enterobacteriaceae Producing OXA-48 Carbapenemase in a Murine Infection Model

Enterobacteriaceae producing the OXA-48 carbapenemase are emerging worldwide, leaving few treatment options. Efficacy has been demonstrated in vivo with ceftazidime against a ceftazidime-susceptible OXA-48 isolate but not with imipenem despite maintaining susceptibility. The relationship between phenotype and in vivo efficacy was assessed for OXA-48 producers using humanized regimens of 2 g doripenem every 8 h (q8h; 4 h infusion), 1 g ertapenem q24h, 2 g ceftazidime q8h (2 h inf), and 500 mg levofloxacin q24h. Each regimen was evaluated over 24 h against an isogenic pair (wild-type and OXA-48 Klebsiella pneumoniae strains) and six clinical OXA-48 isolates with and without other extended-spectrum β-lactamases in immunocompetent and neutropenic murine thigh infection models. Efficacy was determined using the change in bacterial density versus 24-h growth controls in immunocompetent studies and 0-h controls in neutropenic studies. Bacterial reductions of ≥1 log CFU were observed with all agents for the wild-type strain. Consistent with low MICs, ceftazidime and levofloxacin exhibited efficacy against the isogenic OXA-48 strain, whereas doripenem did not, despite having a susceptible MIC; no activity was observed with ertapenem, consistent with a resistant MIC. Similar trends were observed for the clinical isolates evaluated. Ceftazidime, levofloxacin, and ertapenem efficacy against isogenic and clinical OXA-48-producing strains correlated well with phenotypic profiles and pharmacodynamic targets, whereas efficacy with doripenem was variable over the MIC range studied. These data suggest that carbapenems may not be a reliable treatment for treating OXA-48 producers and add to previous observations with KPC and NDM-1 suggesting that genotype may better predict activity of the carbapenems than the phenotypic profile.

Comparative evaluation of a novel chromogenic medium (chromID OXA-48) for detection of OXA-48 producing Enterobacteriaceae

Comparative evaluation of the recently developed chromogenic culture medium chromID OXA-48 (bioMérieux) with chromID CARBA (bioMérieux) and SUPERCARBA showed that chromID OXA-48 and SUPERCARBA media have the highest sensitivity for detection of OXA-48 producing Enterobacteriaceae (91% and 93%) comparatively to chromID CARBA (21 %). The chromID OXA-48 has the highest specificity, with 100%, as compared to 53% and 68% for the SUPERCARBA and chromID CARBA media for detecting those OXA-48 producers.

In vitro activity of temocillin against multidrug-resistant clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp., and evaluation of high-level temocillin resistance as a diagnostic marker for OXA-48 carbapenemase

