Integrated Analysis of Patient Networks and Plasmid Genomes to Investigate a Regional, Multispecies Outbreak of Carbapenemase-Producing Enterobacterales Carrying Both blaIMP and mcr-9 Genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of imipenemase (IMP)-encoding CPE among diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE-positive patients. Genomes of IMP-encoding CPE isolates were overlaid with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, and Escherichia coli); 86% (72 of 84) harbored an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68 of 72). Phylogenetic analysis of IncHI2 plasmids identified 3 lineages showing significant association with patient contacts and movements between 4 hospital sites and across medical specialties, which was missed in initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multimodal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.SummaryThis was an investigation, using integrated pathway networks and genomics methods, of the emergence of imipenemase-encoding carbapenemase-producing Enterobacterales among diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network, which was missed on routine investigations.

Causes of Musculoskeletal Pain in Paget's Disease of Bone.

Paget's disease of bone (PDB) is characterised by increased and disorganised bone remodelling leading to various complications, such as bone deformity, deafness, secondary osteoarthritis, and pathological fracture. Pain is the most common presenting symptom of PDB, but it is unclear to what extent this is due to increased metabolic activity of the disease, complications, or unrelated causes. We conducted a cross-sectional study of 168 people with PDB attending secondary care referral centres in the UK. We documented the presence of musculoskeletal pain and sought to determine its underlying causes. Musculoskeletal pain was reported by 122/168 (72.6%) individuals. The most common cause was osteoarthritis of joints distant from an affected PDB site in 54 (44.3%), followed by metabolically active PDB in 18 (14.7%); bone deformity in 14 (11.4%); osteoarthritis of a joint neighbouring an affected site in 11 (9.0%), neuropathic pain in 10 (8.2%), and various other causes in the remainder. Pain was more common in women (p<0.019) and in older individuals (p<0.001). Circulating concentrations of macrophage colony-stimulating factor (M-CSF) were significantly higher in those with pain (p = 0.008), but there was no difference between groups of patients with and without pain in concentrations of interleukin-6 (IL-6) or biochemical markers of bone turnover. Pain is a common symptom in PDB but is most often due to osteoarthritis at an unaffected site. The study illustrates the importance of fully evaluating people with PDB to determine the underlying cause of pain so that management can be tailored appropriately.

Outbreak of Ralstonia pickettii associated with contamination of saline products distributed internationally, the United Kingdom, 2024.

We describe an outbreak of Ralstonia pickettii in the United Kingdom, with isolates genetically indistinguishable from a 2023 Australian outbreak linked to internationally distributed saline solutions. Confirmed cases (n = 3) had bacteraemia, clinically relevant infection, indwelling venous lines and frequent healthcare contact. Multi-stakeholder intervention was required including product recall and risk communications. We recommend a low threshold for investigating clusters of Ralstonia species and similar opportunistic pathogens, considering contaminated product sources. Effective mitigation requires multi-agency partnership and international collaboration.

NDM-1 carbapenemase resistance gene vehicles emergent on distinct plasmid backbones from the IncL/M family.

OBJECTIVES: To assess the genetic contexts surrounding blaNDM-1 genes carried on IncM plasmids harboured by six carbapenemase-producing Enterobacterales (CPE) isolates referred to the UK Health Security Agency's Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit. METHODS: Between 2014 and 2018, the AMRHAI Reference Unit undertook WGS of CPE isolates using Illumina NGS. Nanopore sequencing was used for selected isolates and publicly available plasmid references were downloaded. Analysis of incRNA, which encodes the antisense RNA regulating plasmidic repA gene expression, was performed and bioinformatics tools were used to analyse whole plasmid sequences. RESULTS: Of 894 NDM-positive isolates of Enterobacterales, 44 NDM-1-positive isolates of five different species (Citrobacter spp., Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca) encoded the IncRNA locus of IncM2 plasmids. Long-read sequencing of six diverse isolates revealed related IncM2, NDM-1-encoding plasmids. Plasmid 'backbone' areas were conserved and contrasted with highly variable resistance regions. Sub-groupings of IncM2 plasmids encoding blaNDM-1 were detected; one sub-group occurred in five different health regions of England in every year. The diversity of NDM-1-encoding resistance gene integrons and transposons and their insertions sites in the plasmids indicated that NDM-1 has been acquired repeatedly by IncM2 variants. CONCLUSIONS: The use of sequencing helped inform: (i) a wide geographical distribution of isolates encoding NDM-1 on emergent IncM2 plasmids; (ii) variant plasmids have acquired NDM-1 separately; and (iii) dynamic arrangements and evolution of the resistance elements in this plasmid group. The geographical and temporal distribution of IncM2 plasmids that encode NDM-1 highlights them as a public health threat that requires ongoing monitoring.

