Jacqueline Findlay

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.

Multicentre evaluation of a real-time PCR assay to detect genes encoding clinically relevant carbapenemases in cultured bacteria

The performance and portability of a multiplex real-time PCR assay to detect KPC, NDM, OXA-48-like and VIM carbapenemase gene families from bacterial isolates was assessed using Rotor-Gene Q and ABI 7500 instruments. Gram-negative bacterial isolates (n=502) were comprised of 100 isolates each with KPC, NDM, VIM or OXA-48-like carbapenemases (including 17 with OXA-181) and 2 isolates with NDM+OXA-48-like enzymes (including 1 with OXA-181) as well as 100 assay-negative isolates comprised of 24 IMP-producers, 24 carbapenem-resistant isolates with no known carbapenemase gene and 52 extended-spectrum β-lactamase-producing carbapenem-susceptible isolates. A multicentre evaluation was carried out in five laboratories using a subset of 100 isolates comprised of 22 isolates each with KPC, NDM, OXA-48-like or VIM alleles and 12 isolates that were negative for the assay targets. Initial validation of the assay on both the Rotor-Gene Q and ABI 7500 instruments demonstrated 100% sensitivity amongst the 402 isolates that were positive for KPC, NDM, OXA-48-like (including OXA-181) and VIM carbapenemase genes, whilst the 100 assay-negative samples were correctly identified indicating 100% specificity. During the multicentre evaluation the same 100% level of sensitivity and specificity was observed in each of the five centres that participated. Neither invalid nor false-positive results were observed. In conclusion, the assay offers a portable and reliable option for the detection of bacteria carrying clinically significant carbapenemases encoded by KPC, NDM, VIM and OXA-48-like carbapenemase genes using either of the two most common real-time PCR instrument platforms.

KPC enzymes in the UK: an analysis of the first 160 cases outside the North-West region

OBJECTIVES Klebsiella pneumoniae carbapenemases (KPCs) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for KPC-positive bacteria confirmed by the national reference laboratory from UK laboratories from August 2003 to August 2014, excluding North-West England, where the epidemiology has previously been studied. METHODS MICs were determined by BSAC agar dilution. Carbapenem-resistant isolates lacking imipenem/EDTA synergy were tested by PCR for blaKPC. MLST and blaKPC sequencing were performed on a subset of isolates. Plasmid analysis was performed by transformation, PCR-based replicon typing and, in some cases, whole-plasmid sequencing. Patient data provided by the sending laboratories were reviewed. RESULTS Two hundred and ten isolates with KPC enzymes were submitted from 71 UK laboratories outside North-West England, representing 160 patients. All were Enterobacteriaceae, predominantly K. pneumoniae (82%; 173/210), and most (91%; 191/210) were from hospitalized patients. Analysis of 100 isolates identified blaKPC-2 (62%), blaKPC-3 (30%) and blaKPC-4 (8%). Clonal group (CG) 258 was dominant among K. pneumoniae (64%; 54/84), but 21 unrelated STs were also identified. Plasmid analysis identified a diverse range of plasmids representing >11 different replicon types and found in multiple STs and species. Most (34/35) plasmids with IncFIB/FIIK replicons exhibited >99% sequence identity to pKpQIL. CONCLUSIONS KPC enzymes are increasingly detected in Enterobacteriaceae in the UK, albeit without the major outbreaks seen in North-West England. K. pneumoniae CG258 are the dominant hosts, but plasmid spread plays a major role in KPC dissemination between other K. pneumoniae STs and enterobacterial species.

SPM-1 metallo-β-lactamase-producing Pseudomonas aeruginosa ST277 in the UK.

Metallo-b-lactamase (MBL)-producing Pseudomonas aeruginosa are increasing in incidence in the UK, largely due to the emergence of several internationally recognized ‘high-risk’ clones (Wright et al., 2015). These predominantly produce VIM carbapenemases, but isolates with IMP and NDM MBLs have also been identified, as have occasional isolates with other, rarer, MBLs such as DIM-1 [Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, unpublished data].