Sir, Temocillin is a narrow-spectrum penicillin active primarily against Enterobacteriaceae and resistant to hydrolysis by penicillinases, extended-spectrum b-lactamases (ESBLs), and AmpC enzymes. It has been shown also to retain some activity in vitro against Enterobacteriaceae with KPC-type carbapenemases, although not against bacteria with OXA-48-like non-metalloenzymes or the metalloenzymes (including IMP, NDM and VIM types). The emergence and spread of bacteria with acquired carbapenemases raises public health concerns, and there is a clear need for reliable diagnostic tests in routine bacteriology laboratories. Simple phenotypic tests with b-lactam/b-lactamase inhibitor combinations can aid the identification of isolates with KPC-type or metallo-carbapenemases, but cannot detect OXA-48 carbapenemases, for which there is currently no good inhibitor. Molecular tests could overcome this shortcoming but are not an option in many laboratories. The Carba NP acido/colorimetric phenotypic test detects carbapenem hydrolysis per se, but its sensitivity is reportedly lower for OXA-48 producers than for isolates with other carbapenemases. High-level temocillin resistance is a characteristic of many OXA-48 producers and has been included in algorithms to aid their recognition. – 8 We investigated the in vitro activity of temocillin against 2280 clinical isolates of Escherichia coli, Klebsiella spp. and Enterobacter spp. submitted to Public Health England’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit for the investigation of unusual resistance between January 2012 and April 2013. They included 1029 producers of KPC, OXA-48 or IMP, NDM and VIM carbapenemases. We also evaluated the diagnostic potential of high-level temocillin resistance (MIC ≥128 mg/L) to predict the production of an OXA-48 carbapenemase. Temocillin (Eumedica, Brussels, Belgium) was tested in the range 1–128 mg/L using agar dilution methodology; MICs were interpreted using BSAC breakpoints (susceptibility: systemic infections, MICs≤8 mg/L; urinary infections, MICs≤32 mg/L). Isolates were also tested against AMRHAI’s standard Gram-negative antibiotic panel, which includes ertapenem, imipenem (tested+EDTA to indicate likely metallo-carbapenemase producers) and meropenem. Carbapenemase production was sought using in-house PCRs and/or a commercial microarray (Check-MDR CT102; Check-Points, Wageningen, The Netherlands). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of temocillin MIC ≥128 mg/L as an indicator of OXA-48 carbapenemase production were determined using an online tool (http://www. medcalc.org/calc/diagnostic_test.php). Temocillin MIC distributions for the 2280 isolates are shown in Table 1. These were submissions to a reference laboratory so were biased towards multiresistance, with proportions of ‘unusual’ isolates far higher than would be observed in most diagnostic laboratories. Allowing for the fact that a larger proportion of Klebsiella spp. isolates produced carbapenemases (760/1133; 67.1%) than did isolates of E. coli (206/701; 29.4%) or Enterobacter spp. (63/446; 14.1%), no major differences were observed in the MIC distributions of temocillin for the three genera. Overall, 920 (40.4%) isolates were susceptible to temocillin at the systemic breakpoint (MICs ≤8 mg/L); those considered resistant included 329/669 (49.2%) KPC producers, 671/1251 (53.6%) isolates without carbapenemases, and all 360 (100%) isolates with other types of carbapenemase (OXA-48, IMP, NDM or VIM). At the higher, urinary breakpoint, 1758 (77.1%) isolates were susceptible to temocillin (MICs ≤32 mg/L), including 628 (93.4%) KPC producers and 1098 (87.8%) isolates without carbapenemases. However, 328 (91.4%) isolates with other carbapenemases (OXA-48, IMP, NDM or VIM) remained resistant with MICs .32 mg/L. Temocillin MICs ≥128 mg/L were observed for 355 (15.6%) isolates overall, most (292; 82.3%) of which had a carbapenemase. However, 63 isolates (17.7%; 30 E. coli, 22 Klebsiella spp. and 11 Enterobacter spp.) lacked these mechanisms and the nature of their high-level temocillin resistance warrants further investigation. Many of them had complex b-lactam antibiograms—for example, 57 were also non-susceptible to ertapenem (MICs .0.5 mg/L)—consistent with ESBL/AmpC production combined with impermeability (data not shown). High-level temocillin resistance (MICs ≥128 mg/L) was recorded for 98/108 (90.7%) OXA-48 producers compared with 257/2172 (11.8%) isolates lacking OXA-48 or with 72/1920 (3.8%) isolates lacking a metallo-carbapenemase. Based on these proportions, the detection of high-level resistance was not a sufficiently robust marker when used as the sole criterion to predict the presence of an OXA-48-like carbapenemase; the PPV of the test was only 27.6% (Table 2). However, if combined with an absence of imipenem/EDTA synergy (i.e. when there was no phenotypic evidence of metallo-carbapenemase production), the PPV of high-level temocillin resistance predicting OXA-48

Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers

To assess the performance of the agar disc diffusion method for the detection of carbapenemase-producing Enterobacteriaceae (CPE) referred to the national reference laboratories (NRLs) in Belgium and France. All Enterobacteriaceae isolates referred to the NRLs for the confirmation of CPE in 2012 were included. The inhibition zone diameters of meropenem, piperacillin/tazobactam and temocillin using CLSI disc diffusion methodology were recorded. Phenotypic and molecular detection of carbapenemases was performed on all isolates. A total of 1354 Enterobacteriaceae isolates, including 435 (32.1%) confirmed CPE isolates [OXA-48 (n = 323), KPC (n = 60), VIM (n = 32) and NDM (n = 20)] and 919 carbapenemase-negative isolates, were tested. Using recommended interpretative criteria, non-susceptibility to meropenem had poor sensitivity (52.0% by CLSI susceptibility breakpoint and 80.0% by EUCAST screening breakpoints), while non-susceptibility to piperacillin/tazobactam (according to CLSI breakpoint) or to temocillin (according to Fuchs, Barry, Thornsberry et al. Eur J Clin Microbiol 1985; 4: 30-3) was highly sensitive (99.8% and 98.2%, respectively) but poorly specific (29.4% and 42.9%, respectively) for the detection of CPE. Temocillin diameters <12 mm alone had high specificity (90.0%) and the combination of temocillin diameters ≥12 mm with piperacillin/tazobactam diameters ≥16 mm observed in 40% of all referred isolates displayed excellent negative predictive value (99.2%). In geographical areas with a high prevalence of OXA-48 producers, recommended meropenem susceptibility or screening breakpoints failed to detect CPE in a large proportion of isolates. The combination of modified zone diameter cut-offs for piperacillin/tazobactam (≥16 mm) and temocillin (≥12 mm) can be used to rule out the presence of carbapenemase and avoid unnecessary additional testing for confirmation of CPE.

Evaluation of the Oxoid Brilliance™ CRE Agar for the detection of carbapenemase-producing Enterobacteriaceae

The adequate detection of carbapenemase-producing Enterobacteriaceae (CPE) is essential for adequate antibiotic therapy and for infection control purposes, especially in an outbreak setting. Selective agars play an important role in the detection of CPE. The Oxoid Brilliance™ CRE Agar (Thermo Fisher Scientific) was evaluated for the detection of CPE using 255 non-repetitive Enterobacteriaceae isolates, including 95 CPE (36 KPC, 4 KPC plus VIM, 4 NDM, 6 GIM, 20 VIM, and 25 OXA-48-producing isolates). The sensitivity of the CRE agar for the detection of CPE was 94 % (89/95), but differed per carbapenemase gene (100 % for KPC, NDM, and GIM, 90 % for VIM, and 84 % for OXA-48-producing isolates). The specificity of the CRE agar was 71 %, due to the growth of AmpC- and/or ESBL-producing isolates. The CRE agar is a sensitive tool for the detection of KPC and metallo-carbapenemase-producing Enterobacteriaceae, although the detection of OXA-48 producers is less optimal. The relatively low specificity requires confirmation of carbapenemase production for isolates recovered from the CRE agar.

Use of Faropenem as an Indicator of Carbapenemase Activity in the Enterobacteriaceae

The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 μg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 μg), temocillin (30 μg), and four carbapenems (each 10 μg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-μg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity, 99%; specificity, 94%). A "double zone" around 10-μg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤ 23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-μg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-μg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 μg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.

Real-time PCR for detection of blaOXA-48 genes from stools

Outbreaks of OXA-48-like carbapenemase producers are increasingly reported in many European countries and are often the result of difficulties in detection, especially for isolates with MICs of carbapenems that remain in the susceptibility range. An in-house real-time quantitative PCR (qPCR) assay using TaqMan chemistry to detect bla(OXA-48-like) genes was compared with bacterial culturing on ChromID ESBL and SUPERCARBA media of spiked stool samples with several species producing OXA-48 variants. qPCR amplification using plasmid DNA was linear over 10 log dilutions (r(2) = 0.998 and slope = -3.14), with an amplification efficiency of 1.10, and the detection limit of the assay was reproducibly estimated at 10 plasmid molecules/PCR. No cross-reaction was detected with DNA extracted from several multidrug-resistant bacteria harbouring other β-lactam resistance genes. The bla(OXA-48) qPCR assay was capable of detecting 10-50 cfu of OXA-48 producers/100 mg of faeces. ChromID ESBL was capable of detecting OXA-48 producers (1 × 10(1) to 3 × 10(2) cfu/100 mg of faeces), as long as the isolates exhibited a high level of resistance to cephalosporins due to an associated extended-spectrum β-lactamase. The SUPERCARBA screening medium was capable of detecting all the OXA-48-like producers (1-3 × 10(1) cfu/100 mg of faeces), except those producing OXA-163, a variant lacking carbapenem-hydrolysing activity. The qPCR is likely to shorten the time for bla(OXA-48) detection from 48 to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

