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The work was funded by the Scottish Executive via the Chief Scientist Office through the provision of a grant to establish the Scottish Healthcare Associated Infection Prevention Institute (SHAIPI). Bacteraemia caused by Escherichia coli is a growing problem with a significant mortality. The factors that influence the acquisition and outcome of these infections are not clear. Here, we have linked detailed genetic data from the whole-genome sequencing of 162 bacteraemic isolates collected in Scotland, UK, in 2013-2015, with clinical data in order to delineate bacterial and host factors that influence the acquisition in hospital or the community, outcome and antibiotic resistance. We identified four major sequence types (STs) in these isolates: ST131, ST69, ST73 and ST95. Nearly 50% of the bacteraemic isolates had a urinary origin. ST69 was genetically distinct from the other STs, with significantly less sharing of accessory genes and with a distinct plasmid population. Virulence genes were widespread and diversely distributed between the dominant STs. ST131 was significantly associated with hospital-associated infections (HAIs), and ST69 with those from the community. However, there was no association of ST with outcome, although patients with HAI had a higher immediate mortality compared to those with community-associated infections (CAIs). Genome-wide association studies revealed genes involved in antibiotic persistence as significantly associated with HAIs and those encoding elements of a type VI secretion system with CAIs. Antibiotic resistance was common, and there were networks of correlated resistance genes and phenotypic antibiotic resistance. This study has revealed the complex interactions between the genotype of E. coli and its ability to cause bacteraemia, and some of the determinants influencing hospital or community acquisition. In part, these are shaped by antibiotic usage, but strain-specific factors are also important. Publisher PDF

L. H., L. A., S. W., P. L. and A. S. A. are current employees of Pfizer, and this work was funded by Pfizer Inc. Neisseria meningitidis serogroup B (MnB) was responsible for two independent meningococcal disease outbreaks at universities in the USA during 2013. The first at University A in New Jersey included nine confirmed cases reported between March 2013 and March 2014. The second outbreak occurred at University B in California, with four confirmed cases during November 2013. The public health response to these outbreaks included the approval and deployment of a serogroup B meningococcal vaccine that was not yet licensed in the USA. This study investigated the use of whole-genome sequencing(WGS) to examine the genetic profile of the disease-causing outbreak isolates at each university. Comparative WGS revealed differences in evolutionary patterns between the two disease outbreaks. The University A outbreak isolates were very closely related, with differences primarily attributed to single nucleotide polymorphisms/insertion-deletion (SNP/indel) events. In contrast, the University B outbreak isolates segregated into two phylogenetic clades, differing in large part due to recombination events covering extensive regions (>30 kb) of the genome including virulence factors. This high-resolution comparison of two meningococcal disease outbreaks further demonstrates the genetic complexity of meningococcal bacteria as related to evolution and disease virulence. Publisher PDF

This work was supported by grants from the UK Clinical Research Collaboration (UKCRC) Translational Infection Research Initiative and the Medical Research Council (grant number G1000803) with contributions to the grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research (NIHR) on behalf of the Department of Health and the Chief Scientist Office of the Scottish Government Health Directorate (to S. J. P.); and by Wellcome Trust grants numbers 098051 and 089472 awarded to the Wellcome Trust Sanger Institute and B. G. S respectively. Antibiotic resistance forms a serious threat to the health of hospitalised patients, rendering otherwise treatable bacterial infections potentially life-threatening. A thorough understanding of the mechanisms by which resistance spreads between patients in different hospitals is required in order to design effective control strategies. We measured the differences between bacterial populations of 52 hospitals in the United Kingdom and Ireland, using whole-genome sequences from 1085 MRSA clonal complex 22 isolates collected between 1998 and 2012. The genetic differences between bacterial populations were compared with the number of patients transferred between hospitals and their regional structure. The MRSA populations within single hospitals, regions and countries were genetically distinct from the rest of the bacterial population at each of these levels. Hospitals from the same patient referral regions showed more similar MRSA populations, as did hospitals sharing many patients. Furthermore, the bacterial populations from different time-periods within the same hospital were generally more similar to each other than contemporaneous bacterial populations from different hospitals. We conclude that, while a large part of the dispersal and expansion of MRSA takes place among patients seeking care in single hospitals, inter-hospital spread of resistant bacteria is by no means a rare occurrence. Hospitals are exposed to constant introductions of MRSA on a number of levels: (1) most MRSA is received from hospitals that directly transfer large numbers of patients, while (2) fewer introductions happen between regions or (3) across national borders, reflecting lower numbers of transferred patients. A joint coordinated control effort between hospitals, is therefore paramount for the national control of MRSA, antibiotic-resistant bacteria and other hospital-associated pathogens. Publisher PDF

