The effect of the proton pump inhibitor pantoprazole on the biology of Campylobacter jejuni

Campylobacter is a major cause of acute bacterial gastroenteritis worldwide, with the highest number of infections being attributed to Campylobacter jejuni. C. jejuni is a Gram negative, spiral, motile bacterium that belongs to the campylobacterales order and is related to both Helicobacter spp. and Wolinella sp.. It has long been established that proton pump inhibitors (PPIs) and other benzimidazole derivatives display anti-Helicobacter activity in vitro. PPIs have in the past been shown to affect Helicobacter pylori growth, survival, motility, morphology, adhesion/invasion potential and susceptibility to conventional antibiotics. PPIs are highly effective drugs that are well tolerated, safe for prolonged daily use and are therefore in high demand. Both the PPIs omeprazole and lansoprazole featured in the top ten drugs prescribed in England in 2014. In 2014 Campylobacter was also the most commonly diagnosed gastrointestinal infection in Scotland, in England and Wales and also in Europe. It has previously been generally accepted that patients who are being treated with PPIs are more susceptible to enteric infections such as Campylobacter than people not taking PPIs. The effect of PPI exposure on H. pylori has been investigated rigorously in the past. A single previous study has hinted that PPIs may also be capable of affecting the related organism C. jejuni,but investigations have been extremely limited in comparison to those investigating the effect of PPIs on H. pylori. This study has investigated the in vitro effects of direct contact with PPIs on the biology ofC. jejuni. Exposure to the PPI pantoprazole was found to affect C. jejuni growth/survival, motility, morphology, biofilm formation, invasion potential and susceptibility to some conventional antibiotics. Microarray studies showed that the cmeA and Cj0561c genes were significantly up-regulated in response to pantoprazole exposure and a CmeABC deficient mutant was found to be significantly more susceptible to killing by pantoprazole than was the parent strain. Proteomic analysis indicated that the oxidative stress response of C. jejuni was induced following exposure to sub-lethal concentrations of pantoprazole. C. jejuni gene expression was assessed using qRT-PCR and the genes encoding for thiol peroxidase and GroEL co-chaperonin (both involved in the C. jejuni oxidative stress response) were found to be around four times higher in response to exposure to sub-lethal concentrations of pantoprazole. Experiments using the oxidative stress inhibitors thiourea (a hydroxyl radical quencher) and bipyridyl (a ferrous iron chelator) showed that killing by pantoprazole was not mediated by hydroxyl radical production.

Studies on Haemonchus contortus infections in Merino sheep

The work described in this thesis was initially directed towards the elucidation of the self-cure phenomenon as a flock phenomenon under natural grazing conditions. As a consequence of these studies it became apparent that several important aspects of the epidemiology and of the clinical syndromes associated with haemonchosis required further investigation. The thesis is therefore essentially a description of haemonchosis of sheep in an endemic area supported, where indicated, by observations obtained by experimental infections. Each of the separate aspects studied is prefaced by a review of the relevant literature and the details of many individual observations which are not included in the text may be found in the various appendices. Some results, for example the individual faecal egg counts and worm burdens at autopsy, were too voluminous to be included and these are available at The Veterinary School of the University of Glasgow.

Mixed genotype Hepatitis C virus infections: incidence in Scotland and methods for detection

The diagnosis of mixed genotype hepatitis C virus (HCV) infection is rare and information on incidence in the UK, where genotypes 1a and 3 are the most prevalent, is sparse. Considerable variations in the efficacies of direct-acting antivirals (DAAs) for the HCV genotypes have been documented and the ability of DAAs to treat mixed genotype HCV infections remains unclear, with the possibility that genotype switching may occur. In order to estimate the prevalence of mixed genotype 1a/3 infections in Scotland, a cohort of 512 samples was compiled and then screened using a genotype-specific nested PCR assay. Mixed genotype 1a/3 infections were found in 3.8% of samples tested, with a significantly higher prevalence rate of 6.7% (p<0.05) observed in individuals diagnosed with genotype 3 infections than genotype 1a (0.8%). An analysis of the samples using genotypic-specific qPCR assays found that in two-thirds of samples tested, the minor strain contributed <1% of the total viral load. The potential of deep sequencing methods for the diagnosis of mixed genotype infections was assessed using two pan-genotypic PCR assays compatible with the Illumina MiSeq platform that were developed targeting the E1-E2 and NS5B regions of the virus. The E1-E2 assay detected 75% of the mixed genotype infections, proving to be more sensitive than the NS5B assay which identified only 25% of the mixed infections. Studies of sequence data and linked patient records also identified significantly more neurological disorders in genotype 3 patients. Evidence of distinctive dinucleotide expression within the genotypes was also uncovered. Taken together these findings raise interesting questions about the evolutionary history of the virus and indicate that there is still more to understand about the different genotypes. In an era where clinical medicine is frequently more personalised, the development of diagnostic methods for HCV providing increased patient stratification is increasingly important. This project has shown that sequence-based genotyping methods can be highly discriminatory and informative, and their use should be encouraged in diagnostic laboratories. Mixed genotype infections were challenging to identify and current deep sequencing methods were not as sensitive or cost-effective as Sanger-based approaches in this study. More research is needed to evaluate the clinical prognosis of patients with mixed genotype infection and to develop clinical guidelines on their treatment.