Biology subjects

Moran, R. A.

Publications and source records attributed to Moran, R. A..

13 recordsLinked to original sources

bioRxiv2025

Genomic rearrangement of the capsule operon in a phylogenetically distinct cluster of the multi-drug resistant Escherichia coli lineage ST131

Escherichia coli ST131 is a dominant cause of multi-drug resistant extra-intestinal infections in humans. The lineage has a three-clade phylogenetic structure of which clade C has gained resistance to multiple classes of antibiotics and is globally disseminated. As well as antimicrobial resistance, a number of virulence factors contribute to the success of ST131 clade C, including the production of capsule. Previous work on a limited number of strains has shown that the capsule operon in ST131 is highly variable. In this study we analysed the capsule genetics of E. coli ST131 by interrogating 3,033 genomes. Our data show a clonal distribution of capsule types and identify a monophyletic group of ST131 clade C strains which are characterised by an atypical capsule operon with highly divergent alleles of the key kpsM and kpsT genes. Our analysis suggests there is ongoing evolution within clade C of ST131 which looks to be driven not by continuing evolution of antimicrobial resistance, but by host-pathogen interactions.

microbiology↗

bioRxiv2025

IS1216 drives the evolution of pRUM-like multidrug resistance plasmids in Enterococcus faecium

pRUM-like plasmids are commonly found in multidrug-resistant Enterococcus faecium, but the evolution of these plasmids has not been characterised in detail. When we analysed the genome sequences of two clinical E. faecium strains isolated in Birmingham, UK, we found two pRUM-like plasmids, pHHEf1 and pHHEf2. They were approximately 25 kb in size and shared the same 10 kb backbone, but contained starkly different accessory regions that were bounded by and interspersed with the IS26 family insertion sequence IS1216. pHHEf1 contained a complete set of vancomycin resistance genes, while pHHEf2 contained aminoglycoside and erythromycin resistance genes along with an integrated small plasmid, pCOLA. It appeared that IS1216 had driven the diversification of these accessory regions. We sought to characterise the role of IS1216 in the broader evolution of pRUM-like plasmids by performing comparative analyses on 152 complete plasmid sequences from five continents. Extensive IS1216-mediated variation included backbone deletions, acquisition and loss of 10 different antibiotic resistance genes, and the formation of cointegrates with plasmids of at least 10 different replicon types. Cointegration events have introduced accessory segments with diverse functions, including horizontal transfer determinants and genes for bacteriocin T8. The derivations of these acquired segments highlight the impact of IS1216 in driving gene exchange between Enterococcus and Staphylococcus species. We traced the emergence of the pRUM-like lineage to a putative ancestor found in a vancomycin-sensitive ST17 E. faecium isolated in 1997. The ancestral plasmid, pCANE, includes the entire pRUM backbone with an additional 44.9 kb in place of the pRUM accessory region. The 44.9 kb segment includes putative conjugation determinants, suggesting that the emergence of the pRUM-like lineage coincided with a loss of transfer functions. We propose an IS1216 driven model for the evolution of pRUM-like plasmids, which appear to have arisen in E. faecium ST17 and contributed to the international success of CC17 as an opportunistic pathogen. Impact StatementExperimental work has shown that the insertion sequence IS1216, found in Enterococcus, shares the cointegrate formation properties of the well-characterised IS26 of Gram-negative bacteria, which is a major contributor to the movement and accumulation of antibiotic resistance genes in important human pathogens. It seems likely that IS1216 plays a similar role in the emergence and spread of multidrug resistance in Enterococcus. However, the actions of IS1216 in the genus have not been the subject of focused comparative studies. Here, we provide genomic evidence for the crucial role of IS1216 in the evolution of the pRUM-like multidrug resistance plasmid lineage that has been found in E. faecium isolated globally. We have traced the evolution of the pRUM-like lineage to a cryptic ancestral plasmid found in a vancomycin-sensitive human clinical strain isolated in the 1990s.

