Abstract Background Resistance to carbapenems and third-generation cephalosporins is increasing in Klebsiella pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, an unusual K. pneumoniae clinical isolate, KpMVR1, was recovered from a hospitalised patient in England, exhibiting resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. To investigate this phenomenon, we characterised the genome and antimicrobial susceptibility of KpMVR1 alongside two clonally related isolates susceptible to all three β-lactam/β-lactamase inhibitor combinations: KpMVS1, collected from the same patient 42 days earlier, and KpMVS2, from another patient in the same hospital. Methods Illumina and MinION whole-genome sequencing were conducted for these three isolates, followed by hybrid genome assembly. Annotated genome assemblies were compared to identify genetic variation. Mutagenesis experiments were performed to verify predicted functional alterations. Results All isolates belonged to clone ST8134 and carried bla KPC-2 alleles (KpMVR1: bla KPC-157; KpMVS1 and KpMVS2: bla KPC-2) in plasmids predicted to be conjugative. Insertion sequence ISEc68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing susceptibility to meropenem-vaborbactam and imipenem-relebactam. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. An intact, iron-transporting fec operon was identified on a novel IncFII(pKP91)/IncFIB(K) plasmid unique to KpMVS2, possibly accounting for the cefiderocol resistance observed in this isolate. Conclusions KpMVR1 carried multiple resistance-associated genetic alterations and likely developed its resistance profile through within-patient evolution. This study highlights the importance of routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy and the need to characterise underlying resistance mechanisms to assess the risk of onward dissemination.

Introduction. Carbapenemase-producing Enterobacterales (CPE) represent a major threat to hospitalised patients worldwide. The dissemination of carbapenemase genes, such as blaIMP, is frequently mediated by mobile genetic elements including plasmids. During a previously described multispecies, healthcare-associated outbreak of blaIMP-positive CPE in North West London, two unusual isolates, IMP47 (Klebsiella grimontii) and IMP76 (K. pneumoniae), recovered in 2019 from routinely collected rectal swabs of inpatients, were predicted to harbour blaIMP-1-carrying IncHI1 megaplasmids. Aims. This study aimed to determine complete genomic sequences of IMP47 and IMP76, resolve the genetic context of blaIMP-1, and assess the conjugative mobility of blaIMP-1-carrying megaplasmids. Methods. Genomic sequences of both isolates were recovered through hybrid assembly of Oxford Nanopore and Illumina sequencing reads. Complete plasmid sequences were characterised to determine replicons, conjugation machinery, and genes encoding resistance to antimicrobials or other stress factors. Integrons and transposable elements (TEs) within flanking regions of blaIMP-1 were resolved through genome annotation and search against public databases. Liquid-mating experiments were performed to assess the mobility of blaIMP-1-carrying plasmids. Results. Completed genome assemblies were generated from both isolates, confirming two blaIMP-1-carrying megaplasmids, pIMP47 (391 kbp) and pIMP76_1 (519 kbp), of the replicon type IncHI1A(pNDM-CIT)/IncHI1B(pNDM-CIT). The blaIMP-1 locus was carried by nearly identical class 1 integrons in both plasmids and a closely related IncHI1 megaplasmid pEB3_IMP1 (361 kbp) previously identified in South West England. Comparative analysis revealed conserved genetic structures linking blaIMP-1 to mercury-resistance genes and TEs Tn6025, TnAs3, IS26, and IS5075, suggesting a history of recombination and potential for TE-mediated mobilisation. Conjugation experiments confirmed transfer of pIMP76_1 into a recipient K. pneumoniae strain, resulting in acquisition of ertapenem resistance, whereas transfer of pIMP47 was not observed under the tested conditions. Conclusion. Two IMP-producing IncHI1 megaplasmids in gut-colonising Klebsiella species revealed TE-mediated blaIMP-1 mobilisation. The co-localisation of blaIMP-1 and metal-resistance genes in both plasmids highlights the potential for co-selection of blaIMP-1 in environments enriched with metal ions. Our findings underscore the importance of longitudinal genomic surveillance of carbapenemase-encoding megaplasmids in healthcare settings.

