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The determination of H. pylori in vitro antibiotic susceptibility to the four key antibiotics and the underlying mechanisms of resistance were investigated. Levels of antibiotic resistance in two independent populations in England and Wales were determined. In the urban London-based population, metronidazole resistance was present in 59% and clarithromycin resistance was in 11% of the 101 H. pylori isolates. Of 363 isolates from rural Bangor, North Wales, 23% were metronidazole resistant and 7% were clarithromycin resistant. Combined clarithromycin and metronidazole resistance occurred in 9% of London and in 4% from Bangor isolates. Analysis of patient questionnaires completed at the London centres identified a significant association between metronidazole resistance and birth outside the UK. Analysis of the method used to determine metronidazole susceptibility status indicated that anaerobic pre-incubation gave results falsely indicating susceptibility. This study would recommend the use of Etest strips on 10% Mueller-Hinton blood agar, incubated microaerophilically at 37°C for 48 h. Comparison of detection methods for 23Sr rDNA mutations associated with H. pylori clarithromycin resistance showed a novel 3’-mismatched reverse primer PCR to be the most sensitive approach. Sequence analysis of 16S rDNA in tetracycline resistant isolates identified the A926g mutation for the first time from a UK H. pylori isolate and a novel A926C mutation. The mechanism of unstable amoxicillin resistance was investigated. Novel PCR-RFLP assays, exploiting sequence variability in the outer membrane protein genes hopB and hopC, indicated no association with antibiotic resistance, but have potential for use as a novel genotyping tool for H. pylori. This study highlights the need for H. pylori antibiotic resistance surveillance at the regional level to help guide treatment regimens.

Introduction: Salmonella Typhi is a globally important pathogen that causes Typhoid fever, responsible for an estimated 11.9-26.9 million cases and 129,000-216,510 typhoid-related deaths per year worldwide. Surveillance of clinical disease based on quality assured diagnostic clinical microbiology services is often not performed, making it difficult to understand the true burden of typhoid. Environmental surveillance (ES) has the potential to be a cheaper alternative to clinical surveillance of Typhoid and S. Typhi in endemic settings that does not rely on patients attending hospitals or consenting to research. Between April 2015 and January 2017, 546 culture-confirmed cases were reported at Queen Elizabeth Central Hospital in Malawi, however it is likely that case numbers are under-reported, and ES data can provide supplementary insight for spatially targeted interventions. Methods: A novel culture method for S. Typhi was developed at the UK Health Security Agency in London and optimized in Blantyre, Malawi with environmental samples collected in 2019, including water (via trap and gran methods), sediments, food, and biofilms. The method used two enrichment broths (2% bile, then selenite F broths) before plating onto modified chromogenic agar for Salmonella esterase agar. Isolates were identified by real-time PCR and confirmed by biochemistry and serology. The method was validated and used for city-wide surveillance in Blantyre, Malawi between May 2021 and April 2022, alongside a method proposed by the Bill and Melinda Gates Foundation S. Typhi ES working group. After findings between these two methods, further refinements to the culture pathway were started. Results: The six-month pilot study, from 2019 to 2020, isolated six S. Typhi cultures from three Moore swabs, two water samples, and one biofilm. PCR positivity was confirmed by biochemistry and serology. Non-typhoidal salmonellae (NTS) accounted for 377 isolates, 16 of which amplified staG. Between 2021 and 2022, 33 samples were S. Typhi positive and 80 positives for NTS by direct PCR detection only, with two S. Typhi and 255 NTS positive isolates cultured. An alternative extraction method for PCR was shown to have good performance under laboratory conditions, but the inclusion of antimicrobial-containing broths required further development. Conclusion: This work demonstrates that ES of Typhoid by culture is achievable in low- and middle-income countries. Moore swabs yielded a higher detection rate than water samples. Direct detection by PCR appeared to be more sensitive than culture, but still had a low positivity rate. This low positivity rate coincided with the lowest rates of blood culture positivity in a decade and the SARS-CoV-2 pandemic. Work to integrate a PCR screening tool before culture is ongoing but shows potential viability.

Norovirus has been identified as a common cause of gastroenteritis worldwide, and food as a transmission vehicle has been well documented. Standardised detection methods exist for the detection of norovirus from fresh produce and molluscan bivalves, whilst detection methods for a wider range of food matrices that may be implicated in transmission of norovirus do not currently exist. The detection of norovirus in foods suspected to be implicated in transmission is paramount for appropriate outbreak investigation. The contamination of foods other than shellfish and fresh produce often occurs via food handlers. The proportion of norovirus that is typically transferred from food handlers to food also remains unknown. Understanding this is necessary in order to estimate the risk of infection and the burden of gastroenteritis caused by norovirus that is attributable to food contaminated by food handlers. These questions were addressed by the development of a combined capture, concentration and quantitative detection protocol with the aim to enhanced norovirus recovery from a range of food types. A food surface wash and norovirus capture method that was sensitive, reduced processing time, and increased throughput capacity was applied to a range of ready to eat foods. An automated nucleic acid extraction method which further reduced processing time and increased throughput was validated. Finally the validated method demonstrated that two real time RT-PCR assays currently used for the detection of norovirus in shellfish and fresh produce or in faecal samples were comparable overall, and hence either could be used in combination with the norovirus capture, concentration and extraction protocol described in this thesis. The protocol was applied to a range of food matrices and resulted in < 1% to 55% recovery of norovirus GI and < 1% to 25% recovery of norovirus GII. The optimised protocol was then used to quantify virus transfer between food handlers hands and to food, in simulation experiments where food handlers’ gloved hands were artificially contaminated prior to preparation of a sandwich. This enabled norovirus transfer to food items and to other food handlers to be measured at each stage. Quantitative data demonstrated that 5.9 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GII inoculum, resulted in a percentage recovery of between 3.0% and 0.02% from Food Handlers and 7.8 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GI inoculum resulted in a percentage recovery between 9.6% and 0.004% from Food Handlers. The average percentage recovered from sandwich pieces over six replicates was 0.2% for norovirus GII and 1.2% for norovirus GI. The method and protocols developed could be rolled out to official control laboratories and aid foodborne outbreak investigation by allowing testing of food categories that currently are not investigated. Furthermore, this work demonstrated the extent of norovirus transfer from hands to food ingredients and the environment and could be used in risk assessment models. Further work applying these protocols to quantify the transfer from contaminated hands using a range of viral loads will be useful in determining risk more accurately, and to monitor and investigate food premises by introducing this as an additional food and hand hygiene marker.