OXA-48-like carbapenemases in the UK: an analysis of isolates and cases from 2007 to 2014

Objectives OXA-48-like carbapenemases have spread worldwide since 2001. We analysed patient and microbiological data for UK isolates with these enzymes as confirmed by the national reference laboratory from November 2007 to December 2014. Methods MICs were determined using BSAC agar dilution. Isolates with reduced susceptibility or resistance to at least one carbapenem and high-level resistance to both piperacillin/tazobactam (MICs ≥64 mg/L) and temocillin (MICs ≥128 mg/L) were screened by PCR for bla OXA-48-like genes. The genomes of about half of the isolates were sequenced, with MLST types, resistance genes and plasmid replicon types inferred. Patient data provided by sending laboratories were reviewed. Results Isolates ( n  =   741) with OXA-48-like carbapenemases were submitted from 111 UK laboratories, representing 536 patients. Almost all (99%; 736 of 741) were Enterobacteriaceae, predominantly Klebsiella pneumoniae (55%; 408), and most (80%; 595) were from inpatients. WGS of 351 non-duplicate isolates identified bla OXA-48 as the most common variant, found in two-thirds (235 of 351) of isolates, followed by bla OXA-181 (68), bla OXA-232 (32), bla OXA-244 (10), bla OXA-484 (5) and bla OXA-245 (1). Among K. pneumoniae (163 of 351), Escherichia coli (114 of 351) and Enterobacter cloacae (42 of 351), 119 STs were identified. Mapping analyses revealed that 63% (222 of 351) of isolates harboured plasmids that shared >99% identity to one of four known plasmids [pOXA-48a (44%; 154 of 351), pOXA-232 (10%; 34 of 351), pOXA181 (9%; 30 of 351) and pKP3-A (1%; 4 of 351)]; the remaining 37% of isolates harboured bla OXA-48-like in unknown environments. Conclusions OXA-48-like carbapenemases are an increasing problem in the UK. This study highlights both the role of successful plasmids and the polyclonal nature of their dissemination.