OXA-48-like carbapenemases: the phantom menace

OXA-48-type carbapenem-hydrolysing class D β-lactamases are increasingly reported in enterobacterial species. To date, six OXA-48-like variants have been identified, with OXA-48 being the most widespread. They differ by a few amino acid substitutions or deletions (one to five amino acids). The enzymes hydrolyse penicillins at a high level and carbapenems at a low level, sparing broad-spectrum cephalosporins, and are not susceptible to β-lactamase inhibitors. When combining permeability defects, OXA-48-like producers may exhibit a high level of resistance to carbapenems. OXA-163 is an exception, hydrolysing broad-spectrum cephalosporins but carbapenems at a very low level, and being susceptible to β-lactamase inhibitors. The bla(OXA-48)-type genes are always plasmid-borne and have been identified in association with insertion sequences involved in their acquisition and expression. The current spread of the bla(OXA-48) gene is mostly linked to the dissemination of a single IncL/M-type self-transferable plasmid of 62 kb that does not carry any additional resistance gene. OXA-48-type carbapenemases have been identified mainly from North African countries, the Middle East, Turkey and India, those areas constituting the most important reservoirs; however, occurrence of OXA-48 producers in European countries is now well documented, with some reported hospital outbreaks. Since many OXA-48-like producers do not exhibit resistance to broad-spectrum cephalosporins, or only decreased susceptibility to carbapenems, their recognition and detection can be challenging. Adequate screening and detection methods are therefore required to prevent and control their dissemination.