A case of neck abscess caused by rare hypervirulent Klebsiella pneumoniae, capsular type K20 and sequence type 420.

BACKGROUND: This case report describes a neck abscess caused by a strain of Hypervirulent Klebsiella pneumoniae in a middle aged man with diabetes without a history of travel to East and South East Asia. This case report is of notable significance as Hypervirulent Klebsiella pneumoniae neck abscesses are rarely seen in the UK and are very infrequently documented in individuals who have not first travelled to the high prevalence areas of East and South East Asia. CASE PRESENTATION: This case report describes a 53 year old diabetic man who contracted a Hypervirulent Klebsiella pneumoniae neck abscess which led to the development of sepsis. Klebsiella pneumoniae was cultured from blood cultures and fluid aspirated from the abscess grew the pathogen with same antimicrobial susceptibility. Hypervirulence was demonstrated after the samples were analysed, at the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit Public Health England Colindale, and found to contain the K20 (rmp)A and rmpA2 virulence genes. DISCUSSION: Hypervirulent Klebsiella pneumoniae is a Gram-negative, encapsulated, non-motile bacillus notable for its ability to metastatically spread and cause potentially life threatening infections in otherwise healthy adults, but especially in those with diabetes. Genes responsible for the production of hyperviscous mucoid polysaccharide capsules and siderophores, such as those isolated in this case, enable the bacteria to more efficiently evade the hosts immune system and disseminate and invade surrounding and distant tissues. Data from Public Health England shows Hypervirulent Klebsiella pneumoniae are rare in the UK. A review of current literature also showed Hypervirulent Klebsiella pneumoniae almost exclusively occur in those who have traveled to East and South East Asia. CONCLUSIONS: This case reported a rare Hypervirulent Klebsiella pneumoniae neck abscess outside of, and without travel to, East and South East Asia. This raises concerns about future, potentially life threatening, Hypervirulent Klebsiella pneumoniae infections becoming more widespread without the need for endemic travel. This concern is further exacerbated by the growing global challenge of antimicrobial resistance.

A Multispecies Cluster of GES-5 Carbapenemase-Producing Enterobacterales Linked by a Geographically Disseminated Plasmid.

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.

Oestrogen-deficiency induces bone loss by modulating CD14+ monocyte and CD4+ T cell DR3 expression and serum TL1A levels.

BACKGROUND: Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown. METHODS: To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model. RESULTS: In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control. CONCLUSION: Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

National outbreak of Pseudomonas aeruginosa associated with an aftercare solution following piercings, July to September 2016, England.

We report a national Pseudomonas aeruginosa outbreak from a common source following piercings between July and September 2016 in England. The multi-agency outbreak investigation included active case finding, microbiological testing of environmental samples and case specimens including Variable Number Tandem Repeat (VNTR) typing and a retrospective cohort study. Overall, 162 outbreak cases (29 confirmed, 14 probable and 119 possible) and 14 non-outbreak cases were identified; all confirmed cases had ear piercings (93% cartilage). Outbreak cases were predominantly female (95%) and had a median age of 18 years (interquartile range: 13-56 years). Nineteen outbreak cases required surgery under general anaesthetic The same outbreak VNTR type (11,3,5,3,3,3,6,4,7) was isolated from bottles of an aftercare solution from a single manufacturer and in specimens from confirmed cases who attended eight different piercing studios supplied with this product. In the cohort study, use of aftercare solution was associated with becoming a case (aOR: 4.60, 95% confidence interval: 1.65-12.90). Environmental, microbiological and epidemiological investigations confirmed that contamination during production of aftercare solution was the source of this national outbreak; highlighting challenges in the regulation of a cosmetic products used in the piercing industry and that guidance on piercing aftercare may need to be reviewed.

