Food animals as reservoirs and potential sources of multidrug-resistant diarrheagenic E. coli pathotypes: Focus on intensive pig farming in South Africa.

BACKGROUND:  Diarrheagenic E. coli (DEC) strains are a major cause of diarrheal diseases in both developed and developing countries. Healthy asymptomatic animals may be reservoirs of zoonotic DEC, which may enter the food chain via the weak points in hygiene practices. AIM:  We investigated the prevalence of DEC along the pig production continuum from farm-to-fork. METHODS:  A total of 417 samples were collected from specific points along the pig production system, that is, farm, transport, abattoir and food. E. coli was isolated and enumerated using Colilert. Ten isolates from each Quanti-tray were selected randomly and phenotypically identified using eosin methylene blue agar selective media. Real-time polymerase chain reaction (PCR) was used to confirm the species and to classify them into the various diarrheagenic pathotypes. Antimicrobial susceptibility was determined against a panel of 20 antibiotics using the Kirby-Bauer disk diffusion method and EUCAST guideline. RESULTS:  The final sample size consisted of 1044 isolates, of which 45.40% (474/1044) were DEC and 73% (762/1044) were multidrug-resistant. Enteroinvasive E. coli (EIEC) was the most predominant DEC at all the sampling sites. CONCLUSION:  The presence of DEC in food animal production environments and food of animal origin could serve as reservoirs for transmitting these bacteria to humans, especially in occupationally exposed workers and via food. Adherence to good hygienic practices along the pig production continuum is essential for mitigating the risk of transmission and infection, and ensuring food safety.

Molecular Epidemiology of Salmonella enterica in Poultry in South Africa Using the Farm-to-Fork Approach.

The presence of the zoonotic pathogen Salmonella in the food supply chain poses a serious public health threat. This study describes the prevalence, susceptibility profiles, virulence patterns, and clonality of Salmonella from a poultry flock monitored over six weeks, using the farm-to-fork approach. Salmonella was isolated using selective media and confirmed to the genus and species level by real-time polymerase chain reaction (RT-PCR) of the invA and iroB genes, respectively. Antimicrobial susceptibility profiles were determined using Vitek-2 and the Kirby-Bauer disk diffusion method against a panel of 21 antibiotics recommended by the World Health Organisation Advisory Group on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR). Selected virulence genes were identified by conventional PCR, and clonality was determined using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Salmonella was present in 32.1% of the samples: on the farm (30.9%), at the abattoir (0.6%), and during house decontamination (0.6%). A total of 210 isolates contained the invA and iroB genes. Litter, faeces, and carcass rinsate isolates were classified as resistant to cefuroxime (45.2%), cefoxitin (1.9%), chloramphenicol (1.9%), nitrofurantoin (0.4%), pefloxacin (11.4%), and azithromycin (11%). Multidrug resistance (MDR) was observed among 3.8% of the isolates. All wastewater and 72.4% of carcass rinsate isolates were fully susceptible. All isolates harboured the misL, orfL, pipD, stn, spiC, hilA, and sopB virulence genes, while pefA, spvA, spvB, and spvC were absent. In addition, fliC was only present among the wastewater isolates. Various ERIC-PCR patterns were observed throughout the continuum with different subtypes, indicating the unrelated spread of Salmonella. This study concluded that poultry and the poultry environment serve as reservoirs for resistant and pathogenic Salmonella. However, there was no evidence of transmission along the farm-to-fork continuum.

Staphylococcus aureus in Intensive Pig Production in South Africa: Antibiotic Resistance, Virulence Determinants, and Clonality.