This study was funded by National Health and Medical Research Council (Australia) grants 1046812 and 1098337. E. P. P. is in receipt of a University of the Sunshine Coast Research Fellowship. Melioidosis is a tropical disease caused by the bacterium Burkholderia pseudomallei. Outbreaks are uncommon and can generally be attributed to a single point source and strain. We used whole-genome sequencing to analyse B. pseudomallei isolates collected from an historical 2-year long case cluster that occurred in a remote northern Australian indigenous island community, where infections were previously linked to a contaminated communal water supply. We analysed the genome-wide relatedness of the two most common multilocus sequence types (STs) involved in the outbreak, STs 125 and 126. This analysis showed that although these STs were closely related on a whole-genome level, they demonstrated evidence of multiple recombination events that were unlikely to have occurred over the timeframe of the outbreak. Based on epidemiological and genetic data, we also identified two additional patients not previously associated with this outbreak. Our results confirm the previous hypothesis that a single unchlorinated water source harbouring multiple B. pseudomallei strains was linked to the outbreak, and that increased melioidosis risk in this community was associated with Piper methysticum root (kava) consumption. Publisher PDF

This study was funded by the National Health and Medical Research Council via awards 1046812 and 1098337, and the Wellcome Trust Sanger Institute via award 098051. S.J.P. receives funding from the NIHR Cambridge Biomedical Research Centre. Melioidosis, caused by the highly recombinogenic bacterium Burkholderia pseudomallei, is a disease with high mortality. Tracing the origin of melioidosis outbreaks and understanding how the bacterium spreads and persists in the environment are essential to protecting public and veterinary health and reducing mortality associated with outbreaks. We used whole-genome sequencing to compare isolates from a historical quarter-century outbreak that occurred between 1966 and 1991 in the Avon Valley, Western Australia, a region far outside the known range of B. pseudomallei endemicity. All Avon Valley outbreak isolates shared the same multilocus sequence type (ST-284), which has not been identified outside this region. We found substantial genetic diversity among isolates based on a comparison of genome-wide variants, with no clear correlation between genotypes and temporal, geographical or source data. We observed little evidence of recombination in the outbreak strains, indicating that genetic diversity among these isolates has primarily accrued by mutation. Phylogenomic analysis demonstrated that the isolates confidently grouped within the Australian B. pseudomallei clade, thereby ruling out introduction from a melioidosis-endemic region outside Australia. Collectively, our results point to B. pseudomallei ST-284 being present in the Avon Valley for longer than previously recognized, with its persistence and genomic diversity suggesting long-term, low-prevalence endemicity in this temperate region. Our findings provide a concerning demonstration of the potential for environmental persistence of B. pseudomallei far outside the conventional endemic regions. An expected increase in extreme weather events may reactivate latent B. pseudomallei populations in this region. Publisher PDF

Streptococcus equi causes equine ‘strangles’. Hyaluronate lyases, which degrade connective tissue hyaluronan and chondroitins, are thought to facilitate streptococcal invasion of the host. However, prophage-encoded hyaluronate lyases are hyaluronan-specific and are thought to be primarily involved in the degradation of the hyaluronan capsule of streptococci during bacteriophage infection. To understand the role of prophage-encoded hyaluronate lyases further, we have biochemically characterized such a hyaluronate lyase, SEQ2045 from S. equi, and have shown that it is produced during equine infection. Prophage-encoded hyaluronan-specific hyaluronate lyases may therefore play a more direct role in disease pathogenesis than previously thought.

Few strains of Erwinia carotovora subsp. carotovora (Ecc) make carbapenem antibiotics. Strain GS101 makes the basic carbapenem molecule, 1-carbapen-2-em-3-carboxylic acid (Car). The production of this antibiotic has been shown to be cell density dependent, requiring the accumulation of the small diffusible molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) in the growth medium. When the concentration of this inducer rises above a threshold level, OHHL is proposed to interact with the transcriptional activator of the carbapenem cluster (CarR) and induce carbapenem biosynthesis. The introduction of the GS101 carR gene into an Ecc strain (SCRI 193) which is naturally carbapenem-negative resulted in the production of Car. This suggested that strain SCRI 193 contained functional cryptic carbapenem biosynthetic genes, but lacked a functional carR homologue. The distribution of trans-activatable antibiotic genes was assayed in Erwinia strains from a culture collection and was found to be common in a large proportion of fee strains. Significantly, amongst the Ecc strains identified, a larger proportion contained trans-activatable cryptic genes than produced antibiotics constitutively. Southern hybridization of the chromosomal DNA of cryptic Ecc strains confirmed the presence of both the car biosynthetic cluster and the regulatory genes. Identification of homologues of the transcriptional activator carR suggests that the cause of the silencing of the carbapenem biosynthetic cluster in these strains is not the deletion of carR. In an attempt to identify the cause of the silencing in the Ecc strain SCRI 193 the carR homologue from this strain was cloned and sequenced. The SCRI 193 CarR homologue was 94% identical to the GS101 CarR and contained 14 amino acid substitutions. Both homologues could be expressed from their native promoters and ribosome-binding sites using an in vitro prokaryotic transcription and translation assay, and when the SCRI 193 carR homologue was cloned in multicopy plasmids and reintroduced into SCRI 193, antibiotic production was observed. This suggested that the mutation causing the silencing of the biosynthetic cluster in SCRI 193 was leaky and the cryptic Car phenotype could be suppressed by multiple copies of the apparently mutant transcriptional activator.