Presence and Virulence Characteristics of Shiga Toxin Escherichia coli and Non-Shiga Toxin-Producing Escherichia coli O157 in Products from Animal Protein Supply Chain Enterprises in South Africa.

Consumption of food that is contaminated with Shiga toxin-producing Escherichia coli (STEC) has been linked to serious foodborne disease outbreaks. Our aim was to provide a descriptive study on the presence and virulence factors of STEC and non-STEC O157 isolates recovered from 2017 diverse meat and meat product samples from all provinces of South Africa (n = 1758) and imported meat from South Africa's major ports of entry (n = 259). A cross-sectional study was undertaken to analyze raw intact meat, raw processed (nonintact) meat, and ready-to-eat (RTE) meat from cattle, game, sheep, pork, and poultry. Isolation was performed using International Organization for Standardization-based microbiological techniques, while detection and characterization were performed using real-time PCR (RT-PCR) and conventional PCR targeting the stx1, stx2, eae, and ehxA genes. A total of 28 of 1758 (1.59%; confidence interval [CI] 1.1-2) samples from the domestic market tested positive (n = 10 Escherichia coli O157:H7; n = 14 Escherichia coli O157: non-H7; and n = 4 non-O157 STEC), while 4/259 (1.54%; CI 0.4-4) samples from ports of entry tested positive for Escherichia coli O157:H7 based on RT-PCR. On average, diverse samples from domestic meat and meat products from cattle showed the highest number of positive samples (22/1758; 1.3%; CI 0.8-2). RT-PCR detected more positive samples (n = 32) compared with culture (n = 17). Sixteen different virulence factor combinations were observed. Our findings demonstrate a relatively low presence of diverse STEC strains along the meat value chain. To our knowledge, this is the first extensive report in South Africa to analyze STEC and non-STEC O157 from local and imported samples from many animal species. This is important as it reveals virulence factors in STEC strains circulating in meat and meat products in South Africa, which contribute to the risk of infection.

Rooibos Flavonoids, Aspalathin, Isoorientin, and Orientin Ameliorate Antimycin A-Induced Mitochondrial Dysfunction by Improving Mitochondrial Bioenergetics in Cultured Skeletal Muscle Cells.

The current study investigated the physiological effects of flavonoids found in daily consumed rooibos tea, aspalathin, isoorientin, and orientin on improving processes involved in mitochondrial function in C2C12 myotubes. To achieve this, C2C12 myotubes were exposed to a mitochondrial channel blocker, antimycin A (6.25 µM), for 12 h to induce mitochondrial dysfunction. Thereafter, cells were treated with aspalathin, isoorientin, and orientin (10 µM) for 4 h, while metformin (1 µM) and insulin (1 µM) were used as comparators. Relevant bioassays and real-time PCR were conducted to assess the impact of treatment compounds on some markers of mitochondrial function. Our results showed that antimycin A induced alterations in the mitochondrial respiration process and mRNA levels of genes involved in energy production. In fact, aspalathin, isoorientin, and orientin reversed such effects leading to the reduced production of intracellular reactive oxygen species. These flavonoids further enhanced the expression of genes involved in mitochondrial function, such as Ucp 2, Complex 1/3, Sirt 1, Nrf 1, and Tfam. Overall, the current study showed that dietary flavonoids, aspalathin, isoorientin, and orientin, have the potential to be as effective as established pharmacological drugs such as metformin and insulin in protecting against mitochondrial dysfunction in a preclinical setting; however, such information should be confirmed in well-established in vivo disease models.

The Potential Role of Polyphenols in Modulating Mitochondrial Bioenergetics within the Skeletal Muscle: A Systematic Review of Preclinical Models.

