Biology subjects

Pickard, D.

Publications and source records attributed to Pickard, D..

9 recordsLinked to original sources

bioRxiv2024

Presence of phage-plasmids in multiple serovars of Salmonella enterica

Evidence is accumulating in the literature that the horizontal spread of antimicrobial resistance (AMR) genes mediated by bacteriophages and bacteriophage-like plasmid (phage-plasmid) elements is much more common than previously envisioned. For instance, we recently identified and characterised a circular P1-like phage-plasmid harbouring a blaCTX-M-15 gene conferring extended-spectrum beta-lactamase (ESBL) resistance in Salmonella enterica serovar Typhi. As the prevalence and epidemiological relevance of such mechanisms has never been systematically assessed in Enterobacterales, in this study we carried out a follow-up retrospective analysis of UK Salmonella isolates previously sequenced as part of routine surveillance protocols between 2016 and 2021. Using a high-throughput bioinformatics pipeline we screened 47,784 isolates for the presence of the P1 lytic replication gene repL, identifying 226 positive isolates from 25 serovars and demonstrating that phage-plasmid elements are more frequent than previously thought. The affinity for phage-plasmids appears highly serovar-dependent, with several serovars being more likely hosts than others; most of the positive isolates (170/226) belonged to S. Typhimurium ST34 and ST19. The phage-plasmids ranged between 85.8-98.2kb in size, with an average length of 92.1kb; detailed analysis indicated a high amount of diversity in gene content and genomic architecture. 132 phage-plasmids had the p0111 plasmid replication type, and 94 the IncY type; phylogenetic analysis indicated that both horizontal and vertical gene transmission mechanisms are likely to be involved in phage-plasmid propagation. Finally, phage-plasmids were present in isolates that were resistant and non-resistant to antimicrobials. In addition to providing a first comprehensive view of the presence of phage-plasmids in Salmonella, our work highlights the need for a better surveillance and understanding of phage-plasmids as AMR carriers, especially through their characterisation with long-read sequencing. Data SummaryAll of the FASTQ files examined in this study have been uploaded to the Sequence Read Archive under BioProject PRJNA248792. Accessions of individual isolates which were found to contain phage plasmids are listed in Supplementary Table S1. Impact StatementBacteriophage-like plasmids are increasingly being recognised as important mobile elements in many species of bacteria, particularly due to their involvement in the transmission of antimicrobial resistance (AMR); however, few studies of their overall prevalence in clinical datasets have been undertaken to date. In this study we have performed the first large-scale surveillance of human disease-associated Salmonella genomes for the presence of P1-like phage-plasmids, showing that they are more common than previously thought. Furthermore, we highlight how valuable information about the evolution and transmission of phage-plasmids in Salmonella and other Enterobacterales can be revealed by linking phage-plasmid prevalence and genetic diversity to epidemiologically relevant metadata such as S. enterica serovar, outbreak clusters, time, and geography. Our work shows the ability to use sequencing data and scalable bioinformatics workflows for the detection and characterisation of these extrachromosomal elements, highlights the importance of screening for novel mechanisms of AMR transmission, and provides a foundation for further surveillance studies of phage-plasmid prevalence.

microbiology↗

bioRxiv2021

Dissemination and pathogenesis of toxigenic Clostridium perfringens strains linked to neonatal intensive care units and Necrotising Enterocolitis