genomics↗

bioRxiv2025

The Kocurious case of Noodlococcus: genomic insights into Kocuria rhizophila from characterisation of a laboratory contaminant

The laboratory contaminant Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2732108 bp chromosome that shared 97.5% Average Nucleotide Identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt), and genome defence (CRISPR-Cas), and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation, and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic, and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as K. pseudorhizophila, exhibited ANI values of <95% relative to TA68 and constitutes a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy. Impact statementBacterial genome sequence databases are dominated by a relatively small number of medically relevant genera, while most of the global bacterial populations diversity is largely uncharacterised. Kocuria is a widespread bacterial genus with industrial and medical relevance. Despite its ubiquity, only 22 complete and 235 draft Kocuria genomes were publicly available at the outset of this study. Our phylogenetic and pangenomic examination of all available Kocuria genomes was the first for this genus, providing insights into its diversity and taxonomy. Most notably, we found that Kocuria rhizophila is comprised of two clades that are sufficiently divergent to constitute different species, but are frequently used interchangeably in experimental and genomic research. The complete, high-quality Noodlococcus genome generated and characterised here can serve as a reference for true K. rhizophila, particularly while there is only a draft genome sequence available for type strain TA68. Data summarySequencing reads and the assembled Noodlococcus genome are available from NBCI BioProject accession PRJNA835814 and BioSample accession SAMN28111796. The complete sequence of the Noodlococcus chromosome can be found in the GenBank nucleotide database under accession number CP097204.1. Entries for the novel insertion sequences ISKrh4 to ISKrh7 can be found in the ISFinder database (https://isfinder.biotoul.fr).

genomics↗

bioRxiv2025

Staphylococcus haemolyticus is a reservoir of antibiotic resistance genes in the preterm infant gut

Among coagulase-negative staphylococci, Staphylococcus haemolyticus is a primary cause of bloodstream infections in preterm infants, with gut colonisation being recognised as a risk factor for subsequent infection. Through a re-analysis of a 16S rRNA gene sequencing dataset (n=497 preterm infants), we found that S. haemolyticus was abundant and prevalent in the gut in the first month of life. To better understand the diversity of S. haemolyticus among preterm infants, we generated genome sequences of S. haemolyticus strains (n=140), which were isolated from 44 stool samples of 22 preterm infants from four different hospitals in the United Kingdom. Core genome phylogenetic analyses, incorporating 126 publicly available S. haemolyticus genome sequences, showed that 85/140 (60.1%) of the isolates, from three different hospitals, formed a clonal group with 79/85 (92.9%) strains being assigned to Multi-Locus Sequence Type (ST) 49. Antibiotic resistance genes were highly prevalent in the genome sequences. Using logistic regression, we found a strong association between the presence of the gene mecA and phenotypic resistance to oxacillin (odds ratio [OR]: 158.00, p<0.0001), and the aacA-aphD gene and phenotypic resistance to gentamicin aacA-aphD (OR: 162.00, p<0.001). None of the strains from the preterm infant cohort had a complete Staphylococcal Cassette Chromosome mec (SCCmec) element. The aacA-aphD gene was associated with the transposon Tn4001. Using hybrid genome assemblies, we found it to be present on the chromosome (54.5% of strains) or on diverse plasmids (27.3%). Four strains (18.2%) had Tn4001 copies on both plasmid and chromosome. Our data suggest the existence of a distinct sub-population of S. haemolyticus that has adapted to colonise the gut of preterm infants. Prevalent resistance to antibiotics is of clinical concern and the diversity of genetic contexts of mecA and Tn4001 suggests widespread horizontal gene transfer and recombination in this species.

microbiology↗

bioRxiv2024

Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug resistant Gram-negative pathogens