Carbapenemase-producingAcinetobacterspp. in Cattle, France

To the Editor: Multidrug resistance in bacteria isolated from animals is an emerging phenomenon, mirroring what is happening among humans. During the past decade, expanded-spectrum β-lactamases in Enterobacteriaceae from humans (1) and animals (2) worldwide have been reported. Among humans, as a consequence of this high rate, use of carbepenems is increasing selection pressure; carbapenem-resistant gram-negative organisms are increasingly reported, including carbapenemase-producing Enterobacteriaceae and Acinetobacter spp (3). The most commonly acquired carbapenemases identified in Acinetobacter spp. correspond to carbapenem-hydrolyzing class D β-lactamases (3). In particular, the worldwide spread of OXA-23–producing A. baumannii is considered a serious threat; those strains are frequently involved in nosocomial outbreaks for which therapeutic options are extremely limited (3,4). Our study objective was to evaluate the possible occurrence of carbapenemase-producing gram-negative bacteria in dairy cattle in France. In August 2010, at a dairy farm 30 km from Paris, France, rectal swabs were collected from 50 cows. Samples were precultured in buffered peptone water and incubated for 18 h at 37°C. Cultures were inoculated by streaking 100 μL of the suspensions onto Drigalski agar plates (bioMerieux, Balmes-les-Grottes, France) containing 1 μg/mL of imipenem to select for carbapenem-resistant gram-negative isolates. Of the 50 samples, 9 produced growth on imipenem-containing plates. All colonies tested (10 colonies/sample) by using the API 20 NE (bioMerieux) system were first identified as A. lwoffii. Molecular techniques based on sequencing of the gyrA, gyrB, and rpoB genes (5) enabled more precise identification and indicated that all isolates belonged to the Acinetobacter genomospecies (DNA group) 15TU, which is known to be phylogenetically related to A. lwoffii and which has been reportedly isolated from sewage, freshwater aquaculture habitats, trout intestines, and frozen shrimp (6). One colony per sample was retained for further investigation (isolates BY1 to BY9). Susceptibility testing and MIC determinations were performed by disk-diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France) and Etest (AB bioMerieux, Solna, Sweden) (Table). All isolates except 1 were resistant to penicillins, combinations of penicillins and β-lactamase inhibitors, and carbapenems but susceptible to cefotaxime and of reduced susceptibility to ceftazidime. Isolate BY1 showed higher MICs for carbapenems (Table). In addition, all isolates were resistant to tetracycline, kanamycin, and fosfomycin and remained susceptible to fluoroquinolones, chloramphenicol, gentamicin, amikacin, tobramycin, and sulfonamides. Susceptibility profiles of 3 Acinetobacter genomospecies 15TU reference strains showed that they were fully susceptible to penicillins, carbapenems, tetracycline, and kanamycin. Table Antimicrobial drug MICs for Acinetobacter genomospecies 15TU isolates from cows and reference strains, France, August 2010 Clonal diversity between the isolates was assessed by pulsed-field gel electrophoresis (5), which showed 6 distinct genotypes. Isolate BY1 corresponded to a single clone (data not shown), which indicated that the occurrence of Acinetobacter genomospecies 15TU strains among these animals was not the result of dissemination of a single clone. PCR detection and sequencing of genes that encode carbapenem-hydrolyzing class D β-lactamases (5) showed that the 9 Acinetobacter genomospecies 15TU isolates harbored a blaOXA-23 gene, whereas the 3 reference strains remained negative. Sequencing confirmed that all isolates expressed β-lactamase OXA-23, which is known to be widespread in A. baumannii. Mating-out assays and plasmid electroporation assays were performed by using blaOXA-23–positive Acinetobacter spp. isolates as donors and rifampin-resistant A. baumannii BM4547 isolates as a recipient strain (5); however, these assays were unsuccessful. Plasmid DNA analysis (5) gave uninterpretable results, with DNA degradations. The genetic structures surrounding the blaOXA-23 gene were investigated by PCR mapping (7), which identified transposon Tn2008 in isolate BY2 only. Tn2008 is a major vehicle for the spread of the blaOXA-23 gene in A. baumannii in the People’s Republic of China (8) and the United States (9). In the other isolates, the ISAba1 element of Tn2008 had been truncated by a novel insertion sequence termed ISAcsp2 (www-is.biotoul.fr). The dairy farmer indicated that most animals from which OXA-23 producers had been identified had received antimicrobial drugs in the previous weeks. Although 1 animal had received amoxicillin-clavulanate, most of the others had been given oxytetracycline and neomycin to treat mastitis. β-lactamase OXA-23 is a common source of carbapenem resistance in A. baumannii (5). Infections with multidrug-resistant OXA-23–producing A. baumannii or A. junii have been reported from hospitals but not from the community. Our study showed that OXA-23–producers in particular, and carbapenemase producers in general, may be isolated from animals. Among the hypotheses that could explain the selection of this carbapenemase, use of penicillins or penicillin–β-lactamase inhibitor combinations could create selective pressure for β-lactamases because OXA-23 does confer, in addition to decreased susceptibility to carbapenems, a high level of resistance to those compounds. We have previously shown that A. radioresistens, an environmental species, was the progenitor of the blaOXA-23 gene (10). Studies are needed to determine to what extent and at which locations Acinetobacter genomospecies 15TU and A. radioresistens might co-reside and therefore where the blaOXA-23 gene exchange might have occurred.