BackgroundClostridium perfringens is an anaerobic toxin-producing bacterium that has long been associated with intestinal diseases, particularly in neonatal humans and animals. More recently, infant gut microbiome studies have suggested an important link between C. perfringens and the devastating preterm-associated disease Necrotising Enterocolitis (NEC), but in-depth studies on this pathogen (genomics and mechanistic) are lacking. Methods/MaterialsWe isolated and whole-genome sequenced 274 infant-associated C. perfringens isolates from 5 hospitals across the UK between 2011-2016 (including longitudinal samples from 31 individuals). We performed in-depth genomic analyses, phenotypically characterised pathogenic traits of 10 strains (including 4 C. perfringens from NEC patients) and established a novel oral-challenge C57BL/6 mouse infection model for microbe-host studies. ResultsPore-forming toxin encoding genes pfoA and cpb2 were enriched within hypervirulent lineages that exclusively consisted of C. perfringens-associated NEC (CPA-NEC) strains, in addition to overabundance of colonisation factors. Importantly, we identified a circulating C. perfringens variant, eventually linked to a fatal CPA-NEC case. The variant was detected consistently within 6 individuals in two sister hospitals across a 40-day window, demonstrating for the first time the intra- and inter-hospital dissemination of C. perfringens. CPA-NEC isolates were determined phenotypically to be more virulent (linked with overabundance of gene pfoA) than isolates obtained from non-NEC preterm babies. In addition, two pfoA-positive CPA-NEC C. perfringens strains were confirmed to induce clinical inflammatory tissue lesions in vivo. ConclusionsHypervirulent lineages are linked to CPA-NEC, potentially due to the production of pore-forming toxins, coupled with higher metabolic, transmission, and pathogenic capacities. These studies indicate C. perfringens is an important bacterial pathogen in preterm infants and highlights the requirement for further investigation into development of intervention and therapeutic strategies.

microbiology↗

bioRxiv2021

Inhibitory concentrations of ciprofloxacin induce an adaptive response promoting the intracellular survival of Salmonella Typhimurium

Antimicrobial resistance (AMR) is a pressing global health crisis, which has been fuelled by the sustained use of certain classes of antimicrobials, including fluoroquinolones. While the genetic mutations responsible for decreased fluoroquinolone (ciprofloxacin) susceptibility are known, the implications of ciprofloxacin exposure on bacterial growth, survival, and interactions with host cells are not well described. Aiming to understand the influence of inhibitory concentrations of ciprofloxacin in vitro, we subjected three clinical isolates of S. Typhimurium to differing concentrations of ciprofloxacin, dependent on their minimum inhibitory concentrations (MIC), and assessed the impact on bacterial growth, morphology, and transcription. We further investigated the differential morphology and transcription that occurred following ciprofloxacin exposure and measured the ability of ciprofloxacin-treated bacteria to invade and replicate in host cells. We found that ciprofloxacin-exposed S. Typhimurium are able to recover from inhibitory concentrations of ciprofloxacin, and that the drug induces specific morphological and transcriptional signatures associated with the bacterial SOS response, DNA repair, and intracellular survival. In addition, ciprofloxacin-treated S. Typhimurium have increased capacity for intracellular replication in comparison to untreated organisms. These data suggest that S. Typhimurium undergoes an adaptive response under ciprofloxacin perturbation that promotes cellular survival, a consequence that may justify more measured use of ciprofloxacin for Salmonella infections. The combination of multiple experimental approaches provides new insights into the collateral effects that ciprofloxacin and other antimicrobials have on invasive bacterial pathogens. ImportanceAntimicrobial resistance is a critical concern in global health. In particular, there is rising resistance to fluoroquinolones, such as ciprofloxacin, a first-line antimicrobial for many Gram-negative pathogens. We investigated the adaptive response of clinical isolates of Salmonella Typhimurium to ciprofloxacin, finding that the bacteria adapt in short timespans to high concentrations of ciprofloxacin in a way that promotes intracellular survival during early infection. Importantly, by studying three clinically relevant isolates, we were able to show that individual isolates respond differently to ciprofloxacin, and for each isolate, there was a heterogeneous response under ciprofloxacin treatment. The heterogeneity that arises from ciprofloxacin exposure may drive survival and proliferation of Salmonella during treatment and lead to drug resistance.