Although Staphylococcus aureus is a major threat to the veterinary, agricultural, and public health sectors because of its zoonotic potential, studies on its molecular characterisation in intensive animal production are rare. We phenotypically and genotypically characterised antibiotic-resistant S. aureus in intensive pig production in South Africa, using the farm-to-fork approach. Samples (n = 461) were collected from the farm, transport vehicles, and the abattoir using the World Health Organisation on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) sampling protocol. Bacteria were isolated using selective media and identified using biochemical tests and polymerase chain reaction (PCR). Phenotypic resistance was determined using the disk diffusion method. Selected resistance and virulence genes were investigated using PCR. Clonality among the isolates was determined using the repetitive element sequence-PCR. In all, 333 presumptive staphylococcal isolates were obtained, with 141/333 (42.3%) identified as staphylococci biochemically. Ninety-seven (97; 68.8%) were confirmed as S. aureus using PCR, 52.6% of which were identified as methicillin-resistant S. aureus (MRSA) through the mecA gene. All the 97 S. aureus isolates (100%) were resistant to at least one of the antibiotics tested, with the highest resistance observed against erythromycin and clindamycin (84.50% each), and the lowest observed against amikacin (2.10%); 82.47% (80/97) were multidrug-resistant with an average multiple antibiotic resistance index of 0.50. Most of the phenotypically resistant isolates carried at least one of the corresponding resistance genes tested, ermC being the most detected. hla was the most detected virulence gene (38.14%) and etb was the least (1.03%). Genetic fingerprinting revealed diverse MRSA isolates along the farm-to-fork continuum, the major REP types consisting of isolates from different sources suggesting a potential transmission along the continuum. Resistance to antibiotics used as growth promoters was evidenced by the high prevalence of MDR isolates with elevated multiple antibiotic resistance indices >0.2, specifically at the farm, indicating exposure to high antibiotic use environments, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.

From the Farms to the Dining Table: The Distribution and Molecular Characteristics of Antibiotic-Resistant Enterococcus spp. in Intensive Pig Farming in South Africa.

Foodborne pathogens, including antibiotic-resistant species, constitute a severe menace to food safety globally, especially food animals. Identifying points of concern that need immediate mitigation measures to prevent these bacteria from reaching households requires a broad understanding of these pathogens' spread along the food production chain. We investigated the distribution, antibiotic susceptibility, molecular characterization and clonality of Enterococcus spp. in an intensive pig production continuum in South Africa, using the farm-to-fork approach. Enterococcus spp. were isolated from 452 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) using the IDEXX Enterolert®/Quanti-Tray® 2000 system. Pure colonies were obtained on selective media and confirmed by real-time PCR, targeting genus- and species-specific genes. The susceptibility to antibiotics was determined by the Kirby-Bauer disk diffusion method against 16 antibiotics recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. A total of 284 isolates, consisting of 79.2% E. faecalis, 6.7% E. faecium, 2.5% E. casseliflavus, 0.4% E. gallinarum, and 11.2% other Enterococcus spp., were collected along the farm-to-fork continuum. The isolates were most resistant to sulfamethoxazole-trimethoprim (78.8%) and least resistant to levofloxacin (5.6%). No resistance was observed to vancomycin, teicoplanin, tigecycline and linezolid. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. Also, 78% of the isolates were multidrug-resistant. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tetM, aph(3')-IIIa, and ermB genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most detected virulence gene was gelE. Clonality revealed that E. faecalis isolates belonged to diverse clones along the continuum with major REP-types, mainly isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. The results suggest that intensive pig farming could serve as a reservoir of antibiotic-resistant bacteria that could be transmitted to occupationally exposed workers via direct contact with animals or consumers through animal products/food. This highlights the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.

Occurrence, Antimicrobial Resistance, and Molecular Characterization of Campylobacter spp. in Intensive Pig Production in South Africa.