Background Carbapenemase-producing Enterobacterales (CPE) are known to cause sporadic outbreaks in healthcare settings and can be challenging to manage due to environmental and human colonisation. Aim Our aim was to identify common exposures among a cluster of cases with NDM-1-producing Klebsiella pneumoniae in a single ward, using epidemiological, genomic and environmental investigation, to identify possible sources and inform targeted control measures. Methods Cases were patients on the ward with laboratory confirmed K. pneumoniae with NDM-1 sequence type 15 identified via variable number tandem repeat (VNTR) analysis and clustered by whole-genome sequencing. Case exposure information was collected through reviewing patient notes and hospital floor plans. Environmental samples were taken from ward surfaces on two occasions and cultured to isolate CPE. Findings Twelve cases (four infections and eight colonisations) were identified over a 3.5-year period. Of the first nine cases that occurred after the index case, eight were inpatients on the ward at the same time as at least one other case, and six occupied a patient side-room after a positive case had occupied that room.NDM-1-producing K. pneumoniae with the same VNTR profile as the clinical isolates were recovered from the ward environment, including from the drain of a handwash basin which had been installed to replace one that tested positive during the first environmental sampling. Conclusion We demonstrated that persistent, multi-year clusters of CPE in hospitals occur, and long-term environmental reservoirs may persist following remediation measures. Understanding the epidemiology, supplemented with targeted environmental sampling, can help to identify potential sources of CPE in healthcare settings.

BACKGROUND:Species of the Burkholderia (B.) cepacia complex (Bcc) have been implicated in multiple nosocomial outbreaks linked to contaminated water-based products, including liquid soaps, mouthwash, and other non-alcoholic aqueous solutions. OBJECTIVE:We describe two substantial healthcare-associated outbreaks of Bcc (B. cepacia and B. contaminans) in the United Kingdom and Ireland associated with contaminated products. We highlight the challenges during investigation and mitigation, and provide recommendations. METHODS:A multidisciplinary, multi-stakeholder investigation across both outbreaks was adopted, involving interviews, trawling questionnaires, and targeted product sampling. RESULTS:There were 153 and 66 confirmed cases in the B. cepacia and B. contaminans outbreaks, respectively. Cases predominantly comprised hospitalized individuals, and 70% of isolates were recovered from an invasive site. The outbreak strain of B. cepacia was isolated from non-sterile ultrasound gel. An analytical study suggested the B. contaminans cluster was also linked to ultrasound gel; however, a disinfectant wipe product was subsequently identified as the source (0-3 single nucleotide polymorphism differences between case and wipe isolates). OUTBREAK CONTROL MEASURES:The affected disinfectant wipes were withdrawn. The ultrasound gel was not recalled but health system procurement was suspended, rapid clinical guidance was produced and a National Patient Safety Alert was issued. Inter-organizational partnership was required to mitigate risks. CONCLUSION:Identifying the source of outbreaks associated with contaminated products can be challenging, requiring complex multi-stakeholder interventions. We recommend a low threshold for investigation of Bcc clusters, adopting a multidisciplinary approach to investigation and mitigation, implementing interventions focusing on practice, and prompt product removal to protect patients.

In September 2023, the UK Health Security Agency's (UKHSA) South West Health Protection Team received notification of patients with Pseudomonas aeruginosa perichondritis. All five cases had attended the same cosmetic piercing studio and a multi-disciplinary outbreak control investigation was subsequently initiated. An additional five cases attending the same studio were found. Seven of the ten cases had isolates available for Variable Number Tandem Repeat (VNTR) typing at the UKHSA national reference laboratory. Clinical and environmental P. aeruginosa isolates from the patients, handwash sink, tap water and throughout the wall-mounted point-of-use water heater (including outlet water) were indistinguishable by VNTR typing (11,6,2,2,1,3,6,3,11). No additional cases were identified after control measures were implemented, which included replacing the sink and point-of-use heater.The lack of specific recommendations to control for P. aeruginosa within Council-adopted ear-piercing byelaws or national guidance means that a cosmetic piercing artist could inadvertently overlook the risks from this bacterial pathogen despite every intention to comply with the law and follow industry best practice advice. Clinicians, Environmental Health Officers and public health professionals should remain alert for single cases of Pseudomonas perichondritis infections associated with piercings and have a low threshold for notification to local health protection teams.