microbiology↗

bioRxiv2020

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time

BackgroundEnterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 fatalities per year, with the highest rates of burden, ca 75 million cases per year, amongst children under five years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. ResultsWe used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. The plasmids carrying ETEC virulence genes were compared to other available long-read sequenced ETEC strains using blastn. The ETEC reference strains harbour between two and five plasmids, including virulence, antibiotic resistance and phage-plasmids. The virulence plasmids carrying the colonisation factors are highly conserved as shown by comparison with plasmids with other ETEC strains and confirm that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. ConclusionWe confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

genomics↗

bioRxiv2019

Genomic surveillance of Escherichia coli ST131 identifies local expansion and serial replacement of subclones

Escherichia coli sequence type 131 (ST131) is a pandemic clone that is evolving rapidly with increasing levels of antimicrobial resistance. Here, we investigated an outbreak of E. coli ST131 producing extended spectrum {beta}-lactamases (ESBLs) in a long-term care facility (LTCF) in Ireland by combining data from this LTCF (n=69) with other Irish (n=35) and global (n=690) ST131 genomes to reconstruct the evolutionary history and understand changes in population structure and genome architecture over time. This required a combination of short and long-read genome sequencing, de novo assembly, read mapping, ESBL gene screening, plasmid alignment and temporal phylogenetics. We found that clade C was the most prevalent (686 out of 794 isolates, 86%) of the three major ST131 clades circulating worldwide (A, B, C), and was associated with the presence of different ESBL alleles, diverse plasmids and transposable elements. Clade C was estimated to have emerged in [~]1985 and subsequently acquired different ESBL gene variants (blaCTX-M-14 vs blaCTX-M-15). An ISEcp1- mediated transposition of the blaCTX-M-15 gene further increased the diversity within Clade C. We discovered a local clonal expansion of a rare C2 lineage (C2_8) with a chromosomal insertion of blaCTX-M-15 at the mppA gene. This was acquired from an IncFIA plasmid. The C2_8 lineage clonally expanded in the Irish LTCF from 2006, displacing the existing C1 strain (C1_10) and highlighting the potential for novel ESBL-producing ST131 with a distinct genetic profile to cause outbreaks strongly associated with specific healthcare environments.\n\nImportanceExtraintestinal pathogenic E. coli (ExPEC) ST131 is adapting in the context of antibiotic exposure, resulting in a pandemic with distinct genetic subtypes. Here, we track the evolution of antibiotic-resistance gene variants originally discovered in an ExPEC ST131 outbreak that was identified in a LTCF in Ireland. Analysis of 794 global ST131 genomes show that subclade C1 was associated with the initial infection outbreak, but that a new lineage from subclade C2 successfully displaced C1. This genetically distinct C2 subclade with a chromosomal insertion of a key antibiotic-resistance gene had clonally expanded within the LTCF. We provide new insights into the timing of genetic events driving the diversification of C2 subclades to show that that outbreak C2 strain likely evolved elsewhere before spreading to the LTCF. This study highlights the scope of antibiotic-resistance gene rearrangement within ST131, reinforcing the need to integrate genomic, epidemiological and microbiological approaches to understand ST131 transmission.

genomics↗

bioRxiv2019

Genomic analysis on broiler-associated Clostridium perfringens strains and caecal microbiome profiling reveals key factors linked to poultry Necrotic Enteritis

BackgroundClostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens, including identifying caecal microbiota signatures associated with NE.\n\nResultsWe analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993-2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna, cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C. perfringens strains (toxinotype G). We also analysed caecal samples from a sub-set of 11 diseased and healthy broilers using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members.\n\nConclusionsThese data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds.

microbiology↗

bioRxiv2019

Phylogenomic analysis of Clostridium perfringens identifies isogenic strains in gastroenteritis outbreaks, and novel virulence-related features

