Danièle Meunier

Evaluation of three commercial assays for rapid detection of genes encoding clinically relevant carbapenemases in cultured bacteria

OBJECTIVES To assess the performance of three commercial molecular assays for detecting major families of carbapenemases in pure bacterial isolates. METHODS A panel of 450 isolates with previously defined carbapenem resistance mechanisms was tested using the Check-Direct CPE kit, the eazyplex(®) SuperBug complete A kit and the Xpert(®) Carba-R kit. Isolates included 438 Enterobacteriaceae and 12 Pseudomonas spp. comprising 100 isolates each with KPC, NDM, VIM or OXA-48-like enzymes, two isolates producing both an NDM and an OXA-48-like enzyme, 24 IMP producers and 24 isolates without a known carbapenemase gene. Discordant results (commercial versus in-house) were investigated using in-house PCR and amplicons were sequenced to define the carbapenemase allele present. RESULTS All three commercial assays detected all isolates with KPC, VIM, NDM and classic OXA-48 carbapenemases (no false-negatives). Isolates producing the OXA-181 variant (n = 18) were not detected by the Xpert(®) Carba-R kit or the eazyplex(®) SuperBug complete A kit, but were subsequently detected with modified versions of these kits. Only the Xpert(®) Carba-R kit could detect IMP carbapenemases, although this was limited to the IMP-1 subgroup. Invalid or false-positive results were either not observed when following the manufacturers protocols or were eliminated by making simple interpretative adjustments to allow use with bacterial isolates rather than clinical samples. CONCLUSIONS Commercial assays offer a reliable means of detecting bacteria with clinically significant carbapenemases. Coverage of some assays required expansion to maximize the sensitivity for OXA-48-like carbapenemases. Choice will ultimately depend on preferred gene coverage, intended throughput, cost and ability to fit into local workflows.

Prevalence of Fecal Carriage of Acquired Expanded-Spectrum Cephalosporin Resistance in Enterobacteriaceae Strains from Cattle in France

In gram-negative pathogens, extended-spectrum beta-lactamases (ESBLs) confer resistance to penicillins, cephalosporins (including extended-spectrum cephalosporins), and aztreonam, but not to cephamycins and carbapenems, and are inhibited by beta-lactamase inhibitors ([8][1]). Recently, cefotaximases

Antibiotic marker modifications of λ Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains

The Red recombinase system of bacteriophage Lambda has been used to inactivate chromosomal genes in bacteria using PCR products. In this study, we describe the replacement of the ampicillin resistance marker of helper plasmids pKD46 and pCP20 by a gentamicin resistance gene to disrupt chromosomal genes and then to eliminate FRT flanked resistance gene in multiple antibiotic-resistant Salmonella enterica strains.

NDM carbapenemases in the United Kingdom: an analysis of the first 250 cases

OBJECTIVES Gram-negative bacteria with diverse carbapenemases, including New Delhi metallo-β-lactamase (NDM) enzymes, have been increasingly recorded in the UK since 2007. We analysed patient data for NDM-positive isolates confirmed by the national reference laboratory from UK laboratories from February 2008 to July 2013. METHODS Isolates resistant to carbapenems and with imipenem MICs reduced ≥8-fold by EDTA were tested by PCR for genes encoding acquired class B carbapenemases. MICs were determined by BSAC agar dilution methodology. When requested by the sender, or when they were members of apparent clusters, NDM-positive isolates were typed by variable number tandem repeat (VNTR) analysis or PFGE. Data provided by the sending laboratories were collated and reviewed. RESULTS From February 2008 to July 2013 the reference laboratory confirmed 326 NDM-positive isolates from 250 patients, submitted by 83 laboratories. Most (85%, 213/250) patients were already hospitalized when the NDM-positive bacteria were detected, were male (61%, 152/250) and were aged >60 years (58%, 145/250). Travel history was available for only 40% of patients, but 52% (53/101) of these had documented healthcare contact within or travel to the Indian subcontinent. Most NDM-positive isolates (94%, 306/326) were Enterobacteriaceae with just 6% (20/326) non-fermenters; the predominant hosts were Klebsiella spp. (55%, 180/326) and Escherichia coli (25%, 80/326). Almost all NDM-positive isolates were resistant to multiple antibiotic classes, but 90% remained susceptible to colistin. CONCLUSIONS Gram-negative bacteria with NDM carbapenemases are a growing challenge, especially for elderly hospitalized patients, including those with healthcare contact in the Indian subcontinent, and leave few therapeutic options. UK outbreaks remain rare and contained.