Polyphenols are naturally derived compounds that are increasingly being explored for their various health benefits. In fact, foods that are rich in polyphenols have become an attractive source of nutrition and a potential therapeutic strategy to alleviate the untoward effects of metabolic disorders. The last decade has seen a rapid increase in studies reporting on the bioactive properties of polyphenols against metabolic complications, especially in preclinical models. Various experimental models involving cell cultures exposed to lipid overload and rodents on high fat diet have been used to investigate the ameliorative effects of various polyphenols against metabolic anomalies. Here, we systematically searched and included literature reporting on the impact of polyphenols against metabolic function, particularly through the modulation of mitochondrial bioenergetics within the skeletal muscle. This is of interest since the skeletal muscle is rich in mitochondria and remains one of the main sites of energy homeostasis. Notably, increased substrate availability is consistent with impaired mitochondrial function and enhanced oxidative stress in preclinical models of metabolic disease. This explains the general interest in exploring the antioxidant properties of polyphenols and their ability to improve mitochondrial function. The current review aimed at understanding how these compounds modulate mitochondrial bioenergetics to improve metabolic function in preclinical models on metabolic disease.

Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa.

BACKGROUND: Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. RESULTS: In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter's box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. CONCLUSIONS: PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes.

Some South African Rubiaceae Tree Leaf Extracts Have Antimycobacterial Activity Against Pathogenic and Non-pathogenic Mycobacterium Species.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds, which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa, and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium bovis BCG (Bacillus Calmette-Guérin) using a twofold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. Minimum inhibitory concentration values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied, and Oxyanthus speciosus had the most promising selectivity index values.

A Mini-Review of Anti-Listerial Compounds from Marine Actinobacteria (1990-2023).

Among the foodborne illnesses, listeriosis has the third highest case mortality rate (20-30% or higher). Emerging drug-resistant strains of Listeria monocytogenes, a causative bacterium of listeriosis, exacerbate the seriousness of this public health concern. Novel anti-Listerial compounds are therefore needed to combat this challenge. In recent years, marine actinobacteria have come to be regarded as a promising source of novel antimicrobials. Hence, our aim was to provide a narrative of the available literature and discuss trends regarding bioprospecting marine actinobacteria for new anti-Listerial compounds. Four databases were searched for the review: Academic Search Ultimate, Google Scholar, ScienceDirect, and South African Thesis and Dissertations. The search was restricted to peer-reviewed full-text manuscripts that discussed marine actinobacteria as a source of antimicrobials and were written in English from 1990 to December 2023. In total, for the past three decades (1990-December 2023), only 23 compounds from marine actinobacteria have been tested for their anti-Listerial potential. Out of the 23 reported compounds, only 2-allyoxyphenol, adipostatins E-G, 4-bromophenol, and ansamycins (seco-geldanamycin B, 4.5-dihydro-17-O-demethylgeldanamycin, and seco-geldanamycin) have been found to possess anti-Listerial activity. Thus, our literature survey reveals the scarcity of published assays testing the anti-Listerial capacity of bioactive compounds sourced from marine actinobacteria during this period.

Virulence profiles of enterotoxigenic, shiga toxin and enteroaggregative Escherichia coli in South African pigs.

Enterotoxigenic Escherichia coli (ETEC) and shiga toxin E. coli (STEC) are important causes of colibacillosis in piglets. Recently, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST-1) has been implicated in pig diarrhoea. This study investigated the prevalence of enterotoxin [heat-labile toxins (LT), heat-stable toxin a (STa), heat-stable toxin b (STb)], shiga toxins (Stx1, Stx2, Stx2e), enteroaggregative heat-stable E. coli (EAST-1), associated fimbriae (F4, F5, F6, F41, F18ab, F18ac) and non-fimbrial adhesins [adhesin involved in diffuse adherence 1 (AIDA-1), attaching and effacing factor, porcine attaching- and effacing-associated factor] in South African pigs. A total of 263 E. coli strains were isolated from Landrace (n = 24), Large White (n = 126), Duroc (n = 28) and indigenous (n = 85) breeds of piglets aged between 9 and 136 days. PCR was used in the analysis. Virulent genes were detected in 40.3% of the isolates, of which 18.6, 0.4 and 17.5% were classified as ETEC, STEC and enteroaggregative E. coli (EAEC), respectively. Individual genes were found in the following proportions: STb (19.01%), LT (0.4%), STa (3.4%), St2xe (1.1%) and EAST-1 (20.2%) toxins. None of the tested fimbriae were detected in ETEC and STEC isolates. About one third of the ETEC and STEC isolates was tested negative for both fimbrial and non-fimbrial adhesins. Twenty-five pathotypes from ETEC-, EAEC- and STEC-positive strains were identified. Pathotypes EAST-1 (30.2%), STb (13.2%) and STb/AIDA-1 (10.4%) were most prevalent. The study provided insight on possible causes of colibacillosis in South African pigs.

