Submitted in fulfillment of the requirements of the degree of Master of Health Sciences in Environmental Health, Durban University of Technology, Durban, South Africa, 2016. / In South Africa, under-five childhood morbidity and mortality rates have increased due to diarrhoea with acute diarrhoea posing a major public health threat especially, in informal settlements. Therefore this study sought to, a) investigate community knowledge, attitudes, behaviour and practices (KABP) regarding domestic waste and childhood diarrhoeal management, b) to enumerate and identify diarrhoeagenic E.coli species from soil samples extracted from open waste dump sites and c) to investigate any association(s) with diarrhoeagenic E.coli and potential risk of contracting diarrhoea. This two-phased cross-sectional study in six informal settlements in the greater Durban area constituted, respectively, of the administering of questionnaires to 360 primary caregivers and; sampling the prevalence of diarrhoeagenic E.coli (DEC) in waste dumps using quantitative polymerase chain reaction methodologies. Relationships between socio-demographic and educational status to determine potential household risk factors towards under-five diarrhoea prevalence were assessed. The KABP results identified domestic waste and greywater disposal, mother and child method of sanitation, personal and domestic hygiene practices and mechanical vectors as significant contributory risk factors. Of concern is that more than 80% of under-five children played in or near faecally-contaminated waste dump sites. The recovery of four DEC pathotypes including enterohaemorrhagic E.coli, enteropathogenic E.coli, enterotoxigenic E.coli and enteroaggregative E.coli suggest that its persistence in waste-dump soil has the ability to cause under-five diarrhoea in both sporadic and endemic settings. This commonly transmitted hand-to-mouth illness will necessitate and place huge demands on the primary catalysts of change i.e. local governmental role players and caregivers. These change agents have to ensure highly consistent levels of domestic and personal hygiene and implement feasible reduction strategies to waste-dump exposure of diarrhoeal-causing pathogens, particularly among under-five children living in Durban’s informal settlements. / M

Gastrointestinal illnesses have continually become a global public health issue. Exposure to zoonotic food borne pathogens such as Salmonella and diarrhoegenic E. coli either by direct or indirect contact through the consumption of food producing animals is likely an important mode of infection to humans. More so, the use of antibiotics in farm animals similar to those used in humans can select for resistance in bacteria frequently harboured by them. These resistant strains can be passed on to humans through contaminated meat products and water leading to resistant infections with consequences such as prolonged illnesses, treatment failures, and increased morbidity and mortality. In animals, these can lead to reduced productivity. Monitoring the level of resistance among bacteria from animal isolates will help in generating data that could be used to create awareness of their presence in the environment and aid in preventing a potential epidemic in the community. In this study, we investigated the prevalence and antimicrobial resistance profile of Escherichia coli serogroups and Salmonella species in faecal samples collected from pigs in Nkonkobe Municipality in the Eastern Cape Province, South Africa between April – July, 2014. A total of 310 presumptive Shiga toxin producing Escherichia coli (STEC) were confirmed as E. coli spp using polymerase chain reaction (PCR) technique by amplification of the uidA gene, out of which 179 (58%) were confirmed positive. Approximately, serogrougs O157:H7, O145 and O26 made up 24% (n=43), 8% (n=14) and 20% (n=35) of the E. coli population respectively. Only E. coli O26 was positive for stx2 gene in 31% of the isolates harbouring the gene, while the other serogroups were non-pathogenic. Susceptibility of the isolates to 18 antibiotics was carried out in vitro by the standardized agar disc-diffusion method. All the isolates were susceptible to imipenem. Similarly, a relatively high susceptibility was observed in norfloxacin (83-100%), ciprofloxacin (63-100%), gentamycin (77-100%), and chloramphenicol (77-100%). However, all the isolates were resistant to tetracycline and its long acting counterpart oxytetracycline. Resistances observed against other antimicrobials are as follows: ampicillin (84-91%), streptomycin (14-100%), erythromycin (91-100%), ceftazidime (35%). Multiple antimicrobial resistance patterns and indices ranged from 3 to 12 and 0.2 to 0.7 to respectively. Genes encoding resistances to ampicillin (ampC), streptomycin (strA) and tetracycline (tetA) were frequently detected in 50-100%, 22-29% and 40-86% of the resistant isolates respectively. In the other arm of the dissertation, two hundred and fifty eight presumptive isolates of Salmonella were recovered from the faecal samples of pigs. Specific primers targeting serogroups A, B, C1, C2, and D were used to delineate the isolates into different serogroups using PCR. Only serogroup A (n=48) was detected. These isolates were examined for antimicrobial susceptibility by disc diffusion method using 18 antibiotics. The results showed that a large proportion of the isolates were resistant to tetracycline (100%), oxytetracycline (100%), ampicillin (75%), sulphamethoxazole/trimethoprim (75%) and streptomycin (75%). Majority of the isolates exhibited multidrug resistances with the predominant multiple antibiotic resistance (MAR) phenotype being against eleven antibiotics. A high multiple antibiotic resistance (MAR) index in a range of 0.3- 0.6 was observed. The incidence of genes encoding resistance against tetracycline (tetA), streptomycin (stra), and ampicillin (ampC) were 54%, 44% and 61% respectively. These findings reveal that pigs within the Nkonkobe Municipality in the Eastern Cape Province could harbour Shiga toxins and multidrug resistant serogroups of E. coli as well as resistant Salmonella which could be transmitted to humans through the food chain. To ensure public health safety, continuous monitoring and sufficient sanitation in swine industries must be ensured.