Campylobacter spp. are among the leading foodborne pathogens, causing campylobacteriosis, a zoonotic infection that results in bacterial gastroenteritis and diarrheal disease in animals and humans. This study investigated the molecular epidemiology of antibiotic-resistant Campylobacter spp. isolated across the farm-to-fork-continuum in an intensive pig production system in South Africa. Following ethical approval, samples were collected over sixteen weeks from selected critical points (farm, transport, abattoir, and retail) using a farm-to-fork sampling approach according to WHO-AGISAR guidelines. Overall, 520 samples were investigated for the presence of Campylobacter spp., which were putatively identified using selective media with identity and speciation confirmed by polymerase chain reaction (PCR) of specific genes. Resistance profiles were ascertained by the Kirby-Bauer disk diffusion method. Antibiotic resistance and virulence genes were identified using PCR and DNA sequencing. Clonal relatedness was determined using ERIC-PCR. Altogether, 378/520 (72.7%) samples were positive for Campylobacter spp., with Campylobacter coli being the predominant species (73.3%), followed by Campylobacter jejuni (17.7%); 8.9% of the isolates were classified as "other spp". Relatively high resistance was observed in C. coli and C. jejuni to erythromycin (89% and 99%), streptomycin (87% and 93%), tetracycline (82% and 96%), ampicillin (69% and 85%), and ciprofloxacin (53% and 67%), respectively. Multidrug resistance (MDR) was noted in 330 of the 378 (87.3%) isolates. The antibiotic resistance genes observed were tetO (74.6%), blaOXA-61 (2.9%), and cmeB (11.1%), accounting for the resistance to tetracycline and ampicillin. The membrane efflux pump (cmeB), conferring resistance to multiple antibiotics, was also detected in most resistant isolates. Chromosomal mutations in gyrA (Thr-86-Ile) and 23S rRNA (A2075G and A2074C) genes, conferring quinolone and erythromycin resistance, respectively, were also found. Of the virulence genes tested, ciaB, dnaJ, pldA, cdtA, cdtB, cdtC, and cadF were detected in 48.6%, 61.1%, 17.4%, 67.4%, 19.3%, 51%, and 5% of all Campylobacter isolates, respectively. Clonal analysis revealed that isolates along the continuum were highly diverse, with isolates from the same sampling points belonging to the same major ERIC-types. The study showed relatively high resistance to antibiotics commonly used in intensive pig production in South Africa with some evidence, albeit minimal, of transmission across the farm-to-fork continuum. This, together with the virulence profiles present in Campylobacter spp., presents a challenge to food safety and a potential risk to human health, necessitating routine surveillance, antibiotic stewardship, and comprehensive biosecurity in intensive pig production.

From Farm-to-Fork: E. Coli from an Intensive Pig Production System in South Africa Shows High Resistance to Critically Important Antibiotics for Human and Animal Use.

Antibiotic resistance profiles of Escherichia coli were investigated in an intensive pig production system in the uMgungundlovu District, South Africa, using the 'farm-to-fork' approach. Four hundred seventeen (417) samples were collected from pig and pig products at different points (farm, transport, and abattoir). E. coli was isolated and enumerated using the Colilert® 18/Quanti-Tray® 2000 system. Ten isolates from each Quanti-tray were selected randomly and putatively identified on eosin methylene blue agar. Real-time PCR targeting the uidA gene was used to confirm isolates to the genus level. The Kirby-Bauer disc diffusion method was used to determine the isolates' antibiotic susceptibility profiles against 20 antibiotics. A total of 1044 confirmed E. coli isolates were obtained across the three critical points in the food chain. Resistance was observed to all the antibiotics tested with the highest and lowest rates obtained against tetracycline (88.5%) and meropenem (0.2%), respectively. Resistance was also observed to chloramphenicol (71.4%), ampicillin (71.1%), trimethoprim-sulfamethoxazole (61.3%), amoxicillin-clavulanate (43.8%), cephalexin (34.3%), azithromycin (23.9%), nalidixic acid (22.1%), cefoxitin (21.1%), ceftriaxone (18.9%), ciprofloxacin (17.3%), cefotaxime (16.9%), gentamicin (15.5%), cefepime (13.8%), ceftazidime (9.8%), amikacin (3.4%), piperacillin-tazobactam (1.2%), tigecycline (0.9%), and imipenem (0.3%). Multidrug resistance (MDR) was observed in 71.2% of the resistant isolates with an overall multiple antibiotic resistance (MAR) index of 0.25, indicating exposure to high antibiotic use environments at the farm level. A high percentage of resistance was observed to growth promoters and antibiotics approved for veterinary medicine in South Africa. Of concern was resistance to critically important antibiotics for animal and human use and the watch and reserve categories of antibiotics. This could have adverse animal and human health consequences from a food safety perspective, necessitating efficient antibiotic stewardship and guidelines to streamline antibiotic use in the food-animal production chain.

Characterisation of Campylobacter spp. Isolated from Poultry in KwaZulu-Natal, South Africa.