Virulence and Antimicrobial Resistance Profiling of Salmonella Serovars Recovered from Retail Poultry Offal in KwaZulu-Natal Province, South Africa.

As poultry organ meat is widely consumed, especially in low- and middle-income countries, there is reason to investigate it as a source of Salmonella infections in humans. Consequently, the aim of this study was to determine the prevalence, serotypes, virulence factors and antimicrobial resistance of Salmonella isolated from chicken offal from retail outlets in KwaZulu-Natal, South Africa. Samples (n = 446) were cultured for the detection of Salmonella using ISO 6579-1:2017. Presumptive Salmonella were confirmed using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Salmonella isolates were serotyped using the Kauffmann-White-Le Minor scheme and antimicrobial susceptibility was determined by the Kirby-Bauer disk diffusion technique. A conventional PCR was used for the detection of Salmonella invA, agfA, lpfA and sivH virulence genes. Of the 446 offal samples, 13 tested positive for Salmonella (2.91%; CI = 1.6-5). The serovars included S. Enteritidis (n = 3/13), S. Mbandaka (n = 1/13), S. Infantis (n = 3/13), S. Heidelberg (n = 5/13) and S. Typhimurium (n = 1/13). Antimicrobial resistance against amoxicillin, kanamycin, chloramphenicol and oxytetracycline was found only in S. Typhimurium and S. Mbandaka. All 13 Salmonella isolates harboured invA, agfA, lpfA and sivH virulence genes. The results show low Salmonella prevalence from chicken offal. However, most serovars are known zoonotic pathogens, and multi-drug resistance was observed in some isolates. Consequently, chicken offal products need to be treated with caution to avoid zoonotic Salmonella infections.

Evaluation of Antimicrobial Activity by Marine Nocardiopsis dassonvillei against Foodborne Listeria monocytogenes and Shiga Toxin-Producing Escherichia coli.

The emergence of multidrug-resistant pathogens creates public health challenges, prompting a continuous search for effective novel antimicrobials. This study aimed to isolate marine actinomycetes from South Africa, evaluate their in vitro antimicrobial activity against Listeria monocytogenes and Shiga toxin-producing Escherichia coli, and characterize their mechanisms of action. Marine actinomycetes were isolated and identified by 16S rRNA sequencing. Gas chromatography-mass spectrometry (GC-MS) was used to identify the chemical constituents of bioactive actinomycetes' secondary metabolites. Antibacterial activity of the secondary metabolites was assessed by the broth microdilution method, and their mode of actions were predicted using computational docking. While five strains showed antibacterial activity during primary screening, only Nocardiopsis dassonvillei strain SOD(B)ST2SA2 exhibited activity during secondary screening for antibacterial activity. GC-MS identified five major bioactive compounds: 1-octadecene, diethyl phthalate, pentadecanoic acid, 6-octadecenoic acid, and trifluoroacetoxy hexadecane. SOD(B)ST2SA2's extract demonstrated minimum inhibitory concentration and minimum bactericidal concentration, ranging from 0.78-25 mg/mL and 3.13 to > 25 mg/mL, respectively. Diethyl phthalate displayed the lowest bacterial protein-binding energies (kcal/mol): -7.2, dihydrofolate reductase; -6.0, DNA gyrase B; and -5.8, D-alanine:D-alanine ligase. Thus, marine N. dassonvillei SOD(B)ST2SA2 is a potentially good source of antibacterial compounds that can be used to control STEC and Listeria monocytogenes.

Antimicrobial Resistance, Enterotoxin and mec Gene Profiles of Staphylococcus aureus Associated with Beef-Based Protein Sources from KwaZulu-Natal Province, South Africa.