Isolation and identification of Acinetobacter spp. from healthy canine skin.

BACKGROUND: Acinetobacter species can exhibit widespread resistance to antimicrobial agents. They are already recognized as important nosocomial pathogens of humans, but are becoming increasingly recognized in opportunistic infections of animals. HYPOTHESIS/OBJECTIVES: This study aimed to determine whether Acinetobacter spp. are carried on skin of healthy dogs and, if present, to identify the species. ANIMALS: Forty dogs were sampled at veterinary practices and rescue centres. They were free from skin disease and receiving no systemic or topical treatments. METHODS: Skin swab samples were collected from four sites on each dog and cultured. Acinetobacter spp. isolates were detected by biochemical tests and gas chromatography. The species was determined by sequencing the RNA polymerase ß-subunit (rpoB) gene. Isolates were screened for OXA carbapenemase genes and class 1 integrons capable of carrying resistance genes, and subjected to antimicrobial susceptibility tests. RESULTS: For 25% dogs sampled (10 of 40), Acinetobacter spp. were isolated at one or more skin sites. Thirteen Acinetobacter spp. isolates were recovered from 160 samples. The most frequently cultured was A. lwoffii (seven of 13), followed by A. baumannii (two of 13), A. junii (one of 13), A. calcoaceticus (one of 13), A. pittii (one of 13) and a novel Acinetobacter species (one of 13). Class 1 integrons and blaOXA-23-like were not detected. Isolates were susceptible to most antibiotics. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms that Acinetobacter spp. can survive on canine skin, where they may be potential reservoirs for infection. This highlights the importance of good hygiene in veterinary practice, adhering to aseptic principles in surgery, and treatment based on culture and susceptibility testing where possible.

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.

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 first occurrence of a CTX-M ESBL-producing Escherichia coli outbreak mediated by mother to neonate transmission in an Irish neonatal intensive care unit.

BACKGROUND: Escherichia coli (E. coli) comprise part of the normal vaginal microflora. Transfer from mother to neonate can occur during delivery resulting, sometimes, in neonatal bacterial disease. Here, we aim to report the first outbreak of CTX-M ESBL-producing E. coli with evidence of mother-to-neonate transmission in an Irish neonatal intensive care unit (NICU) followed by patient-to-patient transmission. METHODS: Investigation including molecular typing was conducted. Infection was defined by clinical and laboratory criteria and requirement for antimicrobial therapy with or without positive blood cultures. Colonisation was determined by isolation without relevant symptoms or indicators of infection. RESULTS: Index case was an 8-day-old baby born at 34 weeks gestation who developed ESBL-producing E. coli infections at multiple body sites. Screening confirmed their mother as colonised with ESBL-producing E. coli. Five other neonates, in the NICU simultaneously with the index case, also tested positive. Of these, four were colonised while one neonate developed sepsis, requiring antimicrobial therapy. The second infected neonate's mother was also colonised by ESBL-producing E. coli. Isolates from all eight positive patients (6 neonates, 2 mothers) were compared using pulsed-field gel electrophoresis (PFGE). Two distinct ESBL-producing strains were implicated, with evidence of transmission between mothers and neonates for both strains. All isolates were confirmed as CTX-M ESBL-producers. There were no deaths associated with the outbreak. CONCLUSIONS: Resources were directed towards control interventions focused on hand hygiene and antimicrobial stewardship, which ultimately proved successful. Since this incident, all neonates admitted to the NICU have been screened for ESBL-producers and expectant mothers are screened at their first antenatal appointment. To date, there have been no further outbreaks.

