Whole-genome sequencing to investigate two concurrent outbreaks of Salmonella Enteritidis in South Africa, 2018.

Salmonella enterica serotype Enteritidis (Salmonella Enteritidis) is a major cause of foodborne disease outbreaks worldwide. In 2018, two concurrent outbreaks of Salmonella Enteritidis gastroenteritis in one district of South Africa were investigated. We describe the use of whole-genome sequencing (WGS) analysis of bacterial isolates to assist with the investigation of these outbreaks. Outbreak A affected children (n=27) attending a day-care centre, while outbreak B affected adults (n=16) who ate breakfast at the same restaurant. Salmonella Enteritidis was isolated from stool samples in both outbreaks (four children in outbreak A; 12 restaurant customers and three restaurant food-handlers in outbreak B). In outbreak B, Salmonella Enteritidis was isolated from three food retention samples (raw chicken egg, hollandaise sauce and rocket-herb). Available isolates from both outbreaks (n=13) were investigated using WGS analysis. Sequencing data for isolates were analysed at the EnteroBase web-based platform and included core-genome multi-locus sequence typing (cgMLST). Isolates with epidemiological links to the restaurant (n=10) and day-care centre (n=3), were shown by cgMLST to be highly genetically related, with no more than five allele differences when comparing one isolate against another. On food history, eggs and hollandaise sauce were the common food items consumed by ill restaurant customers. Unfortunately, Salmonella Enteritidis isolated from the egg and hollandaise sauce were not available for WGS analysis. Our investigation concluded that the two concurrent outbreaks were caused by a highly related strain of Salmonella Enteritidis, suggesting the possibility of a common contaminated food source, of which contaminated eggs are strongly implicated.

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.

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.

Outbreak of Listeria monocytogenes in South Africa, 2017-2018: Laboratory Activities and Experiences Associated with Whole-Genome Sequencing Analysis of Isolates.

In South Africa, a progressive increase in listeriosis cases was noted from mid-June 2017, heralding what was to become the world's largest listeriosis outbreak. A total of 1060 cases were reported for the period January 1, 2017 to July 17, 2018. We describe laboratory activities, experiences, and results of whole-genome sequencing (WGS) analysis of Listeria monocytogenes isolates associated with this outbreak. Bacteria were identified using the VITEK-2 COMPACT 15 microbial identification system. WGS was performed using Illumina MiSeq technology. WGS data were analyzed using CLC Genomics Workbench Software and free-to-use on-line analysis tools/pipelines. Multilocus sequence typing (MLST) showed that 91% of clinical isolates were sequence type 6 (ST6), determining that the outbreak was largely associated with L. monocytogenes ST6. Epidemiological and laboratory findings led to investigation of a large ready-to-eat processed meat production facility in South Africa, named Enterprise Foods. L. monocytogenes ST6 was found in environmental sampling swabs of the production facility and in ready-to-eat processed meat products (including polony, a product similar to bologna sausage) manufactured at the facility. ST6 isolates, sourced at the Enterprise Foods production facility and from Enterprise food products, were shown by single nucleotide polymorphism (SNP) analysis to be highly related to clinical isolates; these nonclinical ST6 isolates showed <10 SNP differences when compared to clinical ST6 isolates. Core-genome MLST showed that clinical ST6 isolates and Enterprise-related ST6 isolates had no more than 4 allele differences between each other, suggestive of a high probability of epidemiological relatedness. WGS data interpreted together with epidemiological data concluded that the source of the listeriosis outbreak was ready-to-eat processed meat products manufactured by Enterprise Foods. Listeriosis has now been added to the South African list of mandatory notifiable medical conditions. Surveillance systems have been strengthened to facilitate prevention and early detection of listeriosis outbreaks.

Shiga toxin-producing Escherichia coli O26:H11 associated with a cluster of haemolytic uraemic syndrome cases in South Africa, 2017.

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that may cause diarrhoeal outbreaks and occasionally are associated with haemolytic-uraemic syndrome (HUS). We report on STEC O26:H11 associated with a cluster of four HUS cases in South Africa in 2017. METHODOLOGY: All case-patients were female and aged 5 years and under. Standard microbiological tests were performed for culture and identification of STEC from specimens (human stool and food samples). Further analysis of genomic DNA extracted from bacterial cultures and specimens included PCR for specific virulence genes, whole-genome sequencing and shotgun metagenomic sequencing. RESULTS: For 2/4 cases, stool specimens revealed STEC O26:H11 containing eae, stx2a and stx2b virulence genes. All food samples were found to be negative for STEC. No epidemiological links could be established between the HUS cases. Dried meat products were the leading food item suspected to be the vehicle of transmission for these cases, as 3/4 case-patients reported they had eaten this. However, testing of dried meat products could not confirm this. CONCLUSION: Since STEC infection does not always lead to severe symptoms, it is possible that many more cases were associated with this cluster and largely went unrecognized.

The Burden of Typhoid Fever in South Africa: The Potential Impact of Selected Interventions.

