A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, 2017 / In South Africa, Salmonella Enteritidis has become a significant pathogen and the numbers of cases reported to the Centre for Enteric Diseases (CED) have increased. Pulsed-field gel electrophoresis (PFGE) is a primary for molecular subtyping of Salmonella. However, this technique has poor discrimination for serotypes with high homogeneity such as Salmonella Enteritidis. Multi-locus variable-number tandem-repeats analysis (MLVA) has shown higher discriminatory power for Salmonella Enteritidis compared to PFGE. In this study, MLVA was used to investigate the molecular epidemiology and relatedness of human Salmonella Enteritidis strains from Gauteng and Western Cape, South Africa. Furthermore, MLVA was also used to investigate the relatedness of human and non-human Salmonella Enteritidis strains. MLVA included analysis of five VNTR loci, with varying degrees of diversity. A total of 1221 human isolates and 43 non-human isolates were included in the study. Eighty-six MLVA profiles were obtained; MLVA profiles 7, 21, 22 and 28 were the predominant MLVA profiles. MLVA profile 28 was the most common MLVA profile amongst both the human and non-human isolates. Isolates had low prevalence of antimicrobial resistance, however sulfamethoxazole resistance was notable amongst both the human (348; 29%) and non-human (10; 23%) isolates. During the study period, seven Salmonella Enteritidis outbreaks were investigated from six provinces and isolates from each individual outbreak showed an identical MLVA profile. MLVA was shown to be a successful molecular subtyping tool for Salmonella Enteritidis, for both surveillance purposes and outbreak investigations. Salmonella Enteritidis strains circulating within the human and non-human population were clonal. The study emphasizes the need for the one health approach, in order to curb the spread of Salmonella Enteritidis in South Africa. / MT2017

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of requirements of the degree of Master of Science. Johannesburg, 2017 / The typhoid fever causing Salmonella Typhi remains an important public health problem in Africa. More importantly, the emergence of the highly antimicrobial resistant H58 Salmonella Typhi haplotype is of greater concern. Rapid and highly discriminatory molecular methods are essential for prompt and effective epidemiological investigation of typhoid fever outbreaks. Traditional methods, such as pulsed-field gel electrophoresis (PFGE) are time-consuming and offer subjective discrimination of highly homologous isolates. On the contrary, molecular subtyping based on multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) is a rapid, PCR-based method which has been successfully used for subtyping homogenous isolates of the Salmonella genus. This study describes the development and application of a MLVA assay for molecular characterization of Salmonella Typhi isolates from sub-Saharan Africa (SSA). This involved evaluation of thirteen VNTR loci using a validation panel consisting of 50 diverse Salmonella Typhi isolates. A MLVA assay consisting of five highly variable VNTR loci was adopted. The developed MLVA assay was used, along with PFGE, to characterize 316 Salmonella Typhi isolates from SSA. A total of 226 MLVA types were identified as compared to 143 PFGE fingerprint types. MLVA typing results indicated intracontinental spread of Salmonella Typhi. For the rapid identification of H58 Salmonella Typhi, a conventional PCR targeting a mutation that is exclusive to the H58 haplotype was employed on 105 isolates from South Africa as well as 121 isolates from other SSA countries. Approximately 54% (105/214) of the Salmonella Typhi isolates from South Africa and 62% (75/121) of the isolates from other SSA countries were identified as H58 Salmonella Typhi. The MLVA tool was able to discriminate among H58 Salmonella Typhi isolates. MLVA is viable alternative to PFGE for subtyping Salmonella Typhi and can be used as first-line assay for routine screening of Salmonella Typhi isolates in SSA, providing excellent discrimination of isolates. / MT2017