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

Prevalence and Antimicrobial Resistance of Campylobacter jejuni and Campylobacter coli Isolated from Cattle between 2002 and 2006 in France

Feces from 2,255 cattle (calves, young beef cattle, and culled cows) were collected at slaughter from nine departments across France. Campylobacter was recovered from 16.5% of the 2,255 samples (C. jejuni from 12.8% and C. coli from 3.7%), predominantly from calves. Antimicrobial resistance to six antibiotics of medical and/or veterinary interest was tested with the E-test. Resistance to tetracycline was found in most isolates (52.8% of C. jejuni isolates and 88.1% of C. coli isolates) in contrast to low but consistent resistance to ampicillin and erythromycin. Only two C. coli isolates were resistant to gentamicin. Multiple resistance was frequently detected in C. jejuni and C. coli isolates, and 0.8% (3 of 372) of the isolates were resistant to five of the six antimicrobials. An upward trend in the resistance to quinolones and fluoroquinolones in C. jejuni from calves was found; resistance to nalidixic acid reached 70.4% in 2006 and fluoroquinolone resistance increased from 29.7 to 70.4% during 2002 through 2006. All data were analyzed in parallel using clinical breakpoints or epidemiological cutoff values, and the results overlapped largely, except those for gentamicin. This 5-year survey (2002 through 2006) gives the first overview of the prevalence and antimicrobial resistance of C. jejuni and C. coli in cattle in France and documents to what extent cattle may contribute to the environmental reservoir of Campylobacter in France in the context of recurrent reports on links between human campylobacterioses and livestock. The results underline a notable increase in the resistance to fluoroquinolones in C. jejuni from cattle that may be of significant importance for public health.

Persistence of Klebsiella pneumoniae clones with OXA-48 or NDM carbapenemases causing bacteraemias in a Riyadh hospital.

We characterized nine carbapenem-resistant Klebsiella pneumoniae collected over 9 months during 2011 in Riyadh; 8 from Hospital A and 1 from Hospital B. Variable number tandem repeat (VNTR) defined three strains at Hospital A, each with 2 or 3 representatives recovered from separate patients over periods of 6-24 weeks; 2 strains had OXA-48 and 1 had NDM. The single isolate from Hospital B also had OXA-48 but was distinct by VNTR from the Hospital A strains. Two strains with OXA-48 were colistin resistant; the third included a colistin-resistant representative from a colistin-treated patient.

Activity of ceftolozane/tazobactam against surveillance and ‘problem’ Enterobacteriaceae, Pseudomonas aeruginosa and non-fermenters from the British Isles