Broad-Spectrum β-Lactam Antibiotics for Treating Experimental Peritonitis in Mice Due to Klebsiella pneumoniae Producing the Carbapenemase OXA-48

A lethal peritonitis model was induced in mice with a Klebsiella pneumoniae isolate producing the carbapenemase OXA-48. Administration of a single dose (up to 100 mg/kg) of the antibiotic piperacillin-tazobactam, imipenem-cilastatin, ertapenem, or cefotaxime had little or no impact on lethality. Ceftazidime had the highest efficacy in vivo, which mirrored its in vitro activity; this was not the case for carbapenems. Therefore, ceftazidime may be recommended for the treatment of infections due to OXA-48 producers if they do not coproduce an extended-spectrum β-lactamase or a plasmid-mediated AmpC cephalosporinase.

Evidence of Diversity among Epidemiologically Related Carbapenemase-Producing Acinetobacter baumannii Strains Belonging to International Clonal Lineage II

Carbapenem-resistant Acinetobacter baumannii strains belonging to international clonal lineage II (ICL-II) have become predominant in intensive care units (ICUs) throughout Italy. Between 2005 and 2009, the carbapenem-hydrolyzing class D β-lactamase (CHDL) bla(OXA-23) gene became more prevalent than bla(OXA-58) among epidemic ICL-II strains showing extensive genetic similarity. These findings posed the question of whether CHDL gene replacement occurred in the homogeneous ICL-II population or a new OXA-23 clone(s) emerged and spread in ICUs. In this study, the changes in the ICL-II A. baumannii population and CHDL gene carriage were investigated in 30 genetically related isolates collected during the bla(OXA-58)-to-bla(OXA-23) transition period. Pulsotyping, randomly amplified polymorphic DNA (RAPD) analysis, and multilocus sequence typing (MLST) results were combined with multilocus variable-number tandem-repeat (VNTR) analysis (MLVA-8), siderotyping, and plasmid profiling to improve genotype-based discrimination between isolates. Pulsotyping, RAPD analysis, and MLST clustered isolates into a single type. MLVA-8 identified 19 types that clustered into three complexes. All OXA-23-producing isolates formed a single complex, while OXA-58 producers were split into two complexes. Southern blot analysis of the physical localization and genetic context of the CHDL genes showed that bla(OXA-58) was invariably located on plasmids, while bla(OXA-23) was present within Tn2006 on the chromosome or both the chromosome and plasmids. These data indicate that the apparently homogeneous population of CHDL-producing ICL-II strains was composed of several independent strains and that, between 2005 and 2009, distinct OXA-23 producers displaced the preexisting OXA-58 producers. Thus, MLVA-8 appears to be a suitable tool not only for investigating A. baumannii population structure but also for high-resolution epidemiological typing.

Genetic Features of the Widespread Plasmid Coding for the Carbapenemase OXA-48

Complete sequencing of plasmid pOXA-48a carrying the bla(OXA-48) gene from a Klebsiella pneumoniae isolate was performed. Its backbone corresponded to that of an IncL/M-type plasmid, in which the bla(OXA-48) gene had been integrated through the acquisition of the Tn1999 composite transposon without any other antibiotic resistance gene. Molecular epidemiology using a collection of international OXA-48 producers revealed the wide diffusion of pOXA-48a or closely related plasmids.

Coexistence of blaOXA48andaac(6′)-Ib-crGenes inKlebsiella PneumoniaeIsolates from Istanbul, Turkey

This study evaluated the presence of carbapenem hydrolysing β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) determinants in 22 Klebsiella pneumoniae isolates collected from the Istanbul Medical Faculty, Turkey, which reduced the susceptibility or resistance to carbapenem. The VITEK(®) 2 system and E-tests were used to determine the minimum inhibitory concentrations needed to inhibit bacterial growth. Genes were screened by polymerase chain reaction, and gene transferability was evaluated by transconjugation. Strain clonality was investigated by pulsed-field gel electrophoresis (PFGE). All strains were OXA-48 β-lactamase producers and three (13.6%) were also positive for the aac(6')-Ib-cr gene. Most of the strains harboured other β-lactamase (bla) genes such as bla(TEM), bla(SHV), bla(CTX-M) and bla(VEB-1). The transconjugants mostly harboured bla(OXA-48) and other β-lactamases separately. PFGE revealed eight pulsotypes among the isolates. The coexistence of bla(OXA-48) and PMQR in K. pneumoniae isolates may present a significant threat to health, especially in the nosocomial setting.