Annually, approximately 23,000 cases of food poisoning by Staphylococcus aureus enterotoxins are reported worldwide. The aim of this study was to determine the occurrence and characterize S. aureus on beef and beef products in South Africa. Organ meats (n = 169), raw processed meat (n = 110), raw intact (n = 53), and ready-to-eat meats (n = 68) were obtained from 25 retail outlets. S. aureus was isolated and enumerated according to the ISO 6888-1 method. Identification of the strains was performed by MALDI-TOF MS. The antimicrobial resistance was determined using the disc diffusion test. The presence of methicillin-resistance genes and the staphylococcal enterotoxin genes was determined by PCR. Prevalence was low (13/400; CI 1.7-5) and all but one positive sample were from organ meats. Eight isolates were resistant to at least one antibiotic. Two isolates carried the mecC gene. All the isolates tested positive for seg, seh, sei, and sep, whilst 53.8% were positive for sea. None of the isolates was positive for ser, sej, seb, sec, or sed. The prevalence of S. aureus was low, with organ meats being the most contaminated. The presence of mecC-positive MRSA and of enterotoxins warrants further investigation and risk assessment.

Experimental models of lipid overload and their relevance in understanding skeletal muscle insulin resistance and pathological changes in mitochondrial oxidative capacity.

It remains essential to decipher some of the pathological mechanisms that link obesity with deteriorating human health. Insulin resistance, due to enhanced free fatty acid substrate delivery, results in disrupted glucose homeostasis and altered mitochondrial oxidative capacity, which is a characteristic feature of an obese state. In fact, as a major site for regulating glucose homeostasis and energy production in response to insulin, the skeletal muscle has become an interesting target tissue to understand the impact of lipid overload on the development of insulin resistance and impaired mitochondrial respiratory function. In addition to systematically retrieving the discussed data, the current review brings an essential perspective in understanding the relevance of experimental models of lipid overload such as high fat diets in understanding the pathological link between insulin resistance and pathological changes in mitochondrial oxidative capacity. Importantly, inclusion of evidence from transgenic model highlights some of the unique molecular targets that are implicated in the development of insulin resistance and inefficient mitochondrial respiration processes within an obese state. Importantly, saturation with lipid products such as ceramides and diacylglycerols, especially within the skeletal muscle, appears to be instrumental in paving the path leading to worsening of metabolic complications. These metabolic consequences mostly interfere with the efficiency of the mitochondrial electron transport chain, leading to overproduction of toxic reactive oxygen species. Therefore, therapeutic agents that reverse the effects of lipid overload by improving insulin sensitivity and mitochondrial oxidative capacity are crucial for the management or even treatment of metabolic diseases.

Disease control tools to secure animal and public health in a densely populated world.

Animal health is a prerequisite for global health, economic development, food security, food quality, and poverty reduction, while mitigating against climate change and biodiversity loss. We did a qualitative review of 53 infectious diseases in terrestrial animals with data from DISCONTOOLS, a specialist database and prioritisation model focusing on research gaps for improving infectious disease control in animals. Many diseases do not have any appropriate control tools, but the prioritisation model suggests that we should focus international efforts on Nipah virus infection, African swine fever, contagious bovine pleuropneumonia, peste des petits ruminants, sheeppox and goatpox, avian influenza, Rift Valley fever, foot and mouth disease, and bovine tuberculosis, for the greatest impact on the UN's Sustainable Development Goals. Easy to use and accurate diagnostics are available for many animal diseases. However, there is an urgent need for the development of stable and durable diagnostics that can differentiate infected animals from vaccinated animals, to exploit rapid technological advances, and to make diagnostics widely available and affordable. Veterinary vaccines are important for dealing with endemic, new, and emerging diseases. However, fundamental research is needed to improve the convenience of use and duration of immunity, and to establish performant marker vaccines. The largest gap in animal pharmaceuticals is the threat of pathogens developing resistance to available drugs, in particular for bacterial and parasitic (protozoal, helminth, and arthropod) pathogens. We propose and discuss five research priorities for animal health that will help to deliver a sustainable and healthy planet: vaccinology, antimicrobial resistance, climate mitigation and adaptation, digital health, and epidemic preparedness.

Prevalence and Characteristics of Staphylococcus aureus Associated with Meat and Meat Products in African Countries: A Review.