Typhoid fever is notifiable in South Africa but clinical notification is notoriously poor. South Africa has an estimated annual incidence rate of 0.1 cases per 100,000 population of culture-confirmed typhoid fever, decreased from 17 cases per 100,000 population in the 1980s. This work was undertaken to identify the reasons for this decrease and identify potential weaknesses that may result in an increase of observed cases. Culture-confirmed cases, with additional demographic and clinical data have been collected from selected sentinel sites since 2003. Data on contextual factors (gross domestic product [GDP], sanitation, female education, and childhood diarrhea mortality) were collected. National incidence rates of culture-confirmed typhoid fever have remained constant for the past 13 years, with the exception of an outbreak in 2005: incidence was 0.4 per 100,000 population. Paratyphoid fever remains a rare disease. Antimicrobial susceptibility data suggest resistance to ciprofloxacin and azithromycin is emerging. The South African population increased from 27.5 million in 1980 to 55.0 million in 2015: urbanization increased from 50% to 65%, GDP increased from United States Dollar (USD) $2,910 to USD $6,167, access to sanitation improved from 64.4% to 70.0% in the urban population and 26.4% to 60.5% in rural areas. Female literacy levels improved from 74.8% to 92.6% over the period. Improved socioeconomic circumstances in South Africa have been temporally associated with decreasing incidence rates of typhoid fever over a 35-year period. Ongoing challenges remain including potential for large outbreaks, a large immigrant population, and emerging antimicrobial resistance. Continued active surveillance is mandatory.

Genome Sequence for Shiga Toxin-Producing Escherichia coli O26:H11, Associated with a Cluster of Hemolytic-Uremic Syndrome Cases in South Africa, 2017.

Shiga toxin-producing Escherichia coli (STEC) strains are primarily foodborne pathogens that may cause diarrheal outbreaks and are associated with severe complications, specifically hemolytic-uremic syndrome (HUS). We report here genome sequence data for STEC O26:H11, which is associated with a cluster of cases of HUS, a rarely described syndrome in South Africa.

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.

Typhoid Fever in South Africa in an Endemic HIV Setting.

BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

Clinical and Microbiological Features of Salmonella Meningitis in a South African Population, 2003-2013.

BACKGROUND: The clinical and microbiological characteristics of nontyphoidal Salmonella (NTS) meningitis in South Africa, where human immunodeficiency virus (HIV) prevalence is high (approximately 15% in persons ≥15 years of age), were reviewed. METHODS: From 2003 through 2013, 278 cases were identified through national laboratory-based surveillance. Clinical information (age, sex, outcome, Glasgow Coma Scale [GCS], and HIV status) was ascertained at selected sites. Isolates were serotyped; susceptibility testing and multilocus sequence typing on Salmonella enterica serovar Typhimurium isolates was performed. Multivariable logistic regression was used to determine factors associated with mortality outcome, using Stata software, version 13. RESULTS: Where age was ascertained, 139 of 256 (54.3%) patients were <15 years. Males represented 151 of 267 (56.6%). Mortality outcome was recorded for 112 of 146 (76.7%) enhanced surveillance patients; 53 of 112 (47.3%) died. Death was associated with GCS ≤13 (adjusted odds ratio [OR], 18.7; 95% confidence interval [CI], 3.0-118.5; P = .002) on multivariable analysis. Where data were available, all 45 patients aged >15 years were HIV infected, compared with 24 of 46 (52.2%) patients aged <5 years. Neonates were less likely to be HIV infected than infants aged 2-12 months (OR, 4.8; 95% CI, 1.1-21.1; P = .039).Salmonella Typhimurium represented 106 of 238 (44.5%) serotyped isolates: 65 of 95 (68.4%) were ST313 vs ST19, respectively, and significantly associated with HIV-infected patients (P = .03) and multidrug resistance (OR, 6.6; 95% CI, 2.5-17.2; P < .001). CONCLUSIONS: NTS meningitis in South Africa is highly associated with HIV in adults, with neonates (irrespective of HIV status), and with Salmonella Typhimurium ST313. GCS is the best predictor of mortality: early diagnosis and treatment are critical. Focused prevention requires further studies to understand the sources and transmission routes.

Cholera outbreak in South Africa, 2008-2009: laboratory analysis of Vibrio cholerae O1 strains.

BACKGROUND: A total of 720 Vibrio cholerae O1 strains were recovered for investigation from an outbreak of cholera in South Africa between November 2008 and April 2009. METHODS: Strains were characterized by serotype testing. Antimicrobial susceptibility testing. Genetic diversity of 248 strains was investigated using pulsed-field gel electrophoresis (PFGE) analysis. Extended characterization was performed on 90 strains. Molecular analysis included: polymerase chain reaction (PCR) identification of ctxA and tcpA genes, sequencing the ctxAB gene, and investigation of molecular mechanisms conferring antimicrobial resistance. RESULTS: The majority of strains were characterized as serotype Ogawa. Strains showed multidrug resistance. Approximately 1.0% of strains displayed extended-spectrum ß-lactamase (ESBL) activity. Strains showed very similar PFGE patterns. Ninety strains selected for extended characterization showed the following results: Strains possessed the cholera toxin (CT) and all were PCR positive for the tcpA-El Tor variant. Sequencing of the ctxB gene matched the B-1 allele. Strains harbored the SXT element. Strains that displayed ESBL activity possessed a 140-kilobase-pair plasmid that produced the TEM-63 ß-lactamase. Nalidixic acid-resistant strains harbored mutations in GyrA (Ser83-Ile) and ParC (Ser85-Leu). CONCLUSIONS: These data highlight the rapid development of antimicrobial resistance and spread of V. cholerae O1 El Tor variants expressing the classical CT within South Africa.

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.