Carbapenem-resistant Enterobacterales are a significant threat to global public health. Here, we characterize bla OXA-23-positive Proteus mirabilis (n=8) and Escherichia coli (n=3) isolates from human clinical samples collected between 2021 and 2024 in the UK. Whole-genome sequencing (WGS) was used to generate data, and a phylogenetic tree inferred from SNPs filtered for recombination was constructed to assess the genomic relatedness among the isolates. To provide an international context, we included publicly available genomes. Short-read mapping to a reference genome enabled reconstruction of the genomic neighbourhood around bla OXA-23. Minimum inhibitory concentration (MIC) determination was performed using broth microdilution and results interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. UK P. mirabilis isolates belonged to ST142 and formed a sub-clade descending from an ancestral cluster of French isolates with relatively few SNPs between them (9-39). E. coli ST38 isolates harboured bla OXA-23 and showed close genetic relatedness (12-15 SNPs) among themselves. In P. mirabilis, bla OXA-23 was associated with transposon Tn6703, while E. coli harboured a novel composite transposon, designated Tn7816, bordered by two copies of IS26 and with three copies of bla OXA-23. bla OXA-23 was integrated into the chromosome in all isolates. All isolates were resistant to amoxicillin/clavulanic acid (>32 mg l-1) and with meropenem MICs above the EUCAST screening cut-off (0.5-1 mg l-1). In conclusion, UK bla OXA-23-positive P. mirabilis isolates belong to the same clonal lineage (ST142) previously reported in Belgium, Germany, Switzerland and France, suggesting introduction of this lineage into the UK. This is the first report of an E. coli ST38 lineage with chromosomally encoded bla OXA-23 located within a novel transposon Tn7816. WGS plays an important role in identifying the mechanism(s) of transmission of emerging carbapenemase genes.

This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile—KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital—were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried bla KPC-2-like alleles (KpMVR1: bla KPC-157; KpMVS1 and KpMVS2: bla KPC-2) in presumptively conjugative plasmids. IS Ec68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summary Illumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information ([www.ncbi.nlm.nih.gov][1]) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institute’s database for K. pneumoniae species complex ([bigsdb.pasteur.fr/klebsiella/][2]) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statement This is the first bla KPC-positive K. pneumoniae isolate referred to the UK’s national reference laboratory with resistance to three last-resort β-lactam/β-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne bla KPC-2-like allele bla KPC-157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an IS Ec68 -induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission. ### Competing Interest Statement The authors have declared no competing interest. UK Health Security Agency, https://ror.org/018h10037 University of Liverpool, https://ror.org/04xs57h96, UGG10057 Wellcome Trust, PSN109 National Institute for Health and Care Research, NIHR200876 [1]: https://www.ncbi.nlm.nih.gov [2]: http://bigsdb.pasteur.fr/klebsiella/

The incidence of carbapenemase-producing Enterobacterales (CPE) is rising globally, yet Imipenemase (IMP) carbapenemases remain relatively rare. This study describes an investigation of the emergence of IMP-encoding CPE amongst diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network. A network analysis approach to patient pathways, using routinely collected electronic health records, identified previously unrecognised contacts between patients who were IMP CPE positive on screening, implying potential bacterial transmission events. Whole genome sequencing of 85 Enterobacterales isolates from these patients revealed that 86% (73/85) were diverse species (predominantly Klebsiella spp, Enterobacter spp, E. coli) and harboured an IncHI2 plasmid, which carried both blaIMP and the putative mobile colistin resistance gene mcr-9. Detailed phylogenetic analysis identified two distinct IncHI2 plasmid lineages, A and B, both of which showed significant association with patient movements between four hospital sites and across medical specialities. Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard microbiology and infection control investigations. With whole genome sequencing (WGS) technologies and large-data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Analysing outbreaks at the plasmid level reveals that resistance may be wider spread than suspected, allowing more targetted interventions to stop the transmission of resistance within hospital networks.