Antimicrobial resistance has been increasing globally, which negatively affects food safety, veterinary, and human medicine. Ineffective antibiotics may cause treatment failure, which results in prolonged hospitalisation, increased mortality, and consequently, increased health care costs. Staphylococcus aureus causes a diverse range of infections including septicaemia and endocarditis. However, in food, it mainly causes food poisoning by the production of enterotoxins. With the discovery of methicillin-resistant S. aureus strains that have a separate reservoir in livestock animals, which were termed as livestock-associated methicillin-resistant S. aureus (LA-MRSA) in 2005, it became clear that animals may pose another health risk. Though LA-MRSA is mainly transferred by direct contact, food transmission cannot be excluded. While the current strains are not very pathogenic, mitigation is advisable, as they may acquire new virulence genes, becoming more pathogenic, and may transfer their resistance genes. Control of LA-MRSA poses significant problems, and only Norway has an active mitigation strategy. There is limited information about LA-MRSA, MRSA in general, and other S. aureus infections from African countries. In this review, we discuss the prevalence and characteristics of antimicrobial susceptible and resistant S. aureus (with a focus on MRSA) from meat and meat products in African countries and compare it to the situation in the rest of the world.

Antimycin A-induced mitochondrial dysfunction is consistent with impaired insulin signaling in cultured skeletal muscle cells.

Insulin resistance and mitochondrial dysfunction are characteristic features of type 2 diabetes mellitus. However, a causal relationship between insulin resistance and mitochondrial dysfunction has not been fully established in the skeletal muscle. Accordingly, we have evaluated the effect of antimycin A (AA), a mitochondrial electron transport chain complex III inhibitor, on mitochondrial bioenergetics and insulin signaling by exposing C2C12 skeletal muscle cells to its concentrations of 3.125, 6.25, 12.5, 25, and 50 µM for 12 h. Thereafter, metabolic activity, ROS production, glucose uptake, Seahorse XF Real-time ATP and Mito Stress assays were performed. Followed by real-time polymerase chain reaction (RT-PCR) and Western blot analysis. This study confirmed that AA induces mitochondrial dysfunction and promote ROS production in C2C12 myotubes, culminating in a significant decrease in mitochondrial respiration and downregulation of genes involved in mitochondrial bioenergetics (TFAM, UCP2, PGC1α). Increased pAMPK and extracellular acidification rates (ECAR) confirmed a potential compensatory enhancement in glycolysis. Additionally, AA impaired insulin signaling (protein kinase B/AKT) and decreased insulin stimulated glucose uptake. This study confirmed that an adaptive relationship exists between mitochondrial functionality and insulin responsiveness in skeletal muscle. Thus, therapeutics or interventions that improve mitochondrial function could ameliorate insulin resistance as well.

Microbial Communities of Meat and Meat Products: An Exploratory Analysis of the Product Quality and Safety at Selected Enterprises in South Africa.

Consumption of food that is contaminated by microorganisms, chemicals, and toxins may lead to significant morbidity and mortality, which has negative socioeconomic and public health implications. Monitoring and surveillance of microbial diversity along the food value chain is a key component for hazard identification and evaluation of potential pathogen risks from farm to the consumer. The aim of this study was to determine the microbial diversity in meat and meat products from different enterprises and meat types in South Africa. Samples (n = 2017) were analyzed for Yersinia enterocolitica, Salmonella species, Listeria monocytogenes, Campylobacter jejuni, Campylobacter coli, Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, and Clostridium botulinum using culture-based methods. PCR was used for confirmation of selected pathogens. Of the 2017 samples analyzed, microbial ecology was assessed for selected subsamples where next generation sequencing had been conducted, followed by the application of computational methods to reconstruct individual genomes from the respective sample (metagenomics). With the exception of Clostridium botulinum, selective culture-dependent methods revealed that samples were contaminated with at least one of the tested foodborne pathogens. The data from metagenomics analysis revealed the presence of diverse bacteria, viruses, and fungi. The analyses provide evidence of diverse and highly variable microbial communities in products of animal origin, which is important for food safety, food labeling, biosecurity, and shelf life limiting spoilage by microorganisms.