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug resistant organisms (MDRO) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by and transmission of MDRO, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focussing primarily on E. coli as both a widespread commensal and a globally disseminated multidrug resistant pathogen. Samples were taken during highly restrictive COVID-19 control measures from May - December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain level colonisation of patients, focussing on E. coli sequence types (STs), their colonisation dynamics, and antimicrobial resistance (AMR) gene carriage. We found a lack of multidrug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying blaKPC-2. There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli. Whilst 22 different E. coli STs were identified, only one strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result which may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR. Impact statementThis study contributes to current literature on the potential routes for AMR spread in a healthcare setting. This study used whole genome sequencing (WGS) to investigate at strain-level bacterial species (including E. coli) colonising the gut of long-stay patients in the ICU. WGS in combination with patient ward movement and prescribing information was used to assess any links or driving factors in strain acquisition and AMR spread in the ICU. Our study gives an insight at a point in time where infection and prevention control restrictions and awareness were high due to the COVID-19 pandemic, combined with local and national travel restrictions and isolation criteria. Consequently, it provides a novel longitudinal dataset that gives a picture of colonising E. coli in a sheltered ICU patient population. Trends seen in this E. coli population are likely linked to the United Kingdom in 2021 rather than the global picture that may have been seen prior to the COVID-19 pandemic. Data summaryAll supporting data, code and protocols have been provided within the article or through supplementary data files. All genomic and metagenomic data are available from NCBI under BioProject accession PRJNA1136496. All relevant metadata is provided in supplementary data files.

microbiology↗

bioRxiv2023

Differential development of antibiotic resistance and virulence between Acinetobacter species

The two species that account for most cases of Acinetobacter-associated bacteraemia in the UK are Acinetobacter lwoffii, often a commensal but also an emerging pathogen, and A. baumannii, a well-known antibiotic-resistant species. While these species both cause similar types of human infection and occupy the same niche, A. lwoffii (unlike A. baumannii) has thus far remained susceptible to antibiotics. Comparatively little is known about the biology of A. lwoffii and this is the largest study on it conducted to date, providing valuable insights into its behaviour and potential threat to human health. This study aimed to explain the antibiotic susceptibility, virulence, and fundamental biological differences between these two species. The relative susceptibility of A. lwoffii, was explained as it encoded fewer antibiotic resistance and efflux pump genes than A. baumannii (9 and 30 respectively). While both species had markers of horizontal gene transfer, A. lwoffii encoded more DNA defence systems and harboured a far more restricted range of plasmids. Furthermore, A. lwoffii displayed a reduced ability to select for antibiotic resistance mutations, form biofilm and infect both in vivo and in vitro models of infection. This study suggests that the emerging pathogen A. lwoffii has remained susceptible to antibiotics because mechanisms exist to make it highly selective about the DNA it acquires, and we hypothesise that the fact that it only harbours a single RND system restricts the ability to select for resistance mutations. This provides valuable insights into how development of resistance can be constrained in Gram negative bacteria. ImportanceAcinetobacter lwoffii is often a harmless commensal but is also an emerging pathogen and is the most common cause of Acinetobacter-derived blood stream infections in England and Wales. In contrast to the well-studied, and often highly drug resistant A. baumannii, A. lwoffii has remained susceptible to antibiotics. This study explains why this organism has not evolved resistance to antibiotics. These new insights are important to understand why and how some species develop antibiotic resistance, while others do not and could inform future novel treatment strategies.

microbiology↗

bioRxiv2023

Non-antibiotic pharmaceuticals exhibit toxicity against Escherichia coli at environmentallyrelevant concentrations with no evolution of cross-resistance to antibiotics

Antimicrobial resistance can arise in the natural environment via prolonged exposure to the effluent surrounding manufacturing facilities. These facilities also produce non-antibiotic pharmaceuticals, and the effect of these on the surrounding microbial communities is less clear; whether they have inherent toxicity, or whether long-term exposure might select for cross-resistance to antibiotics. To this end, we screened four non-antibiotic pharmaceuticals (acetaminophen, ibuprofen, propranolol, met formin) and titanium dioxide for toxicity against Escherichia coli K-12 MG1655 and conducted a 30 day selection experiment to assess the effect of long-term exposure. All compounds reduced the maximum optical density reached by E. coli at a range of concentrations including one of environmental relevance, with transcriptome analysis identifying upregulated genes related to stress response and multidrug efflux in response ibuprofen treatment. The non-antibiotic pharmaceuticals did not select for significant genetic changes following a 30 day exposure, and no evidence of selection for cross-resistance to antibiotics was observed for population evolved in the presence of ibuprofen in spite of the differential gene expression after exposure to this compound. This work suggests that these non-antibiotic pharmaceuticals, at environmental concentrations, do not select for cross-resistance to antibiotics in E. coli.