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.

107 isolates of Enterobacterales consisting of Klebsiella pneumoniae (n=90), Escherichia coli (n=7), Enterobacter cloacae complex (n=6), Klebsiella oxytoca complex (n=3) and Citrobacter freundi (n=1) and additionally an isolate of Acinetobacter baumannii carrying genes encoding NDM (NDM-1, NDM-5 and NDM-14), KPC (KPC-2 and KPC-3), OXA-48-like (OXA-48, OXA-181 and OXA-232) and IMP (IMP-1 and IMP-4) carbapenemases were sequenced using q20 nanopore chemistry to provide complete/near-complete assemblies and relevant plasmids compared. Investigation of potential ‘plasmid outbreaks’ in individual hospitals among isolates of different types and species revealed a mixed situation with some isolates carrying similar plasmids, but with segments missing/added and some plasmids that were clearly distinct. While most plasmids carrying bla OXA-48 were typical IncL plasmids of approximately 60 kb that are widely described, there was some variation among these. One isolate carried an IncR plasmid that had only limited homology with the others. Identical 51,479 bp ColKP3/IncX3 plasmids carrying bla OXA-181 were found from isolates from different hospitals that exactly matched those on GenBank from other countries. In other isolates bla OXA-181 was carried on IncFII plasmids. bla OXA-232 was found in highly conserved small ColKP3 plasmids that matched those found in other countries and continents. These observations highlight the importance of understanding the wider distribution of plasmids of concern. IncHI2/IncHI2A plasmids were important vehicles for carbapenemase genes and were found with bla KPC-2, bla IMP-1, bla IMP-4 or bla NDM-1, sometimes with the colistin resistance gene mcr-9 in addition. Representatives of K. pneumoniae sequence type (ST) 147 from seven hospitals carried IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) hybrid virulence resistance plasmids of 325 to 352 kb that combined bla NDM-5 and other resistance genes with genes found in virulence plasmids. A similar plasmid was also found in an isolate of K. pneumoniae ST1558 and has been described in representatives of ST383. Nanopore sequencing has been instrumental in improving our knowledge of plasmids carrying carbapenemase genes leading to a better understanding of their epidemiology. ### Competing Interest Statement The authors have declared no competing interest.

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-β-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.