by Norman Wai-sing Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 145-160). / Chapter Chapter 1 --- Introduction --- p.1 / Chapter A. --- Classification of Salmonella --- p.3 / Chapter B. --- Enteric fever --- p.4 / Chapter C. --- Chloramphenicol and multiple resistance in S. Typhi --- p.7 / Chapter D. --- Resistance plasmids in the typhoid bacillus --- p.11 / Chapter E. --- In vivo acquisition of resistance plasmids by S. Typhi --- p.18 / Chapter F. --- Worldwide distribution of typhoid fever --- p.23 / Chapter G. --- Epidemiology of typhoid fever in Hong Kong --- p.27 / Chapter H. --- Principles of methods used in the epidemiological typing of S. Typhi --- p.29 / Chapter 1. --- Phage typing --- p.29 / Chapter 2. --- Antibiotics sensitivity pattern --- p.32 / Chapter 3. --- Plasmid analyses --- p.32 / Chapter a. --- Genetic methods --- p.33 / Chapter i. --- Phenotypic expression --- p.33 / Chapter ii. --- Transferability of plasmids --- p.33 / Chapter iii. --- Incompatibility --- p.34 / Chapter b. --- Molecular methods --- p.34 / Chapter i. --- Estimation of molecular size by agarose gel electrophoresis --- p.34 / Chapter ii. --- Plasmid fingerprinting --- p.35 / Chapter iii. --- Localization of resistance genes by DNA-DNA hybridization --- p.35 / Chapter 4. --- Ribotyping --- p.36 / Chapter 5. --- Total DNA fingerprinting --- p.37 / Chapter 6. --- Chromosomal insertion sequence IS200 profile analysis --- p.38 / Chapter 7. --- Others --- p.39 / Chapter a. --- Multilocus enzyme electrophoresis --- p.39 / Chapter b. --- Phenotype of lipopolysaccharide (LPS) --- p.39 / Chapter c. --- Envelope protein profiles and immunoblotting --- p.40 / Chapter I. --- Epidemiological typing of S. Typhi --- p.40 / Chapter J. --- Objectives --- p.46 / Chapter Chapter 2 --- Materials and Methods --- p.48 / Chapter A. --- Materials --- p.48 / Chapter I. --- Bacterial strains --- p.48 / Chapter II. --- Materials --- p.51 / Chapter B. --- Methods --- p.56 / Chapter I. --- Sensitivity testing --- p.56 / Chapter II. --- Characterization of plasmids --- p.58 / Chapter 1. --- Genetic studies --- p.58 / Chapter a. --- Transferability of resistance plasmids --- p.58 / Chapter b. --- Mobilization of resistances --- p.59 / Chapter c. --- Incompatibility grouping --- p.59 / Chapter 2. --- Molecular studies --- p.60 / Chapter a. --- Plasmid profile analysis --- p.60 / Chapter i. --- Plasmid extraction --- p.60 / Chapter ii. --- Agarose gel electrophoresis --- p.61 / Chapter b. --- Molecular characterization of plasmid --- p.61 / Chapter i. --- Extraction of purified plasmid DNA --- p.62 / Chapter ii. --- Restriction endonuclease digestion of plasmid DNA --- p.63 / Chapter III. --- Characterization β-lactamases --- p.63 / Chapter 1. --- Extraction of β-lactamases --- p.63 / Chapter 2. --- Determination of isoelectric points (pIs) --- p.64 / Chapter IV. --- Localization of resistance genes --- p.64 / Chapter 1. --- TEM-1 and TEM-2 genes --- p.65 / Chapter a. --- Oligonucleotide probes --- p.65 / Chapter b. --- Total DNA preparation --- p.66 / Chapter c. --- DNA-DNA-hybridization --- p.66 / Chapter i. --- Transfer of DNA to membrane filters --- p.66 / Chapter ii. --- Labelling of oligonucleotide probes (TEM-1 and TEM-2) --- p.67 / Chapter iii. --- Hybridization --- p.68 / Chapter 2. --- Chloramphenicol- and tetracycline-resistance genes --- p.69 / Chapter a. --- Preparation of probes --- p.69 / Chapter b. --- Hybridization --- p.73 / Chapter i. --- Transfer of DNA to membrane filters --- p.73 / Chapter ii. --- Labelling of chloramphenicol- and tetracycline- resistance probes --- p.73 / Chapter V. --- Epidemiological typing --- p.75 / Chapter 1. --- Ribotyping --- p.75 / Chapter 2. --- DNA fingerprinting --- p.77 / Chapter C. --- Plan for achieving objectives --- p.77 / Chapter Chapter 3 --- Results --- p.79 / Chapter A. --- Antimicrobial susceptibilities --- p.79 / Chapter B. --- Characterization of resistance plasmids --- p.83 / Chapter C. --- β-lactamases produced by ampicillin-resistant S. Typhi --- p.85 / Chapter D. --- Localization of resistance genes --- p.85 / Chapter E. --- Plasmid profile analysis --- p.87 / Chapter F. --- Plasmid fingerprinting --- p.90 / Chapter G. --- Ribotyping --- p.93 / Chapter H. --- Chromosomal DNA fingerprinting --- p.107 / Chapter I. --- "Correlation of PstI, ClaI and KpnI ribotypes and NarI, EcoRV and MluI chromosomal types" --- p.124 / Chapter J. --- Correlation of PstI ribotypes and NarI chromosomal types --- p.126 / Chapter K. --- Epidemiology of S. Typhi in Hong Kong --- p.129 / Chapter Chapter 4 --- Discussion --- p.131 / Chapter A. --- Antimicrobial susceptibilities --- p.131 / Chapter B. --- Characterization of resistances --- p.132 / Chapter C. --- Plasmid profile analysis --- p.135 / Chapter D. --- Epidemiological analysis of S. Typhi --- p.136 / Chapter E. --- Area for future research --- p.143 / References --- p.145 / Appendix --- p.161 / Chapter A. --- Buffer and Stock solutions --- p.161 / Chapter B. --- Epidemiological information of S. Typhi isolates --- p.167

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Thesis (M.Med.Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 35-45). Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 81-86).

Extended-spectrum beta-lactamases (ESBL) mediate resistance to 3rd generation cephalosporins and aztreonam in Enterobacteriaceae and pose major clinical problems. Screened Enterobacteriaceae were collected from PHW Microbiology ABM Swansea laboratory and were demonstrated phenotypically to be ESBL- producers by BSAC methods. Isolates were identified using the BD Phoenix Automated system and Bruker Daltonics MALDI Biotyper. 138 isolates were genetically defined as ESBL-producers (103 Escherichia coli, 32 Klebsiella spp., 2 Enterobacter cloacae and 1 Citrobacter freundii) and 4 isolates (2 E. coli, 1 Enterobacter cloacae and 1 Morganella morganii) were genetically confirmed as AmpC-producers. PCR analysis revealed that the most prevalent ESBLs were CTX-M (n=133), predominantly Group 1 (n=128), of which 51% (66/128) contained the I526-CTX- M-15 link region, which is characteristic for epidemic E. coli strain A. PFGE confirmed that these isolates had a clonal relatedness to epidemic E. coli strain A. Allele-specific PCR revealed that all E. coli positive for I526-CTX-M-15 belonged to clone 025b-ST131 (found internationally), which has a high virulence potential and encompasses diverse PFGE patterns. With the molecular epidemiology established; the sensitivity and performance of phenotypic screening and confirmatory assays were analysed so that optimal strategies to handle difficult-to-identify ESBL resistance traits could be determined. In this study, the sensitivity of ESBL screening increased to 100% when ceftazidime was used alongside cefpodoxime. Isolates harbouring ESBL genes are often difficult to treat, as options are greatly limited. Susceptibility to various well-established antibiotics, along with temocillin and tigecycline, were investigated. Temocillin and tigecycline were effective against 98% and 89% of all isolates tested. The carbapenems were the most active antibiotics with 100% of isolates susceptible to imipenem and meropenem and 99% susceptible to ertapenem. Biofilm production in E. coli was also investigated. The pgaABCD gene locus was detected in all ESBL and AmpC-producing E. coli isolates (n=105); however, only 38% of these produced a phenotypic biofilm.

No description available.