Waterborne Elizabethkingia meningoseptica in Adult Critical Care.

Elizabethkingia meningoseptica is an infrequent colonizer of the respiratory tract; its pathogenicity is uncertain. In the context of a 22-month outbreak of E. meningoseptica acquisition affecting 30 patients in a London, UK, critical care unit (3% attack rate) we derived a measure of attributable morbidity and determined whether E. meningoseptica is an emerging nosocomial pathogen. We found monomicrobial E. meningoseptica acquisition (n = 13) to have an attributable morbidity rate of 54% (systemic inflammatory response syndrome ≥2, rising C-reactive protein, new radiographic changes), suggesting that E. meningoseptica is a pathogen. Epidemiologic and molecular evidence showed acquisition was water-source-associated in critical care but identified numerous other E. meningoseptica strains, indicating more widespread distribution than previously considered. Analysis of changes in gram-negative speciation rates across a wider London hospital network suggests this outbreak, and possibly other recently reported outbreaks, might reflect improved diagnostics and that E. meningoseptica thus is a pseudo-emerging pathogen.

Association of Novel Nonsynonymous Single Nucleotide Polymorphisms in ampD with Cephalosporin Resistance and Phylogenetic Variations in ampC, ampR, ompF, and ompC in Enterobacter cloacae Isolates That Are Highly Resistant to Carbapenems.

InEnterobacter cloacae, the genetic lesions associated with derepression of the AmpC ß-lactamase include diverse single nucleotide polymorphisms (SNPs) and/or indels in theampDandampRgenes and SNPs inampC, while diverse SNPs in the promoter region or SNPs/indels within the coding sequence of outer membrane proteins have been described to alter porin production leading to carbapenem resistance. We sought to define the underlying mechanisms conferring cephalosporin and carbapenem resistance in a collection ofE. cloacaeisolates with unusually high carbapenem resistance and no known carbapenemase and, in contrast to many previous studies, considered the SNPs we detected in relation to the multilocus sequence type (MLST)-based phylogeny of our collection. Whole-genome sequencing was applied on the most resistant isolates to seek novel carbapenemases, expression ofampCwas measured by reverse transcriptase PCR, and porin translation was detected by SDS-PAGE. SNPs occurring inampC,ampR,ompF, andompCgenes (and their promoter regions) were mostly phylogenetic variations, relating to the isolates' sequence types, whereas nonsynonymous SNPs inampDwere associated with derepression of AmpC and cephalosporin resistance. The additional loss of porins resulted in high-level carbapenem resistance, underlining the clinical importance of chromosomal mutations among carbapenem-resistantE. cloacae.

ESBL-producing Enterobacteriaceae in 24 neonatal units and associated networks in the south of England: no clustering of ESBL-producing Escherichia coli in units or networks.

OBJECTIVES: The objectives of this study were to characterize ESBL-producing Enterobacteriaceae present in 24 neonatal units (NNUs) in eight networks participating in a multicentre probiotic study and to test the hypothesis that specific strains would cluster within individual units and networks. METHODS: We performed analysis of stool samples for the presence of ESBL-producing Enterobacteriaceae at 2 weeks post-natal age and 36 weeks post-menstrual age. ESBL-producing Enterobacteriaceae were characterized and typed using molecular methods. RESULTS: ESBL-producing Enterobacteriaceae (n = 71) were isolated from 67/1229 (5.5%) infants from whom we received a sample at either sampling time or both sampling times, and from infants in 18 (75%) of the 24 recruiting NNUs. Thirty-three Escherichia coli, 23 Klebsiella spp. and 6 Enterobacter spp. strains were characterized. ESBL-producing E. coli were all distinguishable within individual NNUs by antibiotic resistance genotype, serogroup (O25b), phenotype, phylotype or ST. Ten of the 33 were ST131 and 9 of the 10 ST131 isolates were ciprofloxacin resistant. Seven of the 10 ST131 isolates carried genes encoding CTX-M group 1 enzymes. ST131 isolates were isolated from centres within five of the eight NNU networks. There were clusters of indistinguishable ESBL-producing Klebsiella and Enterobacter isolates associated with specific NNUs. CONCLUSIONS: Strains of E. coli ST131 were distributed across neonatal networks in the south of England. There was no evidence of clustering of clonally related ESBL-producing E. coli strains, by contrast with Klebsiella spp. and Enterobacter spp., which did cluster within units. The possibility that ESBL-producing E. coli strains are spread by vertical transmission requires further investigation.

