Diversity of carbapenemase-producing Enterobacterales in England as revealed by whole-genome sequencing of isolates referred to a national reference laboratory over a 30-month period.

Introduction. Increasing numbers of carbapenemase-producing Enterobacterales (CPE), which can be challenging to treat, have been referred to the national reference laboratory in England since the early 2000s.Gap Statement/Aim. Previous studies on CPE in the UK have focussed on localized outbreaks. We applied whole-genome sequencing (WGS) to isolates referred to the national reference laboratory over 30 months to inform our understanding of CPE epidemiology in England.Methodology. The first confirmed CPE from each new patient referred by an English diagnostic laboratory between 1 January 2014 and 30 June 2016 was sequenced on an Illumina HiSeq 2500. Multiple isolates from the same patient were included from either different species or the same species with different carbapenemase genes. The data were analysed using an in-house bioinformatics pipeline that determines species identification, multi-locus sequence typing (MLST) profile and antimicrobial resistance gene content.Results. A total of 2658 non-duplicate CPE were sequenced amongst which three host organisms belonging to diverse sequence types (STs) predominated: Klebsiella pneumoniae (1380/2658, 51.9 %; 177 STs), Escherichia coli (723/2658, 27.2 %; 133 STs) and Enterobacter cloacae (294/2658, 11.1 %; 88 STs). Thirty different carbapenemase gene variants were identified, although bla OXA-48-like (1122/2658, 42.2%), bla NDM (692/2658, 26.0 %), bla KPC (571/2658, 21.5 %), bla VIM (100/2658, 3.8 %) and bla IMP (33/2658, 1.2 %) predominated. ST/carbapenemase gene pairings represented widely distributed high-risk clones or clusters at a regional or hospital level.Conclusion. CPE referred to the national reference laboratory are diverse, suggesting multiple introductions to England and a role for horizontal transfer of carbapenemase genes in English CPE epidemiology.

Sustained transmission of high-level azithromycin-resistant Neisseria gonorrhoeae in England: an observational study.

BACKGROUND: Between Nov 3, 2014, and Feb 24, 2017, 70 cases of high-level azithromycin-resistant (HL-AziR; minimum inhibitory concentration [MIC] ≥256 mg/L) Neisseria gonorrhoeae were reported from across England. Whole-genome sequencing was done to investigate this outbreak to determine whether the ongoing outbreak represented clonal spread of an HL-AziR N gonorrhoeae strain identified in Leeds. We also wanted to elucidate the molecular mechanisms of azithromycin resistance in N gonorrhoeae in the UK. METHODS: In this observational study, whole-genome sequencing was done on the HL-AziR N gonorrhoeae isolates from England. As comparators, 110 isolates from the UK and Ireland with a range of azithromycin MICs were also sequenced, including eight isolates from Scotland with azithromycin MICs ranging from 0·12 mg/L to 1·00 mg/L that were N gonorrhoeae multi-antigen sequence type 9768 (ST9768), which was the sequence type initially responsible for the outbreak. The presence of mutations or genes associated with azithromycin resistance was also investigated. FINDINGS: 37 of the 60 HL-AziR isolates from England belonged to ST9768, and were genetically similar (mean 4·3 single-nucleotide polymorphisms). A 2059A→G mutation was detected in three or all four alleles of the 23S rRNA gene. Five susceptible ST9768 isolates had one mutated 23S rRNA allele and one low-level resistant ST9768 isolate had two mutated alleles. INTERPRETATION: Sustained transmission of a successful HL-AziR clone was seen across England. Mutation 2059A→G was found in isolates with lower azithromycin MICs. Azithromycin exposure might have provided the selection pressure for one or two mutated copies of the 23S rRNA gene to recombine with wild-type copies, leading to three or four mutated copies and the HL-AziR phenotype. HL-AziR could emerge in isolates with low azithromycin MICs and eliminate the effectiveness of azithromycin as part of dual therapy for the treatment of gonorrhoea. FUNDING: Public Health England.

Fracture Risk Assessment Tool (FRAX®) Results Calculated With and Without Bone Mineral Density Values for the Evaluation of Fracture Risk in Postmenopausal Women With Osteopenia.

The aim of this study was to evaluate the agreement between fracture risk predictions based on calculations made with and without bone mineral density (BMD) values using the Fracture Risk Assessment Tool (FRAX®) in Turkish postmenopausal women with osteopenia and to compare the treatment recommendations. This descriptive, cross-sectional study included postmenopausal women aged 50-79 yr with a diagnosis of osteoporosis who were not receiving any treatment. A questionnaire was administered to the participants face-to-face to obtain sociodemographic characteristics, medical history, and fracture history. Fracture risk was calculated with FRAX® separately with and without BMD. The study included 230 postmenopausal patients with osteopenia. The mean age of the patients was determined as 63.16 ± 7.59 yr, and the mean body mass index was 30.61 ± 5.02. The intraclass correlation coefficient values of the 10-yr major osteoporotic (MO) fracture and hip fracture score agreement with FRAX® with and without BMD were mean 0.486 and 0.462, respectively. The risk of MO fracture with an intervention threshold of ≥20 was determined in 227/230 patients (98.7%), and the risk of hip fracture with treatment recommendations of ≥3 was determined in 204/230 patients (88.7%). Treatment recommendations in patients with no fracture history and secondary osteoporosis were 100% for MO fracture and 94.7% (123/130) for hip fracture risk. The treatment recommendation rates of FRAX® with and without BMD were similar for the majority of postmenopausal women with osteopenia. The agreement between the values was of a moderate level. When patients with a fracture history and secondary osteoporosis were excluded, the agreement increased. Even though values with BMD are of basic importance for medical treatment in postmenopausal women, the use of measurements evaluating fracture risk, such as FRAX® without BMD, could be useful in postmenopausal women with osteopenia.