Background Pain is the most common symptom of Paget’s Disease of Bone (PDB) and affects between 60% and 70% of patients who present to clinic in the UK, but its mechanisms are incompletely understood [1]. Quantitative sensory testing (QST) has historically been used in the assessment of neuropathic pain [2]. but has precedent in patients with bone metastases and osteoarthritis [3,4]. In this study we used QST to dissect the mechanisms of pain in PDB, taking advantage of the disease’s focal nature which allows the participant to act as their own control by testing at a contralateral site. Objectives To explore the sensory profiles of PDB patients with and without musculoskeletal pain. Methods All study data was collected and stored on the secure web-based software platform Research Electronic Data Capture (REDCap). Quantitative sensory testing (QST) measured the sensory response to various stimuli applied to the skin overlying the Pagetic bone and compared this to an unaffected site in 120 study participants. Differences between QST assessments in sites overlying Pagetic bone and control sites were assessed by the Mann-Whitney test. Results Significantly higher pain scores were reported above the site of PDB when compared to the control site, irrespective of whether the patient complained of musculoskeletal pain. The differences were apparent for hot roller testing (p = 0.047), pain threshold testing (p = 0.007), pin prick testing (p= <0.001) (see Figure 1), and wind-up pinprick testing (p = <0.001). Pain thresholds were significantly reduced above Pagetic bone (p = 0.007). Additionally, temporal summation scores were significantly higher (p = 0.007) in the pain subgroup compared with the no pain subgroup which could indicate higher levels of central sensitisation in patients who experience pain. Conclusion This study illustrates that there are significant differences in the way that sensation is processed above Pagetic bone when compared to unaffected contralateral sites, regardless of the presence of current musculoskeletal pain. Our data suggest that patients who experience pain may do so, in part, because of higher levels of central sensitisation. References [1]Langston AL, Campbell MK, Fraser WD, MacLennan GS, Selby PL, Ralston SH (2010) Randomised Trial of Intensive Bisphosphonate Treatment Versus Symptomatic Management in Paget’s Disease of Bone. J. Bone Miner. Res 25:20-31 [2]Rolke R, Baron, R., Maier, C.A., Tölle, T.R., Treede, R.D., Beyer, A., Binder, A., Birbaumer, N., Birklein, F., Bötefür, I.C. and Braune, S. (2006) Quantitative sensory testing in the German Research Network on Neuropathic Pain (DFNS): standardized protocol and reference values. 123:231-243 [3]Scott AC, McConnell, S., Laird, B., Colvin, L. and Fallon, M. (2012) Quantitative Sensory Testing to assess the sensory characteristics of cancer-induced bone pain after radiotherapy and potential clinical biomarkers of response. 16:123-133 [4]Kosek E, Ordeberg G (2000) Lack of pressure pain modulation by heterotopic noxious conditioning stimulation in patients with painful osteoarthritis before, but not following, surgical pain relief. 88:69-78 Acknowledgements: NIL. Disclosure of Interests Kathryn Berg: None declared, Dervil Dockrell: None declared, Terry Aspray: None declared, Lesley Colvin: None declared, Elaine Dennison Speakers bureau: Speakers/consultancy fees - Viatris, Lilly, UCB, Pfizer, Hrushikesh Divyateja Speakers bureau: Speaker fee from Amgen, Sanofi, and Dianchi Sankyo for talks about their products, Nazim Ghouri Speakers bureau: Honoraria from Boehringer Ingelheim and Novo Nordisk for diabetes related talks over the past 3 years, Richard Keen: None declared, Terence O’Neill Grant/research support from: Financial support to attend conference from UCB, Faiz Rahman Speakers bureau: Have received speakers fees from Sanofi in relation to their product ‘Praluent’ a lipid lowering drug, and from Amgen in relation to their product ‘Repatha’ another lipid lowering drug., Grant/research support from: Department has received grants from Sanofi., Mashood Siddiqi Speakers bureau: Received Speaker fees from, Thornton and Ross, Amgen, Lilly Pharmaceuticals, MSD and Servier Labs, Stephen Tuck Speakers bureau: Speaker fees from UCB to speak about NOGG and romosozumab, Jane Turton Speakers bureau: A speaker fee from AMGEN for a talk about a self-injection service, Grant/research support from: The Bone Research Unit in Cardiff received an educational grant from AMGEN to start up the self-injection service., Stuart Ralston Speakers bureau: Speaker fees from UCB Pharma, Novartis, Janssen (fees paid to University of Edinburgh)., Grant/research support from: Research support from Astra-Zeneca and Kyowa Kirin (Local investigator on clinical studies, fees paid to NHS Lothian). Eli Lilly donated an IMP for clinical trial. Abbvie, Alexion, Amgen, Bristol Myers-Squibb, Celgene, Consilient Health, Janssen, Eli-Lilly, Novartis, Pfizer, Roche, Sandoz, Sanofi-Genzyme, Thornton & Ross, UCB all provided sponsorship of 14th Scientific Symposium on Bone and Joint disease 2022 (Fees paid to University of Edinburgh).