microbiology↗

bioRxiv2023

Growth in a biofilm promotes conjugation of a blaNDM-1-bearing plasmid between Klebsiella pneumoniae strains

Antimicrobial resistance (AMR) is a growing problem, especially in Gram-negative Enterobacteriaceae such as Klebsiella pneumoniae. Horizontal transfer of conjugative plasmids contributes to AMR gene dissemination. Bacteria such as K. pneumoniae commonly exist in biofilms, yet most studies focus on planktonic cultures. Here we studied the transfer of a multidrug resistance plasmid in planktonic and biofilm populations of K. pneumoniae. We determined plasmid transfer from a clinical isolate, CPE16, which carried four plasmids, including the 119-kbp blaNDM-1-bearing F-type plasmid pCPE16_3, in planktonic and biofilm conditions. We found that transfer frequency of pCPE16_3 in a biofilm was orders-of-magnitude higher than between planktonic cells. In 5/7 sequenced transconjugants multiple plasmids had transferred. Plasmid acquisition had no detectable growth impact on transconjugants. Gene expression of the recipient and a transconjugant was investigated by RNA-sequencing in three lifestyles: planktonic exponential growth, planktonic stationary phase, and biofilm. We found that lifestyle had a substantial impact on chromosomal gene expression, and plasmid carriage affected chromosomal gene expression most in stationary planktonic and biofilm lifestyles. Furthermore, expression of plasmid genes was lifestyle-dependent, with unique signatures across the three conditions. Our study shows that growth in biofilm greatly increased the risk of conjugative transfer of a carbapenem resistance plasmid in K. pneumoniae without fitness costs and minimal transcriptional rearrangements, thus highlighting the importance of biofilms in the spread of AMR in this opportunistic pathogen. ImportanceCarbapenem-resistant K. pneumoniae is particularly problematic in hospital settings. Carbapenem resistance genes can transfer between bacteria via plasmid conjugation. Alongside drug resistance, K. pneumoniae can form biofilms on hospital surfaces, at infection sites and on implanted devices. Biofilms are naturally protected and can be inherently more tolerant to antimicrobials than their free-floating counterparts. There have been indications that plasmid transfer may be more likely in biofilm populations, thus creating a conjugation hotspot. However, there is no clear consensus on the effect of the biofilm lifestyle on plasmid transfer. Therefore, we aimed to explore the relationship between plasmid transfer and biofilms, and the impact of plasmid acquisition on the host bacterial cell. Our data show resistance plasmid transfer is greatly increased in a biofilm versus planktonic growth, which may be a significant contributing factor to the rapid dissemination of resistance plasmids in K. pneumoniae.

microbiology↗

bioRxiv2022

Extended-spectrum beta-lactamase genes traverse the Escherichia coli populations of ICU patients, staff and environment.