Abstract Background: We assessed the activity of ceftolozane/tazobactam against consecutive isolates collected in the BSAC Bacteraemia Surveillance from 2011 to 2015 and against ‘problem’ isolates sent to the UK national reference laboratory from July 2015, when routine testing began. Methods: Susceptibility testing was by BSAC agar dilution with resistance mechanisms identified by PCR and interpretive reading. Results: Data were reviewed for 6080 BSAC surveillance isolates and 5473 referred organisms. Ceftolozane/tazobactam had good activity against unselected ESBL producers in the BSAC series, but activity was reduced against ertapenem-resistant ESBL producers, which were numerous among reference submissions. AmpC-derepressed Enterobacter spp. were widely resistant, but Escherichia coli with raised chromosomal AmpC frequently remained susceptible, as did Klebsiella pneumoniae with acquired DHA-1-type AmpC. Carbapenemase-producing Enterobacteriaceae were mostly resistant, except for ceftazidime-susceptible isolates with OXA-48-like enzymes. Ceftolozane/tazobactam was active against 99.8% of the BSAC Pseudomonas aeruginosa isolates; against referred P. aeruginosa it was active against 99.7% with moderately raised efflux, 94.7% with strongly raised efflux and 96.6% with derepressed AmpC. Resistance in P. aeruginosa was largely confined to isolates with metallo-β-lactamases (MBLs) or ESBLs. MICs for referred Burkholderia spp. and Stenotrophomonas maltophilia were 2–4-fold lower than those of ceftazidime. Conclusions: Ceftolozane/tazobactam is active against ESBL-producing Enterobacteriaceae; gains against other problem Enterobacteriaceae groups were limited. Against P. aeruginosa it overcame the two most prevalent mechanisms (up-regulated efflux and derepressed AmpC) and was active against 51.9% of isolates non-susceptible to all other β-lactams, rising to 80.9% if ESBL and MBL producers were excluded.

Resistance to third-generation cephalosporins in human non-typhoidal Salmonella enterica isolates from England and Wales, 2010–12

OBJECTIVES To identify the mechanism(s) underlying cefotaxime resistance in 118 of 21,641 (0.55%) non-typhoidal Salmonella enterica isolates collected from humans throughout England and Wales from January 2010 to September 2012. METHODS Non-duplicate isolates (n = 118) resistant to cefotaxime (MICs >1 mg/L) were screened by PCR for genes encoding CTX-M extended-spectrum β-lactamases (ESBLs) and associated ISEcp1-like elements, and for genes encoding acquired AmpC, SHV, TEM, VEB, PER and GES β-lactamases. Sequencing was used to identify specific alleles in selected isolates. Carbapenem resistance was sought by ertapenem disc screening. RESULTS Seventy-nine isolates (0.37% of all referred S. enterica) produced ESBLs, 37 isolates (0.17%) produced CMY-type AmpC enzymes, and 1 isolate had both enzyme types; the mechanism of cefotaxime resistance in 3 isolates could not be identified. Group 1 CTX-M genes were identified in 57 isolates belonging to 22 serotypes, with CTX-M-1 (n = 11), -15 (n = 9) and -55/57 (n = 8) the most prevalent alleles among the 29 (51%) investigated. CTX-M-2 (n = 5), -14 (n = 5), -8 (n = 1) and -65 (n = 1) were also identified. TEM-52 was identified in two isolates and SHV-12 in seven isolates. There was no evidence of carbapenem resistance. ESBL and AmpC genes were detected in both domestically acquired and travel-associated salmonellae. Eighty-nine isolates (75%) were multidrug resistant (resistant to at least three antimicrobial classes) and 42 (36%) had decreased susceptibility to ciprofloxacin (MICs 0.25-1 mg/L), with a further 13 (11%) isolates resistant (MICs >1 mg/L). CONCLUSIONS The prevalence of CTX-M and acquired AmpC genes in human non-typhoidal S. enterica from England and Wales is still low, but has increased from 0.03% in 2001-03 to 0.49% in 2010-12. Resistance to third-generation cephalosporins requires monitoring as it may reduce therapeutic options.

Plasmid-borne florfenicol and ceftiofur resistance encoded by the floR and blaCMY-2 genes in Escherichia coli isolates from diseased cattle in France

This study was designed to determine the genetic basis of florfenicol and ceftiofur resistance in Escherichia coli isolates recovered from French cattle. In these isolates, ceftiofur resistance was conferred by bla(CMY-2) located on three distinct conjugative plasmids on a specific DNA fragment, ISEcp1-bla(CMY-2)-blc- sugE. Two of the plasmids also carried the floR gene conferring resistance to florfenicol. The floR gene was shown to be associated with the insertion sequence ISCR2. Mobile elements appear to contribute to the mobilization of floR and bla(CMY-2) genes in E. coli. The presence of bla(CMY-2) and floR on the same plasmid highlights the potential risk for a co-selection of the bla(CMY-2) gene through the use of florfenicol in food animal production.