BACKGROUND:We aimed to describe the UK Pseudomonas aeruginosa population structure amongst people with cystic fibrosis (PWCF), and to examine evidence for cross-infection.METHODS:Variable Number Tandem Repeat (VNTR) typing was performed on 4640 isolates from 2619 PWCF received from 55 hospital laboratories between 2017 and 2019. A combination of whole genome sequence (WGS)-based analysis of four clusters from one hospital, and epidemiological analysis of shared strains in twelve hospitals evaluated cross-infection.RESULTS:Of 2619 PWCF, 1324 (51%) harboured common clusters or known transmissible strains, while 1295 carried unique strains/those shared among small numbers of patients. Of the former, 9.5% (250 patients) harboured the Liverpool epidemic strain (LES), followed in prevalence by clone C (7.8%; 205 patients), cluster A (5%;130 patients), and cluster D (3.6%; 94 patients). WGS analysis of 10 LES isolates, 9 of cluster D and 6 isolates each of cluster A and clone C from one hospital revealed LES formed the tightest cluster (between 7 and 205 SNPs), and cluster D the loosest (between 53 and 1531 SNPs). Hospital-specific shared strains were found in some centres, although cross-infection was largely historical, with few new acquisitions. Fifty-nine PWCF (2.3%) harboured "high-risk" clones; one ST235 isolate carried a blaIMP-1 allele.CONCLUSION:Of 2619 PWCF who had P. aeruginosa isolates submitted for VNTR, 51% harboured either common clusters or known transmissible strains, of which LES was the most common. Limited evidence of recent patient-to-patient strain transmission was found, suggesting cross-infection prevention measures and surveillance effectively reduce transmission.

A multidrug- resistant strain of Klebsiella pneumoniae (Kp) sequence type (ST) 1788, an otherwise uncommon ST worldwide, was isolated from 65 patients at 11 hospitals and 11 general practices across South and West Wales, UK, between February 2019 and November 2021. A collection of 97 Kp ST1788 isolates (including 94 from Wales) was analysed to investigate the diversity and spread across Wales and to identify molecular marker(s) to aid development of a strain- specific real- time PCR. Whole genome sequencing (WGS) was performed with Illumina technology and the data were used to perform phylogenetic analyses. Pan- genome analysis of further Kp genome collections was used to identify an ST1788- specific gene target; a real- time PCR was then validated against a panel of 314 strains and 218 broth- enriched screening samples. Low genomic diversity was demonstrated amongst the 94 isolates from Wales. Evidence of spread within and across healthcare facilities was found. A yersiniabactin locus and the KL2 capsular locus were identified in 85/94 (90.4 %) and 94/94 (100 %) genomes respectively; blaSHV-232, blaTEM-1, blaCTX-M-15 and blaOXA-1 were simultaneously carried by 86/94 (91.5 %) isolates; 4/94 (4.3 %) isolates also carried blaOXA-48 carbapenemase. Aminoglycoside and fluoroquinolone resistance markers were found in 94/94 (100 %) and 86/94 (91.5 %) isolates respectively. The ST1788- specific real- time PCR was 100 % sensitive and specific. Our analyses demonstrated recent clonal expansion and spread of Kp ST1788 in the community and across healthcare facilities in South and West Wales with isolates carrying well- defined antimicrobial resistance and virulence markers. An ST1788- specific marker was also identified, enabling rapid and reliable preliminary characterization of isolates by real- time PCR. This study confirms the utility of WGS in investigating novel strains and in aiding proactive implementation of molecular tools to assist infection control specialists.

A hospital outbreak of carbapenem- resistant Enterobacterales was detected by routine surveillance. Whole genome sequencing and subsequent analysis revealed a conserved promiscuous blaOXA-48 carrying plasmid as the defining factor within this outbreak. Four different species of Enterobacterales were involved in the outbreak. Escherichia coli ST399 accounted for 35 of all the 55 isolates. Comparative genomics analysis using publicly available E. coli ST399 genomes showed that the outbreak E. coli ST399 isolates formed a unique clade. We developed a mathematical model of pOXA- 48- like plasmid transmission between host lineages and used it to estimate its conjugation rate, giving a lower bound of 0.23 conjugation events per lineage per year. Our analysis suggests that co- evolution between the pOXA- 48- like plasmid and E. coli ST399 could have played a role in the outbreak. This is the first study to report carbapenem- resistant E. coli ST399 carrying blaOXA- 48 as the main cause of a plasmid- borne outbreak within a hospital setting. Our findings suggest complementary roles for both plasmid conjugation and clonal expansion in the emergence of this outbreak.