Occurrence of the Carbapenem-Hydrolyzing β-Lactamase Gene bla OXA-48 in the Environment in Morocco

The bla OXA-48 gene is plasmid borne and encodes a carbapenem-hydrolyzing class D β-lactamase that was first identified in a Klebsiella pneumoniae clinical isolate in Turkey in 2004 ([11][1]). The high prevalence of OXA-48 producers in Turkey is well established ([3][2]), but there are also

A sensitive and specific phenotypic assay for detection of metallo-β-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin

AbstractEnterobacteriaceae producing carbapenemases, such as KPC or metallo-β-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 μg of EDTA, 1000 μg of dipicolinic acid (DPA), 600 μg of aminophenylboronic acid (APBA), or 750 μg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum β-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added β-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Comparison of BD Phoenix, Vitek 2, and MicroScan Automated Systems for Detection and Inference of Mechanisms Responsible for Carbapenem Resistance in Enterobacteriaceae

We assessed the ability of three commercial systems to infer carbapenem resistance mechanisms in 39 carbapenemase-producing and 16 other carbapenem-resistant Enterobacteriaceae. The sensitivity/specificity values for "flagging" a likely carbapenemase were 100%/0% (BD Phoenix), 82 to 85%/6 to 19% (MicroScan), and 74%/38% (Vitek 2), respectively. OXA-48 producers were poorly detected, but all systems reliably detected isolates with KPC and most with metallo-carbapenemases.

Use of ChromID Extended-Spectrum β-Lactamase Medium for Detecting Carbapenemase-Producing Enterobacteriaceae

ChromID extended-spectrum beta-lactamase (ESBL) culture medium is routinely used for screening ESBL producers. This medium was tested for detecting carbapenemase-producing Enterobacteriaceae isolates from a collection of reference strains and compared to the CHROMagar KPC culture medium previously evaluated for detecting KPC-producing isolates. Producers of IMP-, VIM-, and KPC-type carbapenemases with high levels of resistance to cephalosporins and to carbapenems were detected at 1x10(1) CFU/ml. The OXA-48 producers were not detected on ChromID ESBL medium unless coexpressing ESBLs, whereas carbapenemase-producing isolates with MICs of <4 microg/ml were not detected on CHROMagar KPC medium.

Temporal evolution of carbapenem-resistant Acinetobacter baumannii in Curitiba, southern Brazil

In the last few years, carbapenem-resistant Acinetobacter baumannii isolates (CR-AB) have been identified worldwide. The first description of OXA-23-producing A baumannii in Brazil was from the city of Curitiba in 2003. The aim of the present study was to evaluate the persistence and dissemination of the first OXA-23-producing A baumannii clone isolated from patients in Hospital de Clinicas, Curitiba, Brazil. An antimicrobial susceptibility profile of the isolates was determined by the standard agar dilution method. Molecular detection of beta-lactamase genes was done by polymerase chain reaction. The clonal relationship of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Epidemiologic and clinical features were evaluated as well. Genotypic analysis of 172 CR-AB isolates by PFGE identified 3 distinct major PFGE clusters (A, B, and C, accounting for 36, 69, and 65 isolates, respectively). All isolates carried the bla(OXA-23)-like gene and were multidrug-resistant, but were susceptible to tigecycline and polymixin B. The mortality rate related to CR-AB infection was 45.4%, and ventilator-associated pneumonia and bloodstream infections were the most frequent clinical manifestations. The presence of 3 clones among the CR-AB isolates suggests that cross-transmission was the main mechanism responsible for dissemination of OXA-23 producers. PFGE pattern A was genotypically similar to that of the first OXA-23-producing A baumannii clone identified in Curitiba in 1999. This clone persisted in the same hospital until April 2004. The presence of the bla(OXA-)23-like gene was the main mechanism associated with carbapenem resistance among the isolates studied.