Development and evaluation of new molecular epidemiological methods for analysis of salmonella TYPHI

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, 2017The 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.MT201

Development and evaluation of a multiple-locus variable-number tandem-repeats analysis assay for subtyping Salmonella Typhi strains from sub-Saharan Africa

Purpose: Molecular epidemiological investigations of the highly clonal Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) are important in outbreak detection and in tracking disease transmission. In this study, we developed and evaluated a multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) assay for characterization of S. Typhi isolates from sub-Saharan Africa. Methodology: Twelve previously reported VNTR loci were evaluated and an MLVA assay consisting of five polymorphic loci was adopted. The MLVA assay was developed for use on capillary electrophoresis systems by testing a collection of 50 S. Typhi isolates. This S. Typhi strain panel consisted of six outbreak related isolates and 44 epidemiologically unlinked isolates. Amongst these were nine S.Typhi haplotype H58 isolates. Results: The MLVA assay characterized the 50 isolates into 47 MLVA profiles while PFGE analysis of the same isolates revealed 34 pulsotypes. MLVA displayed higher discriminatory power (Simpson’s index of diversity (DI) 0.998 [95% confidence interval (CI) 0.995–1.000)] as compared to pulsed-field gel electrophoresis [Simpson’s DI 0.984 (95% CI 0.974–0.994)]. Conclusion: The MLVA assay presented in this study is a simple, rapid and more accessible tool that serves as a good alternative to other molecular subtyping methods for S. Typhi

Laboratory-acquired infections of Salmonella enterica serotype Typhi in South Africa: phenotypic and genotypic analysis of isolates

BACKGROUND: Workers in clinical microbiology laboratories are exposed to a variety of pathogenic microorganisms. Salmonella species is among the most commonly reported bacterial causes of laboratory-acquired infections. We report on three cases of laboratory-acquired Salmonella enterica serotype Typhi (Salmonella Typhi) infection which occurred over the period 2012 to 2016 in South Africa. METHODS: Laboratory investigation included phenotypic and genotypic characterization of isolates. Phenotypic analysis included standard microbiological identification techniques, serotyping and antimicrobial susceptibility testing. Genotypic analysis included the molecular subtyping methodologies of pulsed-field gel electrophoresis analysis, multilocus sequence typing and whole-genome sequencing (WGS); with WGS data analysis including phylogenetic analysis based upon comparison of single nucleotide polymorphism profiles of isolates. RESULTS: All cases of laboratory-acquired infection were most likely the result of lapses in good laboratory practice and laboratory safety. The following critical issues were highlighted. There was misdiagnosis and misreporting of Salmonella Typhi as nontyphoidal Salmonella by a diagnostic laboratory, with associated public health implications. We highlight issues concerning the importance of accurate fluoroquinolone susceptibility testing and interpretation of results according to updated guidelines. We describe potential shortcomings of a single disk susceptibility screening test for fluoroquinolone susceptibility and suggest that confirmatory minimum inhibitory concentration testing should always be performed in cases of invasive Salmonella infections. These antimicrobial susceptibility testing issues resulted in inappropriate ciprofloxacin therapy which may have been responsible for failure in clearance of pathogen from patients. Salmonella Typhi capsular polysaccharide vaccine was not protective in one case, possibly secondarily to a faulty vaccine. CONCLUSIONS: Molecular subtyping of isolates proved effective to investigate the genetic relatedness of isolates. Molecular subtyping data interpreted together with epidemiological data allowed us to pinpoint the most likely sources for our cases of laboratory-acquired infection

A One Health Perspective on Salmonella enterica Serovar Infantis, an Emerging Human Multidrug-Resistant Pathogen

Salmonella enterica serovar Infantis presents an ever-increasing threat to public health because of its spread throughout many countries and association with high levels of antimicrobial resistance (AMR). We analyzed whole-genome sequences of 5,284 Salmonella Infantis strains from 74 countries, isolated during 1989-2020 from a wide variety of human, animal, and food sources, to compare genetic phylogeny, AMR determinants, and plasmid presence. The global Salmonella Infantis population structure diverged into 3 clusters: a North American cluster, a European cluster, and a global cluster. The levels of AMR varied by Salmonella Infantis cluster and by isolation source; 73% of poultry isolates were multidrug resistant, compared with 35% of human isolates. This finding correlated with the presence of the pESI megaplasmid; 71% of poultry isolates contained pESI, compared with 32% of human isolates. This study provides key information for public health teams engaged in reducing the spread of this pathogen