MSc (Microbiology) / See the attached abstract below

PhD (Microbiology) / Department of Microbiology / Diarrhoea is still a major problem worldwide. Enteric pathogens such as Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC) and Enterotoxigenic E. coli (ETEC) have been reported to cause diarrhoea in children under the age of 5 years. The incidences of these pathogens are due to factors such as poor water quality, sanitation and hygiene practices. Infections with these pathogens result in diarrhoea and have been reported to result in severe disease outcomes more especially in children under 2 years of age. EPEC infections have been well studied using in vitro analyses, with studies highlighting the adherence traits, proteins and virulence genes involved in pathogenesis and inflammatory responses. EPEC is characterized by localized adherence with microcolony formation at the site of infection. In vivo studies have reported on human EPEC infection. However, the current animal models have not been able to replicate clinical outcomes (such as diarrhoea and weigh loss) of EPEC infection similar to humans. Therefore, there is still a need for a suitable small animal model that mimic clinical outcomes of human EPEC infections in vivo. Children living in poor environmental conditions are more susceptible to diarrhoeal pathogens. Furthermore, the incidences of children being exposed to co-infections (more than one pathogen at the same time) is relatively high. The EAEC/EPEC (A/P) and EPEC/ETEC (P/T) co-infections have been increasingly detected in children with and without diarrhoea. It has been suggested that patients infected with these co-infections might result in severe disease outcome than those infected with single pathogens. Pathogens are constantly evolving and the microbe-microbe interaction in the host can result in these pathogens competing for the same niche and thus result in increased virulence. Interaction of co-infections can lead to increased inflammatory responses thus affecting the infected host. The first objective of this study was to develop an EPEC murine model using weaned C57BL/6 mice that have been pretreated with antibiotic cocktail. Mice were orally infected with wild-type (WT) typical EPEC, bfp- and escN mutant strains. The WT had transient weight loss and wet stools with mucous; and the bfp- infected mice also had transient weight loss and bloody stool appearance. Increase in inflammatory biomarkers MPO, LCN-2, CRP, IL-6 and SAA were observed in the WT and bfp- infected mice. The mice infected with escN mutant did not exhibit any weight changes and the stools were similar to the uninfected mice. Furthermore, no inflammatory biomarkers were observed in mice infected with the escN mutant. Metabolic perturbations were observed in WT EPEC infected mice at day 3 post infection with the TCA cycle metabolites (reduced succinate, citrate, fumarate, cis-aconitate) being excreted at lower quantities indicating that the energy production in the infected mice was greatly affected. The second objective of this study was to determine the interaction between the P/T coinfections using in vitro and in vivo analyses. In vitro, human colorectal tumour 8 (HCT-8) cells were infected with single strains of ETEC, EPEC and both the pathogens and incubated for 3 hours. After infection the cells were analysed for bacterial adherence using real-time PCR. The single strains adhered at the same rate similar to the P/T coinfected cells. IL-8, as a marker of inflammatory response, was measured using ELISA. The results indicated that the P/T co-infected cells had a significant increase in IL-8 response higher than the single infections. The P/T co-infections were further analysed in vivo using the EPEC murine model developed in this study. Interestingly, mice infected with P/T co-infections developed severe diarrhoea accompanied with significant increased weight loss and some mice died during the 3-day infection period. The inflammatory responses MPO, LCN-2 and SAA were higher in the co-infected mice indicating a synergistic effect. The bfp and eltA virulence genes were significantly increased in the P/T co-infections. The third objective of this study was to determine the interaction between A/P coinfections using in vitro and in vivo analyses. HeLa cells and HCT-8 cells were infected with EAEC, EPEC and both the pathogens at the same time in order to determine adherence and inflammatory responses. EAEC adherence was higher than EPEC and A/P co-infections adherence. A/P co-infections did not have increased IL-8 response in HCT-8 cells when compared to EAEC alone. The virulence genes involved in EPEC adherence and Type 3 Secretion System (bfp, eae, tir, ler, per, espB and espA) were significantly reduced in A/P co-infected cells. An interesting adherence trait was observed between the A/P co-infections in HeLA cells, EAEC was found to adhere around EPEC altering the localized adherence pattern. The A/P co-infections were further analysed using the EPEC murine model developed in this study. The A/P infected mice had diminished weight changes and EAEC shedding was enhanced when EPEC was present. Faecal inflammatory biomarkers MPO and LCN-2 in A/P infected mice did not have any additive effect. The findings of this study contributed significantly to the knowledge of human EPEC infection in weaned C57BL/6 mice, highlighting clinical outcomes, inflammatory responses and metabolic perturbations. Furthermore, this study also highlighted the interaction of P/T and A/P co-infections using in vitro and in vivo analyses in order to determine the disease severity and outcomes. It was observed in this study that coinfections can result in either synergistic or antagonistic effects. Further studies are therefore, required in order to understand the underlying mechanisms that are involved during co-infections and this can further assist in the development of therapeutic interventions. / NRF

