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Background Early and accurate treatment of infections due to carbapenem-resistant organisms is facilitated by rapid diagnostics, but rare resistance mechanisms can compromise detection. One year after a Guiana Extended-Spectrum (GES)-5 carbapenemase–positive Klebsiella oxytoca infection was identified by whole-genome sequencing (WGS; later found to be part of a cluster of 3 cases), a cluster of 11 patients with GES-5–positive K. oxytoca was identified over 18 weeks in the same hospital. Methods Bacteria were identified by matrix-assisted laser desorption/ionization–time of flight mass spectrometry, antimicrobial susceptibility testing followed European Committee on Antimicrobial Susceptibility Testing guidelines. Ertapenem-resistant isolates were referred to Public Health England for characterization using polymerase chain reaction (PCR) detection of GES, pulsed-field gel electrophoresis (PFGE), and WGS for the second cluster. Results The identification of the first GES-5 K. oxytoca isolate was delayed, being identified by WGS. Implementation of a GES-gene PCR informed the occurrence of the second cluster in real time. In contrast to PFGE, WGS phylogenetic analysis refuted an epidemiological link between the 2 clusters; it also suggested a cascade of patient-to-patient transmission in the later cluster. A novel GES-5–encoding plasmid was present in K. oxytoca , Escherichia coli , and Enterobacter cloacae isolates from unlinked patients within the same hospital group and in human and wastewater isolates from 3 hospitals elsewhere in the United Kingdom. Conclusions Genomic sequencing revolutionized the epidemiological understanding of the clusters; it also underlined the risk of covert plasmid propagation in healthcare settings and revealed the national distribution of the resistance-encoding plasmid. Sequencing results also informed and led to the ongoing use of enhanced diagnostic tests for detecting carbapenemases locally and nationally. [ABSTRACT FROM AUTHOR]

Objectives: To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population.Methods: Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors.Results: Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population.Conclusions: Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

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., (© Crown copyright 2017.)