Norovirus (NoV) is a major cause of infectious human viral gastroenteritis. Detection is important to understanding the epidemiology of NoV and the viral dynamics of NoV infection which is poorly understood. In 2006, a marked increase in gastroenteritis outbreaks occurred worldwide. During this period, a total of 231 stool samples were obtained from patients with acute gastroenteritis from Australia and New Zealand. A total of 186 isolates of NoV were detected and sequenced to determine the genotype and relatedness to known epidemic NoV GII.4 variants. Two GII.4 variants, 2006a and 2006b, were identified in 61.8% and 11.3%, in these cases, respectively. Thus, the increase in NoV gastroenteritis in 2006 was linked to the emergence of two novel co-circulating GII.4 variants, 2006a and 2006b. During an outbreak in an aged-care facility, stool samples were collected from the onset of illness to cessation of viral excretion. Here, viral shedding peaked in the acute stage of illness and continued for an average of 28.7 days. The viral decay rate was 0.76 per day. Prolonged asymptomatic shedding of NoV was detected in the elderly. A quality control for the assessment of molecular based viral assays for NoV and other RNA viruses is necessary to meet current testing requirements. Available controls only monitor the RNA and DNA amplification steps. An MS2 bacteriophage BioBallTM with 100 pfu was evaluated and applied as a multi-purpose phage control. It was assessed as a quality control, in comparison to MS2 phage stock, to validate MS2 phage assays. Furthermore, MS2 BioBallTM was used as a process control for the molecular detection of RNA viruses. It validated every performed step, determined if the assay worked and its sensitivity. Thus, MS2 BioBallTM offered uniformity, stability and reproducibility across molecular based viral detection systems. Overall, this thesis provided valuable insight into the molecular epidemiology of NoV in the southern hemisphere and nature of NoV infections in the elderly. The MS2 BioBallTM provides standardisation and quality control of viral RNA assays. Understanding the genetic diversity and viral dynamics of NoV will be crucial to developing effective intervention and treatment strategies, and ultimately lead to reduced viral gastroenteritis worldwide.

Hymenolepis nana is a ubiquitous parasite, found throughout many developing and developed countries. Globally, the prevalence of H. nana is alarmingly high, with estimates of up to 75 million people infected. In Australia, the rates of infection have increased substantially in the last decade, from less than 20% in the early 1990’s to 55 - 60% in these same communities today. Our knowledge of the epidemiology of infection of H. nana is hampered by the confusion surrounding the host specificity and taxonomy of this parasite. The suggestion of the existence of two separate species, Hymenolepis nana von Siebold 1852 and Hymenolepis fraterna Stiles 1906, was first proposed at the beginning of the 20th century. Despite ongoing discussions in the subsequent years it remained unclear, some 90 years later, whether there were two distinct species, that are highly host specific, or whether they were simply the same species present in both rodent and human hosts. The ongoing controversy surrounding the taxonomy of H. nana has not yet been resolved and remains a point of difference between the taxonomic and medical literature. The epidemiology of infection with H. nana in Australian communities is not well understood as the species present in these communities has never been identified with certainty. It is not clear which form of transmission commonly occurs in Australia, whether the H. nana ‘strain/species’ present in the north-west of Western Australia is present in human and rodent hosts, or whether humans harbour their own ‘strain/sub-species’ of Hymenolepis. Furthermore, it is not known whether mice are a potential zoonotic source for transmission of Hymenolepis to human hosts. In this study, 51 human isolates of H. nana were inoculated into highly susceptible laboratory rodent species. However, these failed to develop into adult worms in all instances, including when rodent species were chemically and genetically immunosuppressed.In addition, 24 of these human isolates were also cross-tested in the flour beetle intermediate host, Tribolium confusum. Of these, only one isolate developed to the cysticercoid stage in beetles, yet when inoculated into laboratory rodents, the cysticercoids also failed to develop into adult stage. Since isolates of H. nana infecting humans and rodents are morphologically indistinguishable, the only way they can be reliably identified is by comparing the parasite in each host using molecular criteria. In the current study, three regions of ribosomal DNA, the small subunit (18S), the first internal transcribed spacer (ITS1) and the intergenic spacer (IGS) were chosen for genetic characterisation of Hymenolepis spp. from rodent and human hosts from a broad geographic range. In addition, a mitochondrial gene, the cytochrome c oxidase subunit 1 (C01) gene and a non-ribosomal nuclear gene, paramyosin, were characterised in a number of Hymenolepis isolates from different hosts. A small PCR fragment of 369 bp, plus a larger fragment of 1223 bp, were sequenced from the 18S gene of reference