Escherichia coli O104 Associated with Human Diarrhea, South Africa, 2004–2011

To determine the origin of >4,000 suspected diarrheagenic Escherichia coli strains isolated during 2004–2011 in South Africa, we identified 7 isolates as serotype O104; 5 as enteroaggregative E. coli O104:H4, and 2 as enteropathogenic E. coli O104:non-H4. Pulsed-field gel electrophoresis showed that these isolates were unrelated to the 2011 E. coli O104:H4 outbreak strain from Germany

Prevalence and antibiotic susceptibility patterns of enteric bacterial pathogens in human and non-human sources in an urban informal settlement in Cape Town, South Africa

Abstract Background In light of rampant childhood diarrhoea, this study investigated bacterial pathogens from human and non-human sources in an urban informal settlement. Meat from informal abattoirs (n = 85), river water (n = 64), and diarrheic stool (n = 66) were collected between September 2015 and May 2016. A duplex real-time PCR, gel-based PCR, and CHROMagar™STEC were used to screen Tryptic Soy Broth (TSB) for diarrheic E. coli. Standard methods were used to screen for other selected food and waterborne bacterial pathogens. Results Pathogens isolated from stool, meat, and surface water included Salmonella enterica (6, 5, 0%), Plesiomonas shigelloides (9, 0, 17%), Aeromonas sobria (3, 3, 0%), Campylobacter jejuni (5, 5, 0%), Shigella flexneri (17, 5, 0%), Vibrio vulnificus (0, 0, 9%), and diarrheic E. coli (21, 3, 7%) respectively. All the isolates were resistant to trimethoprim–sulphamethoxazole. Conclusions There was a high burden of drug resistant diarrheal pathogens in the stool, surface water and meat from informal slaughter. Integrated control measures are needed to ensure food safety and to prevent the spread of drug resistant pathogens in similar settings

Typhoid Fever in South Africa in an Endemic HIV Setting.

Typhoid fever remains an important disease in Africa, associated with outbreaks and the emerging multidrug resistant Salmonella enterica serotype Typhi (Salmonella Typhi) haplotype, H58. This study describes the incidence of, and factors associated with mortality due to, typhoid fever in South Africa, where HIV prevalence is high.Nationwide active laboratory-based surveillance for culture-confirmed typhoid fever was undertaken from 2003-2013. At selected institutions, additional clinical data from patients were collected including age, sex, HIV status, disease severity and outcome. HIV prevalence among typhoid fever patients was compared to national HIV seroprevalence estimates. The national reference laboratory tested Salmonella Typhi isolates for antimicrobial susceptibility and haplotype. Unadjusted and adjusted logistic regression analyses were conducted determining factors associated with typhoid fever mortality. We identified 855 typhoid fever cases: annual incidence ranged from 0.11 to 0.39 per 100,000 population. Additional clinical data were available for 369 (46.8%) cases presenting to the selected sites. Among typhoid fever patients with known HIV status, 19.3% (29/150) were HIV-infected. In adult females, HIV prevalence in typhoid fever patients was 43.2% (19/44) versus 15.7% national HIV seroprevalence (P < .001); in adult males, 16.3% (7/43) versus 12.3% national HIV seroprevalence (P = .2). H58 represented 11.9% (22/185) of Salmonella Typhi isolates tested. Increased mortality was associated with HIV infection (AOR 10.7; 95% CI 2.3-50.3) and disease severity (AOR 9.8; 95% CI 1.6-60.0) on multivariate analysis.Typhoid fever incidence in South Africa was largely unchanged from 2003-2013. Typhoid fever mortality was associated disease severity. HIV infection may be a contributing factor. Interventions mandate improved health care access, including to HIV management programmes as well as patient education. Further studies are necessary to clarify relationships between HIV infection and typhoid fever in adults