Carbapenem resistance mediated by blaOXA-181 in Pseudomonas aeruginosa

Sir, The emergence of carbapenemase-producing bacteria worldwide is primarily associated with five acquired carbapenemase families, which belong to Ambler classes A, B and D. Classes A and B are globally dominated by KPC non-metallo-carbapenemases and IMP, NDM and VIM metallo-carbapenemases, respectively. Amongst class D carbapenemases, five subtypes of acquired OXA are restricted mostly to Acinetobacter spp., whereas OXA-48-like enzymes are widespread among the Enterobacteriaceae. OXA-48-like genes have been described in Acinetobacter baumannii, but have not been reported in Pseudomonas aeruginosa. Besides the intrinsic oxacillinase OXA-50, OXA-40 and OXA-198 are the only OXA-type carbapenemases that have been reported in P. aeruginosa to date. OXA-48-like carbapenemases do not confer resistance to third-generation cephalosporins and may confer only low levels of carbapenem resistance or reduced susceptibility, which makes laboratory detection difficult, potentially allowing them to spread undetected. An isolate of P. aeruginosa was submitted for investigation of carbapenem resistance to PHE’s Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit in 2012. It had been isolated from sputum of a patient who was transferred to the ICU of a UK hospital after a 6 month stay in an ICU in India. MICs were determined by agar dilution and interpreted using BSAC criteria. Carbapenemase genes were sought by commercial microarray (Check-MDR CT102; Check-Points, Wageningen, The Netherlands) and in-house PCRs. Nine-locus variable-number tandem-repeat (VNTR) typing was performed as previously described. WGS was performed using a HiSeq sequencing system (Illumina, Little Chesterford, UK) and data analysed using an in-house bioinformatics pipeline; resistance genes were identified by mapping reads against a library curated in-house from publically accessible databases. The P. aeruginosa isolate was resistant to meropenem (MIC 32 mg/L) and imipenem (MIC 64 mg/L), but without imipenem/ EDTA synergy, ruling out a metallo-carbapenemase. Nevertheless, resistance to carbapenems and piperacillin/tazobactam (MIC 32 mg/L), but with aztreonam relatively spared (MIC 8 mg/L) and susceptibility to ceftazidime (MIC 2 mg/L), raised suspicions that the isolate may harbour a carbapenemase, not least because six other carbapenemase-producing bacteria had been confirmed from the same patient in the preceding 3 weeks, as follows: a blaVIM-positive P. aeruginosa (isolated from sputum); a blaNDM-positive Providencia rettgeri (isolated from a hip wound); and, from rectal swabs, a blaOXA-181-positive Escherichia coli and blaNDM-positive isolates of Citrobacter freundii, Klebsiella pneumoniae and Providencia stuartii. The suspect P. aeruginosa isolate was also screened for acquired carbapenemase genes, with blaOXA-181 detected by microarray, PCR and Sanger sequencing. Carbapenemase activity was detected using a Rapid CARB Screen kit (BioConnections, Knypersley, UK), but the result from the modified Hodge test was negative. Analysis of WGS data identified blaOXA-181 on an 114391 bp contig, where it was located within a 4260 bp transposon that shared 100% identity with nucleotides 2357 to 6616 of pKP3-A (GenBank accession number JN205800.1), indicating that blaOXA-181 was located in Tn2013 as previously described. ISEcp1 was located upstream of blaOXA-181, with truncated lysR, ereA and repA genes located downstream. ISEcp1 has been reported to play a role in the mobilization of blaOXA-181 and other b-lactamase genes via a one-ended transposition process, thus facilitating their wider dissemination.Rather than being located on a ColE-type or IncTplasmid as previously reported in Enterobacteriaceae, blaOXA-181 was integrated into the chromosome between genes encoding a membrane protein and a transcriptional regulator of the PbsX family. Isolation of blaOXA-181-positive P. aeruginosa strains from the same patient in 2014, from a percutaneous endoscopic gastronomy tube insertion site and sputum, indicated that the blaOXA-181 was stably maintained. VNTR typing indicated that the 2012 and 2014 isolates had highly related profiles, which correspond to multilocus ST773, whilst the VIM-positive P. aeruginosa belonged to ST235. Both STs are known as international ‘high-risk clones’, which are commonly associated with multidrug resistance. The gene blaOXA-181 has previously been identified in Enterobacteriaceae originating mainly from the Indian subcontinent, East Asia and the Middle East. Nevertheless, this is the first report of this or any other OXA-48-like carbapenemase in P. aeruginosa and also only the second report of an OXA-48-like carbapenemase outside of the Enterobacteriaceae. The dominant carbapenemases in P. aeruginosa are metallo-enzymes, and most producers can be detected readily using EDTA or dipicolinic acid, which show synergy with carbapenems. However, inferring the presence of a non-metallo-carbapenemase in this species is more difficult owing to its frequent resistance to multiple b-lactams, including carbapenems. Although high-level temocillin resistance (MIC ≥128 mg/L) can serve as a diagnostic marker for OXA-48-like carbapenemases in Enterobacteriaceae, P. aeruginosa is intrinsically resistant to temocillin. This study highlights both the ability of acquired carbapenemase genes to spread beyond their ‘traditional’ host species

Characterization of carbapenemase-producing Enterobacteriaceae in the West Midlands region of England: 2007-14.

Objectives Carbapenemase-producing Enterobacteriaceae (CPE) have been increasingly reported in the UK since 2003. We analysed patient and isolate data for CPE confirmed by the national reference laboratory from laboratories in the West Midlands region from November 2007 to December 2014. Methods MICs were determined by BSAC agar dilution methodology and isolates exhibiting resistance to one or more carbapenems were screened for carbapenemase genes by PCR. Plasmid analyses were performed after electro-transformation of carbapenemase-encoding plasmids. WGS was performed on both transformants and clinical isolates. Patient data provided by the sending laboratories were reviewed. Results During the study period, CPE ( n  = 139) were submitted from 13 laboratories in the West Midlands region, originating from 108 patients and including one environmental isolate. CPE submissions increased significantly from 2009 onwards. Isolates were predominantly Klebsiella pneumoniae (89/139) obtained from inpatients. WGS was performed on all clinical isolates and transformants. After deduplication 119 isolates and 96 transformants remained for analysis. Within these, four families of carbapenemase genes were identified: bla NDM (69/119), bla KPC (26/119), bla OXA-48-like (16/119) and bla VIM (7/119); one isolate carried both bla NDM and bla OXA-48-like . Isolates represented diverse STs and plasmid replicon types. Plasmid analyses identified plasmids of different replicon types encoding bla KPC , bla NDM and bla OXA-48-like genes, found across several species and STs. Conclusions CPE have been reported increasingly in the West Midlands region over a 7 year period. bla NDM , bla KPC and bla OXA-48-like were the dominant carbapenemase genes and were found in a range of diverse genomic/plasmid environments, highlighting their ability to mobilize across different plasmids, often impeding the detection of outbreaks.