Introduction. The New Delhi metallo-β-lactamase (NDM) variant NDM-5 was first described in 2011 in an isolate of Escherichia coli . We noted that a high proportion of isolates of E. coli positive for bla NDM carbapenemase genes submitted to the UK Health Security Agency (formerly Public Health England) between 2019 and mid-2021 carried the bla NDM-5 allele, with many co-harbouring rmtB, rendering them highly resistant to aminoglycosides as well as to most β-lactams. Hypothesis/Gap Statement. This observation suggested that a common plasmid may be circulating. Aim. To compare these isolates and describe the plasmids carrying these resistance elements. Methodology. All isolates were sequenced on an Illumina platform, with five also subjected to long-read nanopore sequencing to provide complete assemblies. The locations of bla NDM-5, rmtB and other associated genetic elements were identified. Susceptibility testing to a wide range of antibiotics was carried out on representative isolates. Results. The 34 isolates co-harbouring bla NDM-5 and rmtB were from 14 hospital groups and six different regions across England and consisted of 11 distinct sequence types. All carried IncF plasmids. Assembly of the NDM plasmids in five isolates revealed that they carried rmtB and bla NDM-5 in an IncF conjugative plasmid ranging in size from 85.5 to 161 kb. All carried a highly conserved region, previously described in E. coli plasmid pHC105-NDM, that included bla TEM-1B and rmtB followed by sequence bounded by two IS26 elements containing ΔISAba125, bla NDM-5, ble, trpF and tat followed by ISCR1 and an integron with sul1, aadA2 and dfrA12 cassettes. This arrangement has been described in isolates from other countries and continents, suggesting that such plasmids are widely distributed, at least in E. coli , with similar plasmids also found in Klebsiella pneumoniae . Tested isolates were resistant to most antibiotics except colistin, fosfomycin and tigecycline. Conclusion. These observations suggest that conjugative plasmids carrying a highly conserved resistance gene segment have become widespread in England and elsewhere. This study highlights the value of routine whole-genome sequencing in identifying genetic elements responsible for resistance dissemination.

Introduction. Klebsiella species are some of those most implicated in neonatal sepsis. However, many isolates from infections appear unremarkable; they are generally susceptible to antibiotics and often of sporadic types not associated with virulence. Hypothesis/Gap Statement. Investigation is needed to identify if such isolates have virulence characteristics. Aim. To sequence multiple isolates of a range of types from cases of neonatal invasive disease to identify elements that may explain their virulence, and to determine if such elements are more common among these isolates than generally. Methodology. In total, 14 isolates of K. pneumoniae/K. variicola belonging to 13 distinct types from blood or CSF from neonatal infections were sequenced using long -read nanopore technology. PCR assays were used to screen a general set of isolates for heavy metal resistance genes arsC, silS and merR. Results. Overall, 12/14 isolates carried one or more plasmids. Ten carried a large plasmid (186 to 310 kb) containing heavy metal resistance genes associated with hypervirulence plasmids, with most (nine) carrying genes for resistance to copper, silver and one other heavy metal (arsenic, tellurite or mercury), but lacking the genes encoding capsule-upregulation and siderophores. Most isolates (9/14) lacked any additional antibiotic resistance genes other than those intrinsic in the species. However, a representative of an outbreak strain carried a plasmid containing bla(CTX-M-15), qnrS1, aac3_IIa, dfrA17, sul1, mph(A), tet(A), bla(TEM1B) and aadA5, but no heavy metal resistance genes. arsC, silS and merR were widely found among 100 further isolates screened, with most carbapenemase- gene-positive isolates (20/27) carrying at least one. Conclusion. Plasmids containing heavy metal resistance genes were a striking feature of isolates from neonatal sepsis but are widely found. They share elements in common with virulence and antibiotic resistance plasmids, perhaps providing a basis from which such plasmids evolve.

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.