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.

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.

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.

In vitro selection of ceftazidime-avibactam resistance in Enterobacteriaceae with KPC-3 carbapenemase.

Ceftazidime-avibactam is active against most Enterobacteriaceae isolates with KPC carbapenemases. We investigated whether this activity could be compromised by mutation. Single-step and multistep selections were attempted using ceftazidime-avibactam (avibactam fixed at 1 or 4 µg/ml) versus two strains each of Enterobacter cloacae and Klebsiella pneumoniae, all with the KPC-3 enzyme. Mutant bla KPC alleles were sequenced, and their parentage was confirmed by typing. Ceftazidime-avibactam selected mutants at up to 16× MIC, with frequencies of ca. 10(-9). This contrasted with previous experience for ceftaroline-avibactam, where mutant frequencies under similar conditions were <10(-9). The MICs of ceftazidime with 1 µg/ml avibactam for the ceftazidime-avibactam-selected mutants rose from 1 to 8 µg/ml to 16 to >256 µg/ml and those of ceftazidime with 4 µg/ml avibactam from 0.25 to 1 µg/ml to 4 to 128 µg/ml; ceftaroline-avibactam MICs rose less, typically from 0.5 to 1 µg/ml to 1 to 8 µg/ml. The MICs of carbapenems and cephalosporins except ceftazidime and piperacillin-tazobactam were reduced for many mutants. Sequencing of blaKPC revealed point and insertion changes in 12/13 mutants investigated, representing all four parents; one mutant lacked bla KPC changes and possibly had reduced permeability. Amino acid changes commonly involved Ω loop alterations or 1 to 6 amino acid insertions immediately C-terminal to this loop. The most frequent change, seen in four mutants from three strains, was Asp179Tyr, replacing a residue that ordinarily forms a salt bridge to stabilize the Ω loop. Since ceftaroline-avibactam was less affected than ceftazidime-avibactam, we postulate that these mutations increase ceftazidimase specificity rather than conferring avibactam resistance. The clinical relevance remains uncertain.

Clusters of genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the UK.

Variable number tandem repeat (VNTR) analysis at nine loci of isolates of Pseudomonas aeruginosa submitted to the national reference laboratory from UK hospitals, from over 2000 patients, between June 2010 and June 2012 revealed four widely found types that collectively were received from approximately a fifth of patients, including from those with cystic fibrosis. These types were also prevalent among related submissions from the clinical environment and were received from up to 54 (out of 143) hospitals. Multi-locus sequence typing and blaOXA-50-like sequencing confirmed the clonal relationship within each cluster, and representatives from multiple centres clustered within about 70 % by pulsed-field gel electrophoresis. Illumina sequencing of 12 isolates of cluster A of VNTR profile 8, 3, 4, 5, 2, 3, 5, 2, x (where the repeat number at the last, most discriminatory locus is variable) revealed a large number of variably present targets in the accessory genome and seven of these were sought by PCR among a larger set of isolates. Representatives from patients within a single centre mostly had distinct accessory gene profiles, suggesting that these patients acquired the strain independently, while those with clear epidemiological links shared the same profile. Profiles also varied between representatives from different centres. Epidemiological investigations of widely found types such as these require the use of finer-typing methods, which increasingly will be informed by next generation sequencing.

Identification of Achromobacter xylosoxidans by detection of the bla(OXA-114-like) gene intrinsic in this species.

Achromobacter xylosoxidans is an emerging pathogen among patients with cystic fibrosis. Here we describe a specific PCR for identification of this organism, based on detection of bla(OXA-114-like). Comparison of isolates by pulsed-field gel electrophoresis revealed evidence of cross-infection in some cases, but most patients harbored their own strain.

Molecular comparison of isolates of Burkholderia multivorans from patients with cystic fibrosis in the United Kingdom.

Burkholderia multivorans strains from 47 cystic fibrosis (CF) patients in 28 hospitals were compared by pulsed-field gel electrophoresis (PFGE) and flagellin (fliC) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. A considerable degree of genetic variation was evident, with each patient harboring a strain with a unique PFGE profile. Four sizes of fliC amplicons were produced, and these amplicons gave 13 RFLP types with restriction enzyme MspI. B. multivorans did not appear to spread between patients, suggesting that most CF patients acquire the organism from the natural environment.