To assess the performance of three commercial molecular assays for detecting major families of carbapenemases in pure bacterial isolates.
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.
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 manufacturer's protocols or were eliminated by making simple interpretative adjustments to allow use with bacterial isolates rather than clinical samples.
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.
•Design of a multiplex PCR detecting genes rmtE- rmtH, npmA and subvariants.•94.5% (762/806) pan-aminoglycoside resistant Enterobacteriaceae had 16S RMTases.•In the UK 16S RMTases are associated with ...carbapenemases (93.4%, 712/762 isolates).
The emergence of 16S rRNA methyltransferases (16S RMTases) worldwide is a growing concern due to their ability to confer high-level resistance (minimum inhibitory concentrations (MICs) >256 mg/L) to all clinically relevant aminoglycosides. As the occurrence of 16S RMTases in the United Kingdom has not been investigated to date, we screened 806 Enterobacteriaceae isolates displaying high-level aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) for 16S RMTases either by analysing whole-genome sequence (WGS) data (which were available for 449 isolates) or by polymerase chain reaction. A total of 94.5% (762/806) pan-aminoglycoside-resistant Enterobacteriaceae were positive for one or more 16S RMTase genes; armA was the most common (340, 44.6%) followed by rmtC (146, 19.2%), rmtF (137, 18.0%), rmtB (87, 11.4%) and various two-gene combinations (52, 6.8%). Most (93.4%; 712/762) 16S RMTase producers also carried acquired carbapenemase genes, with blaNDM the most common (592/712; 83.1%). Additionally, high-risk bacterial clones associated with blaNDM were identified in the subset of isolates with WGS data. These included Escherichia coli sequence types (STs) 405 (21.8%, 19/87), 167 (20.7%, 18/87) 410 (12.6%, 11/87) and K. pneumoniae STs 14 (35.6%, 112/315), 231 (15.6%, 49/315) and 147 (10.5%, 33/315). These accounted for 4.2% (15/358), 5.0% (18/358), 3.1% (11/358), 28.2% (101/358), 3.1% (11/358) and 7.0% (25/358) blaNDM-producing isolates, respectively. This study shows that 16S RMTases occur in the UK and Ireland and carbapenemases are particularly prevalent in 16S RMTase-producing Enterobacteriaceae. This association poses a risk to the treatment of multidrug-resistant Gram-negative infections in the clinical setting.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The epidemiological importance of tracing plasmids conferring drug resistance prompted us to develop a PCR method based on replicons (inc/rep PCR) of the major plasmid incompatibility groups among
...Enterobacteriaceae. Eighteen pairs of primers were designed to perform 5 multiplex- and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. The specificity of the method was tested on a collection of 61 reference plasmids and on 20
Salmonella enterica strains of different serotypes isolated in Italy. Results indicated that the inc/rep PCR method demonstrates high specificity and sensitivity in detecting replicons on reference plasmids and also revealed the presence of recurrent and common plasmids in epidemiologically unrelated
Salmonella isolates of different serotypes. These results suggest that the method is potentially applicable to a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. Here we present associated patient data and molecular, epidemiological and phenotypic characteristics of ...all CPE isolates in Norway from 2007 to 2014 confirmed at the Norwegian National Advisory Unit on Detection of Antimicrobial Resistance. All confirmed CPE isolates were characterized pheno- and genotypically, including by whole genome sequencing (WGS). Patient data were reviewed retrospectively. In total 59 CPE isolates were identified from 53 patients. Urine was the dominant clinical sample source (37%) and only 15% of the isolates were obtained from faecal screening. The majority of cases (62%) were directly associated with travel or hospitalization abroad, but both intra-hospital transmission and one inter-hospital outbreak were observed. The number of CPE cases/year was low (2-14 cases/year), but an increasing trend was observed. Klebsiella spp. (n = 38) and E. coli (n = 14) were the dominant species and blaKPC (n = 20), blaNDM (n = 19), blaOXA-48-like (n = 12) and blaVIM (n = 7) were the dominant carbapenemase gene families. The CPE isolates were genetically diverse except for K. pneumoniae where clonal group 258 associated with blaKPC dominated. All isolates were multidrug-resistant and a significant proportion (21%) were resistant to colistin. Interestingly, all blaOXA-48-like, and a large proportion of blaNDM-positive Klebsiella spp. (89%) and E. coli (83%) isolates were susceptible in vitro to mecillinam. Thus, mecillinam could have a role in the treatment of uncomplicated urinary tract infections caused by OXA-48- or NDM-producing E. coli or K. pneumoniae. In conclusion, the impact of CPE in Norway is still limited and mainly associated with travel abroad, reflected in the diversity of clones and carbapenemase genes.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In response to the first report of transmissible colistin resistance mediated by the mcr-1 gene in Escherichia coli and Klebsiella spp. from animals and humans in China, we sought to determine its ...presence in Enterobacteriaceae isolated in the UK.