A review of Listeria monocytogenes from meat and meat products: Epidemiology, virulence factors, antimicrobial resistance and diagnosis.

Listeria monocytogenes is a zoonotic food-borne pathogen that is associated with serious public health and economic implications. In animals, L. monocytogenes can be associated with clinical listeriosis, which is characterised by symptoms such as abortion, encephalitis and septicaemia. In human beings, listeriosis symptoms include encephalitis, septicaemia and meningitis. In addition, listeriosis may cause gastroenteric symptoms in human beings and still births or spontaneous abortions in pregnant women. In the last few years, a number of reported outbreaks and sporadic cases associated with consumption of contaminated meat and meat products with L. monocytogenes have increased in developing countries. A variety of virulence factors play a role in the pathogenicity of L. monocytogenes. This zoonotic pathogen can be diagnosed using both classical microbiological techniques and molecular-based methods. There is limited information about L. monocytogenes recovered from meat and meat products in African countries. This review strives to: (1) provide information on prevalence and control measures of L. monocytogenes along the meat value chain, (2) describe the epidemiology of L. monocytogenes (3) provide an overview of different methods for detection and typing of L. monocytogenes for epidemiological, regulatory and trading purposes and (4) discuss the pathogenicity, virulence traits and antimicrobial resistance profiles of L. monocytogenes.

Phylogenomic structure of Bacillus anthracis isolates in the Northern Cape Province, South Africa revealed novel single nucleotide polymorphisms.

Bacillus anthracis, the aetiological agent of anthrax, is regarded as a highly monomorphic pathogen that presents a low genetic diversity using standard molecular techniques. Whole genome sequencing and single nucleotide polymorphisms (SNPs) are definitive signatures for subtyping of B. anthracis. Here we employed whole genome single nucleotide polymorphism (wgSNP) analysis to investigate the genetic diversity of B. anthracis in the historically endemic region of Northern Cape Province (NCP), South Africa. Twenty-six isolates from anthrax outbreaks that occurred between 1998 and 2008/9 in NCP as well as from Namibia-South Africa Transfontier Conservation area and Botswana were compared to global B. anthracis genomes. Most NCP B. anthracis strains (n = 22) clustered in the A.Br.003/004 (A.Br.101) branch and are closely related to the Zimbabwe and Mozambique strains (A.Br.102 branch). A total of 4923 parsimony informative-SNPs accurately established the A.Br.003/004 phylogenetic relationships of the NCP isolates into two distinct sub-clades and SNP markers designated as A.Br.172 and A.Br.173 were developed. Other NCP strains (n = 2) grouped in the A.Br.001/002 (Sterne) branch while strains (n = 2) from the Namibia-South Africa Transfontier Conservation area and Botswana clustered in A.Br.005/006 (Ancient A) branch. The sequenced B. anthracis strains (A0094, A0096 and A0097) that clustered in the A.Br.064 (V770) clade were isolated from Vaalbos National Park and similar strains have not been isolated. The B. anthracis A0088 strain cluster with the NCP strains in the A.Br.003/004 (A.Br.172) SNP branch which has been isolated in NCP, South Africa. This study highlights the phylogenetic structure of NCP B. anthracis strains with distinctive SNP branches important for forensic tracing and novel SNP discovery purposes. The sequenced strains will serve as a means to further trace the dissemination of B. anthracis outbreaks in NCP, South Africa, and on the continent, as well as for forensic tracking on a global scale.

Population Structure of Non-ST6 Listeria monocytogenes Isolated in the Red Meat and Poultry Value Chain in South Africa.