isolates of H. nana and the rat tapeworm H. diminuta. Minimal sequence variation was found in the two regions of the 18S between these two morphologically distinct, phylogenetically recognised species, H. nana and H. diminuta, and this indicated that the 18S gene was too conserved for further genetic characterisation of isolates of H. nana from different hosts. A large number of human isolates of H. nana (104) were characterised at the ITS1 using PCRrestriction length fragment polymorphism (PCR-RFLP). The profiles obtained were highly variable and often exceeded the original size of the uncut fragment. This was highly suggestive of the existence of ribosomal spacers that, whilst identical in length, were highly variable in sequence. To overcome the problems of the variable PCR-RFLP profiles, further characterisation of the ITS1, by cloning and sequencing 23 isolates of H. nana, was conducted and this confirmed the existence of spacers which, although similar in length (approximately 646 bp), differed in their primary sequences. The sequence differences led to the separation of the isolates into two clusters when analysed phylogenetically. This sequence variation was not, however, related to the host of origin of the isolate, thus was not a marker of genetic distinction between H. nana from rodents and humans. Indeed, the levels of variability were often higher within an individual isolate than between isolates, regardless of whether they were collected from human or mice hosts, which was problematic for phylogenetic analysis. In addition, mixed parasite infections of H. nana and the rodent tapeworm H. microstoma were identified in four humans in this study, which was unexpected and surprising, as there have been no previous reports in the literature documenting humans as definitive hosts for this parasite. Further studies are required, however, to determine if the detection of H. microstoma in humans reflects a genuine, patent infection or an atypical, accidental occurrence. Sequencing of the mitochondrial cytochrome c oxidase 1 gene (C01) in a number of isolates of Hymenolepis nana from rodents and humans identified a phylogenetically supported genetic divergence of approximately 5% between some mouse isolates compared to isolates of H. nana from humans. This provided evidence that the mitochondrial C01 gene was useful for identifying genetic divergences in H. nana that were not resolvable using nuclear loci. Despite a morphological identity between isolates of H. nana from rodent and human hosts, the genetic divergence observed between isolates at the mitochondrial locus was highly suggestive that H. nana is a species complex, or “cryptic” species (= morphologically identical yet genetically distinct). In addition, whilst not supported by high bootstrap values, a clustering of the Australian human isolates into one uniform genetic group that was phylogenetically separated from all the mouse isolates was well supported by biological data obtained in this study. To confirm the phylogeny of the C01 tree a small segment of the nuclear gene, paramyosin, was sequenced in a number of isolates from humans and rodents. However, this gene did not provide the level of heterogeneity required to distinguish between isolates from rodent and human hosts. The high sequence conservation of the paramyosin gene characterised in this study did not refute the finding that H. nana may be a cryptic species that is becoming host adapted. It simply did not provide additional data to that already obtained. A DNA fingerprinting tool, PCR-RFLP, of the ribosomal intergenic spacer (IGS), was developed in this study in order to evaluate its usefulness in tracing particular genotypes within a community, thus determining transmission patterns of H. nana between rodent and human hosts. Analysis of the IGS of numerous H. nana isolates by PCR-RFLP identified the presence of copies of the IGS that, whilst similar in length, differed in their sequence. Similar to that observed in the ITS1, the existence of different IGS copies was found in both rodent and human isolates of H. nana, thus the variability was not evidence of the existence of a rodent- or humanspecific genotype. Evaluation of the intergenic spacer (IGS) as a fingerprinting tool suggests that this region of DNA is too variable within individuals and thus, cannot be effectively used for the study of transmission patterns of the tapeworm H. nana between different hosts. In summary, it appears that the life cycle of H. nana that exists in remote communities in the north-west of Western Australia is likely to involve mainly ‘human to human’ transmission. This is supported by both the biological and genetic data obtained for the mitochondrial locus in this study. The role of the intermediate hosts, such as Tribolium spp., in the Hymenolepis life cycle is still unclear, however it would appear that it may be greatly reduced in the transmission of this parasite in remote Australian communities. In the future, it is recommended that further genetic characterisation of faster evolving mitochondrial genes, and/or suitable nuclear genes be characterised in a larger number of isolates of H. nana. The use of techniques which can combine the characterisation of genotype and phenotype, such as proteomics, may also be highly valuable for studies on H. nana from different hosts.