Over a three-month period, we monitored the population of extended-spectrum {beta}-lactam-resistant Escherichia coli (ESBL-EC) associated with the patients, staff and environment of an intensive care unit (ICU) in Guangzhou, China. Thirty-four clinical isolates were obtained from the same hospital 12 months later. A total of 165 isolates were characterised and whole-genome sequenced, with 24 isolates subjected to long-read sequencing. The diverse population included representatives of 59 different sequence types (STs). ICU patient and environmental isolates were largely distinct from staff isolates and clinical isolates. We observed five instances of highly similar isolates (0-13 core-gene SNPs) being obtained from different patients or bed unit environments. ESBL resistance in this collection was largely conferred by blaCTX-M genes, which were found in 96.4% of all isolates. The contexts of blaCTX-M genes were diverse, situated in multiple chromosomal positions and in various plasmids. We identified blaCTX-M-bearing plasmid lineages that were present in multiple STs across the surveillance, staff and clinical collections. Closer examination of ISEcp1-blaCTX-M transposition units shed light on the dynamics of their transmission, with evidence for the acquisition of chromosomal copies of blaCTX-M genes from specific plasmid lineages, and for the movement of blaCTX-M-55 from a ST1193 chromosome to a small mobilisable plasmid. A carbapenem-resistant ST167 strain isolated from a patient that had been treated with meropenem and piperacillin-tazobactam contained seven copies of blaCMY-146, which appears to have been amplified by IS1. Our data revealed limited persistence and movement of ESBL-EC strains in the ICU environment, but we observed circulating plasmid lineages playing an essential and ongoing role in shaping the cephalosporin-resistance landscape in the population examined. Impact statementESBL resistance significantly impacts clinical management of E. coli infections in hospitals globally. It is important to understand the structures of ESBL-EC populations carried by hospital patients and staff, their capacity to persist in hospital environments, and the dynamics of mobile genes that drive the spread of ESBL resistance. In our three-month study, ESBL-EC strains found in the ICU environment were strongly associated with patient carriage, but distinct from strains found in staff. However, plasmid lineages carrying blaCTX-M genes were found across the ICU populations and in a collection of clinical isolates obtained one year later. By examining their content and contexts, we have traced the recent histories of chromosomal and plasmid-borne ISEcp1-blaCTX-M transposition units in the ICU population. This allowed us to implicate specific plasmid lineages in the acquisition of chromosomal blaCTX-M genes, even when the plasmids were no longer present, and to detect recent transposition of blaCTX-M-55 from a chromosome to a mobilisable plasmid. Similar high-resolution approaches to the study of mobile genetic elements will be essential if the transmission routes associated with the spread of ESBL resistance are to be understood and subjected to interventions. Data summarySequencing reads are available under NCBI BioProject accession PRJNA907549. The 91 complete plasmid sequences generated in this study are in a supplementary file called pDETEC_collection.fa.

microbiology↗

bioRxiv2022

Integrons, plasmids, and resistance genes in equine faecal bacteria

Antimicrobial resistance in bacteria is a threat to both human and animal health. We aimed to understand the impact of domestication and antimicrobial treatment on the types and numbers of resistant bacteria, antibiotic resistance genes (ARGs), and class 1 integrons (C1I) in the equine gut microbiome. Antibiotic-resistant faecal bacteria were isolated from wild horses, healthy farm horses, and horses undergoing veterinary treatment, and isolates (9,083 colonies) were screened by PCR for C1I; these were found at frequencies of 9.8% (vet horses), 0.31% (farm horses), and 0.05% (wild horses). A collection of 71 unique C1I+ isolates (17 Actinobacteria and 54 Proteobacteria) was subjected to resistance profiling and genome sequencing. Farm horses yielded mostly C1I+ Actinobacteria (Rhodococcus, Micrococcus, Microbacterium, Arthrobacter, Glutamibacter, Kocuria), while vet horses primarily gave C1I+ Proteobacteria (Escherichia, Klebsiella, Enterobacter, Pantoea, Acinetobacter, Leclercia, Ochrobactrum); the vet isolates had more extensive resistance and stronger PC promoters in the C1Is. All integrons in Actinobacteria were flanked by copies of IS6100, except in Micrococcus, where a novel IS5 family element (ISMcte1) was implicated in mobilization. In the Proteobacteria, C1Is were predominantly associated with IS26, and also IS1, Tn21, Tn1721, Tn512, and a putative formaldehyde-resistance transposon (Tn7489). Several large C1I-containing plasmid contigs were retrieved; two of these (plasmid types Y and F) also had extensive sets of metal resistance genes, including a novel copper-resistance transposon (Tn7519). Both veterinary treatment and domestication increase the frequency of C1Is in equine gut microflora, and each of these anthropogenic factors selects for a distinct group of integron-containing bacteria. IMPORTANCEThere is increasing acknowledgement that a One Health approach is required to tackle the growing problem of antimicrobial resistance. This requires that the issue is examined from not only the perspective of human medicine, but also includes consideration of the roles of antimicrobials in veterinary medicine and agriculture, and recognises the importance of other ecological compartments in the dissemination of ARGs and mobile genetic elements such as C1I. We have shown that domestication and veterinary treatment increase the frequency of occurrence of C1Is in the equine gut microflora, and that in healthy farm horses, the C1I are unexpectedly found in Actinobacteria, while in horses receiving antimicrobial veterinary treatments, a taxonomic shift occurs, and the more typical integron-containing Proteobacteria are found. We identified several new mobile genetic elements (plasmids, IS and transposons) on genomic contigs from the integron-containing equine bacteria.