Meat products have been implicated in many listeriosis outbreaks globally, however there is a dearth of information on the diversity of L. monocytogenes isolates circulating in food products in South Africa. The aim of this study was to investigate the population structure of L. monocytogenes isolated in the meat value chain within the South African market. Based on whole-genome sequence analysis, a total of 217 isolates were classified into two main lineage groupings namely lineages I (n = 97; 44.7%) and II (n = 120; 55.3%). The lineage groups were further differentiated into IIa (n = 95, 43.8%), IVb (n = 69, 31.8%), IIb (n = 28, 12.9%), and IIc (n = 25, 11.5%) sero-groups. The most abundant sequence types (STs) were ST204 (n = 32, 14.7%), ST2 (n = 30, 13.8%), ST1 (n = 25, 11.5%), ST9 (n = 24, 11.1%), and ST321 (n = 21, 9.7%). In addition, 14 clonal complex (CCs) were identified with over-representation of CC1, CC3, and CC121 in "Processed Meat-Beef", "RTE-Poultry", and "Raw-Lamb" meat categories, respectively. Listeria pathogenic islands were present in 7.4% (LIPI-1), 21.7% (LIPI-3), and 1.8% (LIPI-4) of the isolates. Mutation leading to premature stop codons was detected in inlA virulence genes across isolates identified as ST121 and ST321. The findings of this study demonstrated a high-level of genomic diversity among L. monocytogenes isolates recovered across the meat value chain control points in South Africa.

Shiga Toxin-Producing Escherichia coli Contamination of Raw Beef and Beef-Based Ready-to-Eat Products at Retail Outlets in Pretoria, South Africa.

ABSTRACT: A cross-sectional study was conducted to determine the prevalence of and factors associated with Shiga toxin-producing Escherichia coli (STEC) in raw beef and ready-to-eat (RTE) beef products sold in 31 retail outlets in Pretoria, South Africa, and nearby areas. A total of 463 beef and RTE samples were screened for four STEC virulence genes (stx1, stx2, eaeA, and hlyA) and seven O-serogroups (O113, O157, O26, O91, O145, O111, and O103) with a multiplex PCR assay. The total aerobic plate count (TAPC) per gram was also determined. A total of 38 STEC isolates were recovered and characterized by conventional PCR assay and serotyping. The overall prevalence of STEC in the beef and RTE samples tested was 16.4% (76 of 463 samples; 95% confidence interval, 13 to 20%). The prevalence of STEC differed significantly by product type (P < 0.0001), with the highest prevalence (35%) detected in boerewors (spicy sausage). The STEC prevalences in minced beef, brisket, RTE cold beef, and biltong were 18, 13, 9, and 5%, respectively. The most frequently detected stx gene was stx2 (13%), and STEC serogroups from recovered isolates were detected at the following prevalences: O2, 15%; O8, 12%; O13, 15%; O20, 8%; O24, 3%; O39, 3%; O41, 8%; O71, 3%; O76, 3%; O150, 12%; and O174, 3%. A high proportion (77%) of the samples had TAPCs that exceeded the South African microbiological standards for meat export (5.0 log CFU/g). The prevalence of O157 STEC (16%) and the diversity of non-O157 STEC serogroups found in five common beef-based products from retail outlets in South Africa suggest exposure of raw beef and beef products to multiple contamination sources during carcass processing and/or cutting and handling at retail outlets. These data provide direct estimates of the potential health risk to consumers from undercooked contaminated products and indicate the need to improve sanitary practices during slaughter and processing of beef and beef-based RTE products. A risk-based surveillance system for STEC may be needed.

Prevalence of enterotoxigenic Escherichia coli virulence genes from scouring piglets in Zimbabwe.

World-wide, enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC)-induced diarrhea are economically important for porcine producers. Our aim was to investigate the prevalence of toxin and fimbrial genes among E. coli isolated from diarrheic piglets from randomly selected piggeries in Zimbabwe. We used multiplex PCR for screening STa, STb, LT, and Stx-2e toxins. Subsequently F4, F5, F6, F18 and F41 fimbriae genes were screened in toxin positive isolates. Toxin positive strains lacking tested fimbriae genes were characterized using transmission electron microscopy, agglutination and agglutination inhibition tests. Approximately 32% of the 1,984 isolates tested positive for STa, STb, LT or Stx-2e genes. Of these, approximately 81% had F4, F5, F6, F18 or F41 fimbriae genes. The remaining toxin positive strains lacked tested fimbriae genes and appeared to either express F1-like fimbriae, or lacked fimbriae. The data constitute an important framework for implementation of prevention measures, such as using relevant fimbriae-based vaccines against ETEC induced diarrhea or VTEC-induced edema.