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, limited Whole Genome Sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of C. perfringens-associated outbreak strains. We performed both genomic and phylogenetic analysis on 109 C. perfringens strains (human and food) isolated from disease cases in England and Wales between 2011-2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of Fluorescent Amplified Fragment Length Polymorphism (fAFLP). Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected the enterotoxin CPE gene was encoded in all but 4 isolates (96.4%; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603- and pCPF4969-family plasmids) extensively distributed (82.6%;90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7%; 50/109), and genes encoding phage proteins were also frequently identified, with additional analysis indicating their contribution to increased virulence determinants within the genomes of gastroenteritis-associated C. perfringens. Overall this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that 3 major sub-types underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid, and (3) strains carrying cpe on transposable element Tn5565(usually integrated into chromosome). Our findings indicate that further studies will be required to fully probe this enteric pathogen, particularly in relation to developing intervention and prevention strategies to reduce food poisoning disease burden in vulnerable patients, such as the elderly.

microbiology↗

bioRxiv2018

Diagnostic host gene signature to accurately distinguish enteric fever from other febrile diseases

Misdiagnosis of enteric fever is a major global health problem resulting in patient mismanagement, antimicrobial misuse and inaccurate disease burden estimates. Applying a machine-learning algorithm to host gene expression profiles, we identified a diagnostic signature which could accurately distinguish culture-confirmed enteric fever cases from other febrile illnesses (AUROC<95%). Applying this signature to a culture-negative suspected enteric fever cohort in Nepal identified a further 12.6% as likely true cases. Our analysis highlights the power of data-driven approaches to identify host-response patterns for the diagnosis of febrile illnesses. Expression signatures were validated using qPCR highlighting their utility as PCR-based diagnostic for use in endemic settings.

molecular biology↗

bioRxiv2018

Emergence of carbapenem, beta-lactamase inhibitor and cefoxitin resistant lineages from a background of ESBL-producing Klebsiella pneumoniae and K. quasipneumoniae highlights different evolutionary mechanisms

Klebsiella pneumoniae is recognised as a major threat to public health, with increasing emergence of multidrug-resistant lineages including strains resistant to all available antibiotics. We present an in-depth analysis of 178 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains, with a high background diversity and two dominant lineages, as well as several equally resistant lineages with less prevalence. Neither the overall resistance profile nor the virulence factors explain the prevalence of some lineages; we observe several putative hypervirulence factors across the population, including a reduced virulence plasmid, but this does not correlate with expansion of one or few highly virulent and resistant lineages. Phenotypic analysis of the profiles of resistance traits shows that the vast majority of the phenotypic resistance profiles can be explained by detailed genetic analyses. The main discrepancies are observed for beta-lactams combined with beta-lactamase inhibitors, where most, but not all, resistant strains carry a carbapenemase or ampC. Complete genomes for six selected strains, including three of the 21 carbapenem-resistant ones, are reported, which give detailed insights into the early evolution of the bla-NDM-1 enzyme, a carbapenemase that was first reported in 2009 and is now globally distributed. Whole-genome based high-resolution analyses of the dominant lineages suggests a very dynamic picture of gene transfer and selection, with phenotypic changes due to plasmid acquisition and chromosomal changes, and emphasize the need to monitor the bacteria at high resolution to understand the rise of high-risk clones, which cannot be explained by obvious differences in resistance profiles or virulence factors.\n\nImportanceCarbapenem-resistant and extended-spectrum beta-lactamase (ESBL) carrying Enterobacteriaceae were recently highlighted as critical priority fo the development of new treatments by the WHO. Klebsiella pneumoniae is a member of the Enterobacteriaceae and has seen a dramatic rise in clinical relevance due to its uncanny ability to accumulate multidrug-resistance plasmids. We present a detailed analysis of a set of ESBL-resistant K. pneumoniae clinical isolates, and our high-resolution whole-genome sequence analyses highlight that acquisition of drug resistances is not a one-way street in K. pneumoniae, but a highly dynamic process of gain and loss, and that the most successful lineages in the clinic are not necessarily the most resistant or most virulent ones. Analysis of the virulence potential also shows that these strains harbour some, but not all, hallmarks of hypervirulent strains, emphasizing that it is not a clear distinction between hypervirulent and other strains, but equally in flux.

genomics↗