microbiology↗

bioRxiv2022

The highly diverse and complex plasmid population found in Escherichia coli colonising travellers to Laos and their role in antimicrobial resistance gene carriage

Increased colonisation by antimicrobial resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL) -producing Escherichia coli that colonised travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 49 isolates obtained from the daily stool samples of 23 travellers over a three-week period. This method revealed a collection of 105 distinct plasmids, 38.1% of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=22) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasised the importance of IS elements, specifically members of the IS6/IS26 family, in the creation of complex multi-drug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying blaCTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks. Impact StatementThe global spread of AMR is closely associated with international travel. AMR is a severe global concern and has compromised treatment options for many bacterial pathogens, among them pathogens carrying ESBL and colistin resistance genes. Colonising MDR organisms have the potential to cause serious consequences. Infections caused by MDR bacteria are associated with longer hospitalisation, poorer patient outcomes, greater mortality, and higher costs compared to infections with susceptible bacteria. This study elucidates the numerous different types of plasmids carrying AMR genes in colonising ESBL-producing E. coli isolates found in faecal samples from in travellers to Vientiane, Laos. Here we add to known databases of AMR plasmids by adding these MDR plasmids found in Southeast Asia, an area of high AMR prevalence. We characterised novel AMR plasmids including complex ESBL (blaCTX-M) and colistin (mcr) resistance co-carriage plasmids, emphasising the potential exposure of travellers to Laos to a wide variety of mobile genetic elements that may facilitate global AMR spread. This in-depth study has revealed further detail of the numerous factors that may influence AMR transfer, therefore potential routes of AMR spread internationally, and is a step towards finding methods to combat AMR spread. Data SummaryLong-read sequencing data is available through National Center for Biotechnology Information under the BioProject PRJNA853172. Complete plasmid sequences have been uploaded to GenBank with accession numbers in supplementary S1. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2022

GR13-type plasmids in Acinetobacter potentiate the accumulation and horizontal transfer of diverse accessory genes

