Susanna Frost

Introduction. Shiga toxin-producing Escherichia coli (STEC) are a group of bacteria that cause severe bloody diarrhoea and haemolytic uraemic syndrome and are associated with neurological complications and potentially life-threatening infections. Children under five and elderly individuals are particularly vulnerable to STEC infections. The majority of STEC infections have been traced back directly or indirectly to ruminants, and there is an unmet need for a vaccine to protect these vulnerable cohorts. Gap Statement. We previously identified a series of STEC proteins that are involved in attachment to human gastrointestinal epithelial cells, some of which are protective in a murine challenge model. However, the prevalence of the genes in dairy herds was unknown. Aim. Due to zoonotic transmission, the development of a successful human vaccine relies on any protective vaccine antigens being prevalent in ruminant herds in different geographical locations. We aimed to examine the potential geographical differences in antigen gene prevalence. Methodology. Faecal isolates collected from a dairy herd in CA, USA, and isolated from dairy cows in Ireland were analysed by quantitative PCR. Bioinformatic analysis of previously sequenced isolates was also performed. Results. Four antigen genes were highly prevalent among STEC shed from cattle in a Californian dairy herd (>95.7%). Genes encoding FkpA, YiaF and GlnH were also highly prevalent in faecal samples from cows sampled in Ireland (93%). The point prevalence of terD among cattle in the Irish cohort appeared low relative to Californian isolates, but this is most likely due to the absence of tellurite in isolation media used in the former. Expansion of the study to examine Irish bovine and ovine Whole genome sequencing datasets increased the detection of terD in Irish isolates. Conclusion. Overall, three antigen genes are prevalent in both geographically distinct cohorts. Discrepancies in terD gene prevalence are likely due to the use of selective agars in isolation protocols.

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.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Verotoxin-producing Escherichia coli (VTEC) causes zoonotic infections, with potentially devastating complications, and children under 5 years old are particularly susceptible. Antibiotic treatment is contraindicated, and due to the high proportion of infected children that suffer from severe and life-changing complications, there is an unmet need for a vaccine to prevent VTEC infections. Bacterial adhesins represent promising candidates for the successful development of a vaccine against VTEC. Using a proteomic approach to identify bacterial proteins interacting with human gastrointestinal epithelial Caco-2 and HT-29 cells, we identified eleven proteins by mass spectrometry. These included a glutamine-binding periplasmic protein, GlnH, a member of the ABC transporter family. The glnH gene was identified in 13 of the 15 bovine and all 5 human patient samples tested, suggesting that it is prevalent. We confirmed that GlnH is involved in the host cell attachment of an O157:H7 prototype E. coli strain to gastrointestinal cells in vitro. Recombinant GlnH was expressed and purified prior to the immunisation of mice. When alum was used as an adjuvant, GlnH was highly immunogenic, stimulating strong serological responses in immunised mice, and it resulted in a modest reduction in faecal shedding but did not reduce colonisation. GlnH immunisation with a T-cell-inducing adjuvant (SAS) also showed comparable antibody responses and an IgG1/IgG2a ratio suggestive of a mixed Th1/Th2 response but was partially protective, with a 1.25-log reduction in colonisation of the colon and caecum at 7 days relative to the adjuvant only (p = 0.0280). It is clear that future VTEC vaccine developments should consider the contribution of adjuvants in addition to antigens. Moreover, it is likely that a combined cellular and humoral response may prove more beneficial in providing protective interventions against VTEC.