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestine and produces the phage-encoded Shiga toxin (Stx) which is absorbed systemically and can lead to hemolytic uremic syndrome (HUS) characterized by hemolytic anemia, thrombocytopenia, and renal failure. EHEC, and two related pathogens, Enteropathogenic E. coli (EPEC), and the murine pathogen, Citrobacter rodentium, are attaching and effacing (AE) pathogens that intimately adhere to enterocytes and form actin “pedestals” beneath bound bacteria. The actin pedestal, because it is a unique characteristic of AE pathogens, has been the subject of intense study for over 20 years. Investigations into the mechanism of pedestal formation have revealed that to generate AE lesions, EHEC injects the type III effector, Tir, into mammalian cells, which functions as a receptor for the bacterial adhesin intimin. Tir-intimin binding then triggers a signaling cascade leading to pedestal formation. In spite of these mechanistic insights, the role of intimin and pedestal formation in EHEC disease remains unclear, in part because of the paucity of murine models for EHEC infection. We found that the pathogenic significance of EHEC Stx, Tir, and intimin, as well as the actin assembly triggered by the interaction of the latter two factors, could be productively assessed during murine infection by recombinant C. rodentium expressing EHEC virulence factors. Here we show that EHEC intimin was able to promote colonization of C. rodentium in conventional mice. Additionally, previous in vitro data indicates that intimin may have also function in a Tir-independent manner, and we revealed this function using streptomycin pre-treated mice. Lastly, using a toxigenic C. rodentium strain, we assessed the function of pedestal formation mediated by Tir-intimin interaction and found that Tir-mediated actin polymerization promoted mucosal colonization and a systemic Stx-mediated disease that shares several key features with human HUS.

MSc (Microbiology) / Department of Microbiology / See the attached abstract below