The PHE archive of whole-genome sequences of isolates from surveillance collections, submissions to reference services and research projects was retrospectively analysed for the presence of mcr-1 using Genefinder. The genetic environment of the gene was also analysed.
Rapid screening of the genomes of ∼24 000 Salmonella enterica, E. coli, Klebsiella spp., Enterobacter spp., Campylobacter spp. and Shigella spp. isolated from food or humans identified 15 mcr-1-positive isolates. These comprised: 10 human S. enterica isolates submitted between 2012 and 2015 (8 Salmonella Typhimurium, 1 Salmonella Paratyphi B var Java and 1 Salmonella Virchow) from 10 patients; 3 isolates of E. coli from 2 patients; and 2 isolates of Salmonella Paratyphi B var Java from poultry meat imported from the EU. The mcr-1 gene was located on diverse plasmids belonging to the IncHI2, IncI2 and IncX4 replicon types and its association with ISApl1 varied. Six mcr-1-positive S. enterica isolates were from patients who had recently travelled to Asia.
Analysis of WGS data allowed rapid confirmation of the presence of the plasmid-mediated colistin resistance gene mcr-1 in diverse genetic environments and plasmids. It has been present in E. coli and Salmonella spp. harboured by humans in England and Wales since at least 2012.
Surveillance of antimicrobial resistance (AMR) in non-typhoidal
(NTS), is essential for monitoring transmission of resistance from the food chain to humans, and for establishing effective treatment ...protocols. We evaluated the prediction of phenotypic resistance in NTS from genotypic profiles derived from whole genome sequencing (WGS). Genes and chromosomal mutations responsible for phenotypic resistance were sought in WGS data from 3,491 NTS isolates received by Public Health England's Gastrointestinal Bacteria Reference Unit between April 2014 and March 2015. Inferred genotypic AMR profiles were compared with phenotypic susceptibilities determined for fifteen antimicrobials using EUCAST guidelines. Discrepancies between phenotypic and genotypic profiles for one or more antimicrobials were detected for 76 isolates (2.18%) although only 88/52,365 (0.17%) isolate/antimicrobial combinations were discordant. Of the discrepant results, the largest number were associated with streptomycin (67.05%,
= 59). Pan-susceptibility was observed in 2,190 isolates (62.73%). Overall, resistance to tetracyclines was most common (26.27% of isolates,
= 917) followed by sulphonamides (23.72%,
= 828) and ampicillin (21.43%,
= 748). Multidrug resistance (MDR), i.e., resistance to three or more antimicrobial classes, was detected in 848 isolates (24.29%) with resistance to ampicillin, streptomycin, sulphonamides and tetracyclines being the most common MDR profile (
= 231; 27.24%). For isolates with this profile, all but one were
. Typhimurium and 94.81% (
= 219) had the resistance determinants
and
(A). Extended-spectrum β-lactamase genes were identified in 41 isolates (1.17%) and multiple mutations in chromosomal genes associated with ciprofloxacin resistance in 82 isolates (2.35%). This study showed that WGS is suitable as a rapid means of determining AMR patterns of NTS for public health surveillance.
Abstract
Carbapenem resistance in Gram-negative bacteria is a public health concern. Consequently, numerous government and agency reports discuss carbapenem-resistant Enterobacterales (CRE) and ...carbapenem-resistant organisms (CROs). Unfortunately, these terms are fuzzy. Do they include (1) Proteeae with inherent imipenem resistance; (2) porin-deficient Enterobacterales resistant to ertapenem but not other carbapenems; (3) Enterobacterales with OXA-48–like enzymes that remain “carbapenem susceptible” at breakpoint; and (4) Pseudomonas aeruginosa that merely lack porin OprD? Counting CPE or CPOs is better but still insufficient, because different carbapenemases have differing treatment implications, particularly for new β-lactam/β-lactamase inhibitor combinations. At the least, it is essential for authors, journals, and regulatory agencies to specify the carbapenemases meant. The future may demand even greater precision, for mutations can alter hydrolytic activity, and the ability to confer resistance, within carbapenemase families.