This study investigated the antibiotic resistance, virulence profiles, and clonality of Campylobacter jejuni and Campylobacter coli isolated from an intensive poultry farming system in KwaZulu-Natal, South Africa. Following ethical approval, samples were collected over six weeks using the farm-to-fork approach. Campylobacter spp. were identified using culture, confirmed and differentiated to species level by PCR, and subjected to antibiotic susceptibility testing. Selected antibiotic resistance (and mutations) and virulence genes were screened by PCR and confirmed by DNA sequencing. Genetic relatedness amongst the isolates was ascertained using pulsed-field gel electrophoresis. In all, 105 isolates were confirmed as belonging to both Campylobacter coli (60; 57%) and C. jejuni (45; 43%). The highest resistance was recorded against erythromycin and clindamycin. The gyrA mutation, A20175C/A2074G point mutation, tet(O), and cmeB, all associated with antibiotic resistance, were detected. All the virulence genes (pldA, ciaB, cdtA, cdtB, cdtC, dnaJ, except for cadF) were also detected. Isolates were grouped into five pulsotypes displaying 85% similarity, irrespective of their resistance profiles. The numerous permutations of clonality, antibiotic resistance, and virulence profiles evident in Campylobacter spp. pose a challenge to food safety and necessitate a comprehensive understanding of the molecular epidemiology of this organism to decrease its spread in the food chain.

Antibiotic Resistance in Staphylococcus aureus from Poultry and Poultry Products in uMgungundlovu District, South Africa, Using the "Farm to Fork" Approach.

Background: This study determined the prevalence and antibiotic susceptibility profiles of Staphylococcus aureus isolated from selected critical control points (farm, transport, abattoir, and retail product) in an intensive poultry production system in the uMgungundlovu District, South Africa, using the "farm to fork" approach. Materials and Methods: Three hundred eighty-four samples from poultry and poultry products were examined across the "farm to fork" continuum for S. aureus using selective media, biochemical tests, and API Staph kit and confirmed by polymerase chain reaction identification of the nuc gene. Antibiotic susceptibility testing of the isolates was determined by the Kirby-Bauer disc diffusion method to 19 antimicrobials and to vancomycin by the broth microdilution technique. Results: The overall prevalence rate of S. aureus was 31.25% (n = 120/384), distributed across the continuum: farm site (40), transport (15), abattoir (30), and retail point (35). The isolates were resistant to tetracycline (61.67%), penicillin G (55.83%), erythromycin (54.17%), clindamycin (43.33%), doxycycline (36.67%), ampicillin (34.17%), moxifloxacin (30.83%), amikacin (30.83%), trimethoprim-sulfamethoxazole (30.00%), and levofloxacin (23.33%). A 100% susceptibility to tigecycline, teicoplanin, vancomycin, nitrofurantoin, chloramphenicol, and linezolid was observed in all isolates. The rate of multidrug resistance and the multiple antibiotic resistance index of the strains were 39.17% and 0.23%, respectively. The isolates showed similar patterns of resistance to commonly used growth promoters and antibiotics in veterinary and human medicine belonging to the same class. Conclusion: It is evident that the different antibiotics and growth promoters used in poultry production are exerting selection pressure for the emergence and co-selection of antibiotic-resistant bacteria in the production system, necessitating efficient antibiotic stewardship guidelines to streamline their use.

Infection of African buffalo (Syncerus caffer) by oryx bacillus, a rare member of the antelope clade of the Mycobacterium tuberculosis complex.

Mycobacterium tuberculosis complex species cause tuberculosis disease in animals and humans. Although they share 99.9% similarity at the nucleotide level, several host-adapted ecotypes of the tubercule bacilli have been identified. In the wildlife setting, probably the most well-known member of this complex is Mycobacterium bovis, the causative agent of bovine tuberculosis. The recently described oryx bacillus is an extremely rare slow-growing member of the antelope clade of the M. tuberculosis complex and is closely related to the dassie bacillus, Mycobacterium africanum and Mycobacterium microti. The antelope clade is a group of strains apparently host adapted to antelopes, as most described infections were associated with deer and antelope, most specifically the Arabian oryx (Oryx leucoryx). In this study, oryx bacillus was isolated from a free-ranging adult female African buffalo (Syncerus caffer), in good physical condition, which tested strongly positive on three consecutive comparative intradermal tuberculin tests. Upon necropsy, a single pulmonary granuloma and an active retropharyngeal lymph node was found. Comprehensive molecular genetic assays were performed, which confirmed that the causative microorganism was not M. bovis but oryx bacillus. Oryx bacillus has never been reported in Southern Africa and has never been found to infect African buffalo. The identification of this microorganism in buffalo is an important observation in view of the large and ever-increasing epidemic of the closely related M. tuberculosis complex species M. bovis in some African buffalo populations in South Africa.