High-Resolution Analysis by Whole-Genome Sequencing of an International Lineage (Sequence Type 111) of Pseudomonas aeruginosa Associated with Metallo-Carbapenemases in the United Kingdom.

Whole-genome sequencing (WGS) was carried out on 87 isolates of sequence type 111 (ST-111) of Pseudomonas aeruginosa collected between 2005 and 2014 from 65 patients and 12 environmental isolates from 24 hospital laboratories across the United Kingdom on an Illumina HiSeq instrument. Most isolates (73) carried VIM-2, but others carried IMP-1 or IMP-13 (5) or NDM-1 (1); one isolate had VIM-2 and IMP-18, and 7 carried no metallo-beta-lactamase (MBL) gene. Single nucleotide polymorphism analysis divided the isolates into distinct clusters; the NDM-1 isolate was an outlier, and the IMP isolates and 6/7 MBL-negative isolates clustered separately from the main set of 73 VIM-2 isolates. Within the VIM-2 set, there were at least 3 distinct clusters, including a tightly clustered set of isolates from 3 hospital laboratories consistent with an outbreak from a single introduction that was quickly brought under control and a much broader set dominated by isolates from a long-running outbreak in a London hospital likely seeded from an environmental source, requiring different control measures; isolates from 7 other hospital laboratories in London and southeast England were also included. Bayesian evolutionary analysis indicated that all the isolates shared a common ancestor dating back ∼50 years (1960s), with the main VIM-2 set separating approximately 20 to 30 years ago. Accessory gene profiling revealed blocks of genes associated with particular clusters, with some having high similarity (≥95%) to bacteriophage genes. WGS of widely found international lineages such as ST-111 provides the necessary resolution to inform epidemiological investigations and intervention policies.

Dominance of international 'high-risk clones' among metallo-ß-lactamase-producing Pseudomonas aeruginosa in the UK.

OBJECTIVES: Carbapenem-resistant isolates of Pseudomonas aeruginosa producing metallo-ß-lactamases (MBLs) are increasingly reported worldwide and often belong to particular 'high-risk clones'. This study aimed to characterize a comprehensive collection of MBL-producing P. aeruginosa isolates referred to the UK national reference laboratory from multiple UK laboratories over a 10 year period. METHODS: Isolates were referred to the UK national reference laboratory between 2003 and 2012 for investigation of resistance mechanisms and/or outbreaks. MBL genes were detected by PCR. Typing was carried out by nine-locus variable-number tandem repeat (VNTR) analysis and MLST. RESULTS: MBL-producing P. aeruginosa isolates were referred from 267 source patients and 89 UK laboratories. The most common isolation sites were urine (24%), respiratory (18%), wounds (17%) and blood (13%). VIM-type MBLs predominated (91% of all MBLs found), but a few IMP- and NDM-type enzymes were also identified. Diverse VNTR types were seen, but 86% of isolates belonged to six major complexes. MLST of representative isolates from each complex showed that they corresponded to STs 111, 233, 235, 357, 654 and 773, respectively. Isolates belonging to these complexes were received from between 9 and 25 UK referring laboratories each. CONCLUSIONS: The incidence of MBL-producing P. aeruginosa is increasing in the UK. The majority of these isolates belong to several 'high-risk clones', which have been previously reported internationally as host clones of MBLs.

Genetic environment of metallo-ß-lactamase genes in Pseudomonas aeruginosa isolates from the UK.