The term “carbapenem-resistant Enterobacterales” has become inadequate with the advent of new therapies. These make it essential for authors and licensing agencies to specify the particular carbapenemase(s). The future may demand greater precision as mutations modulate activity within carbapenemase families.
Extended-spectrum β-lactamase-producing Escherichia coli isolates (ESBL-E coli) cause more than 5000 cases of bacteraemias annually in the UK. The contribution of the food chain to these infections ...is debated. We aimed to identify the most important reservoirs of ESBL-E coli that colonise and infect humans to identify strategic intervention points.
Sampling for ESBL-E coli was done between Aug 1, 2013, and Dec 15, 2014. We used selective media to seek ESBL-E coli in routinely submitted samples from human faeces, and prospectively collected samples from sewage, farm slurry, and retail foodstuffs in London, East Anglia, northwest England, Scotland, and Wales. We sequenced recovered isolates and compared these isolates with 293 bloodstream and 83 veterinary surveillance ESBL-E coli isolates from the same regions.
2157 (11%) of 20 243 human faeces samples contained ESBL-E coli, including 678 (17%) of 3995 in London. ESBL-E coli also were frequent in sewage and retail chicken (104 65% of 159 meat samples), but were rare in other meats and absent from plant-based foods (0 of 400 fruit and vegetable samples). Sequence type (ST) 131 dominated among ESBL-E coli from human blood (188 64% of 293 isolates), faeces (128 36% of 360), and sewage (14 22% of 65) with STs 38 and 648 also widespread; CTX-M-15 was the predominant ESBL in these lineages (319 77% of 416). By contrast, STs 602, 23, and 117—mostly with CTX-M-1 ESBL—dominated among food and veterinary isolates (68 31% of 218), with only two ST131 organisms recovered. ST10 occurred in both animals and humans, being frequent in surveillance bovines (11 22% of 51 cattle) and representing 15 (4%) of 360 human faecal isolates (but only three 1% of 293 from bacteraemias); however, both human and animal ST10 isolates were diverse in serotype.
Most human bacteraemias with ESBL-E coli in the UK involve internationally prevalent human-associated STs, particularly ST131; non-human reservoirs made little contribution to invasive human disease. Any interventions that seek to target food or livestock can affect the numbers of human infections caused by ESBL-E coli; prevention of the spread of resistant lineages among humans is more vital.
NIHR Policy Research.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background
ESBL-producing Escherichia coli have expanded globally since the turn of the century and present a major public health issue. Their in vitro susceptibility to penicillin/inhibitor ...combinations is variable, and clinical use of these combinations against ESBL producers remains controversial. We hypothesized that this variability related to co-production of OXA-1 penicillinase.
Methods
During a national study we collected 293 ESBL-producing E. coli from bacteraemias, determined MICs by BSAC agar dilution, and undertook genomic sequencing with Illumina methodology.
Results
The collection was dominated by ST131 (n = 188 isolates, 64.2%) and blaCTX-M-15 (present in 229 isolates, 78.2%); over half the isolates (159/293, 54.3%) were ST131 with blaCTX-M-15. blaOXA-1 was found in 149 ESBL producers (50.9%) and blaTEM-1/191 in 137 (46.8%). Irrespective of whether all isolates were considered, or ST131 alone, there were strong associations (P < 0.001) between co-carriage of blaOXA-1 and reduced susceptibility to penicillin/inhibitor combinations, whereas there was no significant association with co-carriage of blaTEM-1/191. For piperacillin/tazobactam the modal MIC rose from 2 mg/L in the absence of blaOXA-1 to 8 or 16 mg/L in its presence; for co-amoxiclav the shift was smaller, from 4 or 8 to 16 mg/L, but crossed the breakpoint. blaOXA-1 was strongly associated with co-carriage also of aac(6′)-Ib-cr, which compromises amikacin and tobramycin.
Conclusions
Co-carriage of OXA-1, a penicillinase with weak affinity for inhibitors, is a major correlate of resistance to piperacillin/tazobactam and co-amoxiclav in E. coli and is commonly associated with co-carriage of aac(6′)-Ib-cr, which narrows aminoglycoside options.
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