Carbapenem resistance and other antibiotic resistance genes (ARGs) can be found in plasmids in Acinetobacter, but many plasmid types in this genus have not been well-characterised. Here we describe the distribution, diversity and evolutionary capacity of rep group 13 (GR13) plasmids that are found in Acinetobacter species from diverse environments. Our investigation was prompted by the discovery of two GR13 plasmids in A. baumannii isolated in an intensive care unit (ICU). The plasmids harbour distinct accessory genes: pDETAB5 contains blaNDM-1 and genes that confer resistance to four further antibiotic classes, while pDETAB13 carries putative alcohol tolerance determinants. Both plasmids contain multiple dif modules, which are flanked by pdif sites recognised by XerC/XerD tyrosine recombinases. The ARG-containing dif modules in pDETAB5 are almost identical to those found in pDETAB2, a GR34 plasmid from an unrelated A. baumannii isolated in the same ICU a month prior. Examination of a further 41 complete, publicly available plasmid sequences revealed that the GR13 pangenome consists of just four core but 1086 accessory genes, 123 in the shell and 1063 in the cloud, reflecting substantial capacity for diversification. The GR13 core genome includes genes for replication and partitioning, and for a putative tyrosine recombinase. Accessory segments encode proteins with diverse putative functions, including for metabolism, antibiotic/heavy metal/alcohol tolerance, restriction-modification, an anti-phage system and multiple toxin-antitoxin systems. The movement of dif modules and actions of insertion sequences play an important role in generating diversity in GR13 plasmids. Discrete GR13 plasmid lineages are internationally disseminated and found in multiple Acinetobacter species, which suggests they are important platforms for the accumulation, horizontal transmission and persistence of accessory genes in this genus. Impact statementAcinetobacter species are particularly well-adapted for persistence in hospital environments where they pose a life-threatening infection risk to the most clinically-vulnerable patients. Plasmids with the potential to transfer multiple antibiotic resistance determinants between Acinetobacter species are therefore concerning, but most are not well-characterised. This work sheds further light on the poorly-understood mobile gene pool associated with Acinetobacter. We show here that GR13 plasmids carry a small set of core genes but have access to a highly diverse set of accessory segments that might provide fitness advantages under certain conditions. The complex evolutionary dynamics of GR13 plasmids appear to be driven by the exchange of dif modules and by the actions of a diverse population of insertion sequences. The novel dif modules characterised here emphasise the broader importance of these elements to the dissemination of accessory genes in Acinetobacter. This study has improved our understanding of the diversity and distribution of dif modules, plasmids that carry them, and how both disseminate in the continuum of Acinetobacter populations that link hospitals and the wider environment.

microbiology↗

bioRxiv2021

Microbiota analysis of rural and urban surface waters and sediments in Bangladesh identifies human waste as driver of antibiotic resistance

In many low- and middle-income countries antibiotic resistant bacteria spread in the environment due to inadequate treatment of wastewater and the poorly regulated use of antibiotics in agri- and aquaculture. Here we characterised the abundance and diversity of antibiotic-resistant bacteria and antibiotic resistance genes in surface waters and sediments in Bangladesh through quantitative culture of Extended-Spectrum Beta-Lactamase (ESBL)-producing coliforms and shotgun metagenomics. Samples were collected from highly urbanised settings (n = 7), from rural ponds with a history of aquaculture-related antibiotic use (n = 11) and from rural ponds with no history of antibiotic use (n = 6). ESBL-producing coliforms were found to be more prevalent in urban samples than in rural samples. Shotgun sequencing showed that sediment samples were dominated by the phylum Proteobacteria (on average 73.8% of assigned reads), while in the water samples Cyanobacteria (on average 60.9% of assigned reads) were the predominant phylum. Antibiotic resistance genes were detected in all samples, but their abundance varied 1,525-fold between sites, with the highest levels of antibiotic resistance genes being present in urban surface water samples. We identified an IncQ1 sulphonamide resistance plasmid ancestral to the widely studied RSF1010 in one of the urban water samples. The abundance of antibiotic resistance genes was significantly correlated (R2 = 0.73; P = 8.9 x 10-15) with the abundance of bacteria originating from the human gut, which suggests that the release of untreated sewage is a driver for the spread of environmental antibiotic resistance genes in Bangladesh, particularly in highly urbanised settings. ImportanceLow- and middle-income countries (LMICs) have higher burdens of multidrug-resistant infections than high-income countries and there is thus an urgent need to elucidate the drivers of the spread of antibiotic-resistant bacteria in LMICs. Here we study the diversity and abundance of antibiotic resistance genes in surface water and sediments from rural and urban settings in Bangladesh. We found that urban surface waters are particularly rich in antibiotic resistance genes, with a higher number of them associated with plasmids indicating that they are more likely to spread horizontally. The abundance of antibiotic resistance genes was strongly correlated with the abundance of bacteria that originate from the human gut, suggesting that uncontrolled release of human waste is a major driver for the spread of antibiotic resistance in the urban environment. Improvements in sanitation in LMICs may thus be a key intervention to reduce the dissemination of antibiotic resistant bacteria.

microbiology↗