OBJECTIVES: We sought to characterize the genetic environment of blaVIM and blaIMP genes in Pseudomonas aeruginosa isolates from the UK; these included members of six previously described prevalent complexes, A-F, which correspond to international 'high-risk clones', along with diverse strains. METHODS: Metallo-ß-lactamase (MBL)-encoding class 1 integrons were amplified by PCR from 218 P. aeruginosa isolates producing VIM-type (n = 196) or IMP-type (n = 22) enzymes, referred from UK hospital laboratories between 2003 and 2012. The variable regions of selected integrons were sequenced using a primer walking method. RESULTS: One-hundred-and-nineteen isolates had an MBL-encoding integron with the 3' conserved sequence (3'CS), 65 had Tn5090-like 3' regions and 17 had the sul1 gene, but lacked the qacEΔ1 gene; the 3' region could not be amplified using any primer combinations for the remaining 17 isolates. Six integron profiles were each seen in more than five isolates. Predominant integron types were seen amongst isolates belonging to STs 111, 233, 654/964 and 773 (complexes A, C, D and F, respectively), whereas diverse integron profiles were seen in isolates belonging to ST235 (complex B) and ST357 (complex E). CONCLUSIONS: In UK P. aeruginosa isolates, MBL genes occur in diverse class 1 integron structures, though commonly with 3' regions containing the classical 3'CS or Tn5090-like regions. Four of the six main clonal complexes, referred from multiple laboratories, carried a predominant integron type, whereas the remaining two had more diverse types.

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

OBJECTIVES: Gram-negative bacteria with diverse carbapenemases, including New Delhi metallo-ß-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.

Klebsiella pneumoniae sequence type 147: a high-risk clone increasingly associated with plasmids carrying both resistance and virulence elements.

Introduction. The first hybrid resistance/virulence plasmid, combining elements from virulence plasmids described in hypervirulent types of Klebsiella pneumoniae with those from conjugative resistance plasmids, was described in an isolate of sequence type (ST) 147 from 2016. Subsequently, this type has been increasingly associated with these plasmids.Hypothesis or gap statement. The extent of carriage of hybrid virulence/resistance plasmids in nosocomial isolates of K. pneumoniae requires further investigation.Aim. To describe the occurrence of virulence/resistance plasmids among isolates of K. pneumoniae received by the UK reference laboratory, particularly among representatives of ST147, and to compare their sequences.Methodology. Isolates received by the laboratory during 2022 and the first half of 2023 (n=1278) were screened for virulence plasmids by PCR detection of rmpA/rmpA2 and typed by variable-number tandem repeat analysis. Twenty-nine representatives of ST147 (including a single-locus variant) from seven hospital laboratories were subjected to long-read nanopore sequencing using high-accuracy q20 chemistry to provide complete assemblies.Results. rmpA/rmpA2 were detected in 110 isolates, of which 59 belonged to hypervirulent K1-ST23, K2-ST86 and K2-ST65/375. Of the remainder, representatives of ST147 formed the largest group, with 22 rmpA/rmpA2-positive representatives (out of 47 isolates). Representatives were from 19 hospital laboratories, with rmpA/rmpA2-positive isolates from 10. Nanopore sequencing of 29 representatives of ST147 divided them into those with no virulence plasmid (n=12), those with non-New Delhi metallo-ß-lactamase (NDM) virulence plasmids (n=6) and those carrying bla NDM-5 (n=9) or bla NDM-1 (n=2) virulence plasmids. These plasmids were of IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) replicon types. Most of the non-NDM virulence plasmids were highly similar to the originally described KpvST147L_NDM plasmid. Those carrying bla NDM-5 were highly similar to one another and to previously described plasmids in ST383 and carried an extensive array of resistance genes. Comparison of the fully assembled chromosomes indicated multiple introductions of ST147 in UK hospitals.Conclusion. This study highlights the high proportion of representatives of ST147 that carry IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) hybrid resistance virulence plasmids. It is important to be aware of the high probability that representatives of this type carry these plasmids combining resistance and virulence determinants and of the consequent increased risk to patients.