FRI-2 carbapenemase-producing Enterobacter cloacae complex in the UK

Objectives: Detection of rarer carbapenemases is challenging, as it requires molecular assays with comprehensive coverage or the use of phenotypic methods for the detection of carbapenemase activity. We describe a new class A carbapenemase, FRI‐2, in an Enterobacter cloacae complex isolate following implementation of an in‐house multiplex PCR for the detection of ‘rare’ class A carbapenemases. Methods: MICs were determined by agar dilution. A carbapenem‐resistant E. cloacae complex isolate was tested by PCR for the class A carbapenemases blaKPC, blaFRI, blaIMI, blaGES and blaSME. Carbapenemase activity was assessed using Carba NP and the carbapenem inactivation method. Whole genome and plasmid analyses of the clinical isolate and the FRI‐2 transformant were performed by WGS, respectively. Typing was carried out by PFGE. Results: The E. cloacae complex isolate showed resistance to imipenem (MIC = 16 mg/L), meropenem (MIC = 8 mg/L) and ertapenem (MIC = 8 mg/L), but remained susceptible to piperacillin/tazobactam (MIC = 8 mg/L). Carbapenemase activity was confirmed in the isolate by both phenotypic methods. A blaFRI‐1‐like gene was detected by PCR and analysis of WGS data of the clinical isolate identified an ORF of 885 bp, which showed 97% nucleotide identity with blaFRI‐1 and was named blaFRI‐2. WGS of the transformant indicated blaFRI‐2 was located on a 108 kb IncF/IncR plasmid. The FRI‐2‐positive E. cloacae complex isolate belonged to a novel ST (ST829). Conclusions: The possible circulation of rarer carbapenemases in clinical settings highlights the role of phenotypic tests to detect carbapenemase activity when molecular assays are negative for the ‘big 5’ carbapenemase families.

Covert dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) in a successfully controlled outbreak: long- and short-read whole-genome sequencing demonstrate multiple genetic modes of transmission

Abstract Background Carbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety. Objectives To use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak. Materials and methods Enterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013–14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI. Results Twenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQIL-D2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts. Conclusions WGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts.

Major role of pKpQIL-like plasmids in the early dissemination of KPC-type carbapenemases in the UK

Objectives Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were first seen in the UK in 2003 and have been increasingly reported since 2010, largely owing to an ongoing outbreak in North-West England. We examined the role of clonal spread and plasmid transmission in their emergence. Methods Isolates comprised KPC-positive K. pneumoniae ( n  =   33), Escherichia coli ( n  =   7) and Enterobacter spp. ( n  =   4) referred to the national reference laboratory between 2008 and 2010 from 17 UK centres, including three in North-West England. Isolates were typed by MLST. Plasmids were transferred by electroporation and characterized by PCR or sequencing. PCR screening assays were developed to distinguish plasmid pKpQIL variants. Results The K. pneumoniae isolates included 10 STs, of which three belonged to clonal group (CG) 258. CG258 ( n  =   19) isolates were detected in 13 centres but accounted for only 7/19 (36.8%) of those from North-West England. Most KPC-producers (37/44, 84.1%), including 16/19 CG258 K. pneumoniae , carried bla KPC on IncFII K2 plasmids. Sequencing of a subset of these plasmids ( n  =   11) revealed similarities with published pKpQIL. One variant, pKpQIL-UK [identified in K. pneumoniae CG258 ( n  =   5) and ST468 ( n  =   1) isolates from distinct centres] had only a few nucleotide changes from classical pKpQIL, whereas pKpQIL-D1 ( n  =   1) and pKpQIL-D2 ( n  =   4), from isolates of various species in the North-West, harboured large variations, reflecting replacement of the partitioning and replication functions and potentially thereby facilitating spread. PCR revealed that 36/37 (97.3%) IncFII K2 -type plasmids in KPC-positive isolates had pKpQIL markers. Conclusions pKpQIL-like plasmids played a major role in the early dissemination of KPC enzymes in the UK.