SARS-CoV-2 BA.4/5 infection triggers more cross-reactive FcγRIIIa signaling and neutralization than BA.1, in the context of hybrid immunity.

SARS-CoV-2 variants of concern (VOCs) differentially trigger neutralizing and antibody-dependent cellular cytotoxic (ADCC) antibodies with variable cross-reactivity. Omicron BA.4/5 was approved for inclusion in bivalent vaccination boosters, and therefore the antigenic profile of antibodies elicited by this variant is critical to understand. Here, we investigate the ability of BA.4/5-elicited antibodies following the first documented (primary) infection (n = 13) or breakthrough infection after vaccination (n = 9) to mediate neutralization and FcγRIIIa signaling across multiple SARS-CoV-2 variants including XBB.1.5 and BQ.1. Using a pseudovirus neutralization assay and a FcγRIIIa crosslinking assay to measure ADCC potential, we show that unlike SARS-CoV-2 Omicron BA.1, BA.4/5 infection triggers highly cross-reactive functional antibodies. Cross-reactivity was observed both in the absence of prior vaccination and in breakthrough infections following vaccination. However, BQ.1 and XBB.1.5 neutralization and FcγRIIIa signaling were significantly compromised compared to other VOCs, regardless of prior vaccination status. BA.4/5 triggered FcγRIIIa signaling was significantly more resilient against VOCs (<10-fold decrease in magnitude) compared to neutralization (10- to 100-fold decrease). Overall, this study shows that BA.4/5 triggered antibodies are highly cross-reactive compared to those triggered by other variants. Although this is consistent with enhanced neutralization and FcγRIIIa signaling breadth of BA.4/5 vaccine boosters, the reduced activity against XBB.1.5 supports the need to update vaccines with XBB sublineage immunogens to provide adequate coverage of these highly antibody evasive variants. IMPORTANCE: The continued evolution of SARS-CoV-2 has resulted in a number of variants of concern. Of these, the Omicron sublineage is the most immune evasive. Within Omicron, the BA.4/5 sublineage drove the fifth wave of infection in South Africa prior to becoming the dominant variant globally. As a result this spike sequence was approved as part of a bivalent vaccine booster, and rolled out worldwide. We aimed to understand the cross-reactivity of neutralizing and Fc mediated cytotoxic functions elicited by BA.4/5 infection following infection or breakthrough infection. We find that, in contrast to BA.1 which triggered fairly strain-specific antibodies, BA.4/5 triggered antibodies that are highly cross-reactive for neutralization and antibody-dependent cellular cytotoxicity potential. Despite this cross-reactivity, these antibodies are compromised against highly resistant variants such as XBB.1.5 and BQ.1. This suggests that next-generation vaccines will require XBB sublineage immunogens in order to protect against these evasive variants.

Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana.

BACKGROUND: ESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital. MATERIALS AND METHODS: Twenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny. RESULTS: The prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The ß-lactamase genes, blaTEM-1B (10 isolates) and blaCTX-M-15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX-M-15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. CONCLUSION: We identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

Early assessment of the clinical severity of the SARS-CoV-2 omicron variant in South Africa: a data linkage study.

BACKGROUND: The SARS-CoV-2 omicron variant of concern was identified in South Africa in November, 2021, and was associated with an increase in COVID-19 cases. We aimed to assess the clinical severity of infections with the omicron variant using S gene target failure (SGTF) on the Thermo Fisher Scientific TaqPath COVID-19 PCR test as a proxy. METHODS: We did data linkages for national, South African COVID-19 case data, SARS-CoV-2 laboratory test data, SARS-CoV-2 genome data, and COVID-19 hospital admissions data. For individuals diagnosed with COVID-19 via TaqPath PCR tests, infections were designated as either SGTF or non-SGTF. The delta variant was identified by genome sequencing. Using multivariable logistic regression models, we assessed disease severity and hospitalisations by comparing individuals with SGTF versus non-SGTF infections diagnosed between Oct 1 and Nov 30, 2021, and we further assessed disease severity by comparing SGTF-infected individuals diagnosed between Oct 1 and Nov 30, 2021, with delta variant-infected individuals diagnosed between April 1 and Nov 9, 2021. FINDINGS: From Oct 1 (week 39), 2021, to Dec 6 (week 49), 2021, 161 328 cases of COVID-19 were reported in South Africa. 38 282 people were diagnosed via TaqPath PCR tests and 29 721 SGTF infections and 1412 non-SGTF infections were identified. The proportion of SGTF infections increased from two (3·2%) of 63 in week 39 to 21 978 (97·9%) of 22 455 in week 48. After controlling for factors associated with hospitalisation, individuals with SGTF infections had significantly lower odds of admission than did those with non-SGTF infections (256 [2·4%] of 10 547 vs 121 [12·8%] of 948; adjusted odds ratio [aOR] 0·2, 95% CI 0·1-0·3). After controlling for factors associated with disease severity, the odds of severe disease were similar between hospitalised individuals with SGTF versus non-SGTF infections (42 [21%] of 204 vs 45 [40%] of 113; aOR 0·7, 95% CI 0·3-1·4). Compared with individuals with earlier delta variant infections, SGTF-infected individuals had a significantly lower odds of severe disease (496 [62·5%] of 793 vs 57 [23·4%] of 244; aOR 0·3, 95% CI 0·2-0·5), after controlling for factors associated with disease severity. INTERPRETATION: Our early analyses suggest a significantly reduced odds of hospitalisation among individuals with SGTF versus non-SGTF infections diagnosed during the same time period. SGTF-infected individuals had a significantly reduced odds of severe disease compared with individuals infected earlier with the delta variant. Some of this reduced severity is probably a result of previous immunity. FUNDING: The South African Medical Research Council, the South African National Department of Health, US Centers for Disease Control and Prevention, the African Society of Laboratory Medicine, Africa Centers for Disease Control and Prevention, the Bill & Melinda Gates Foundation, the Wellcome Trust, and the Fleming Fund.

Genomic analysis of antibiotic-resistant Enterococcus spp. reveals novel enterococci strains and the spread of plasmid-borne Tet(M), Tet(L) and Erm(B) genes from chicken litter to agricultural soil in South Africa.

Manure from food animals exposed to antibiotics is often used as soil fertiliser, potentially releasing antibiotic-resistant bacteria (ARB) with diverse antibiotic-resistance genes (ARGs) into the soil. To determine the impact of chicken litter application on the soil resistome, Enterococcus spp. isolated from chicken litter and soil samples collected before and after the soil amendment were characterised, using whole-genome sequencing and bioinformatics tools. Nineteen Enterococcus spp. isolates from the three sources were sequenced on Illumina Miseq platform to ascertain the isolates' resistome, mobilome, virulome, clonality, and phylogenomic relationships. Multilocus sequence typing (MLST) analysis revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). The isolates harboured multiple resistance genes including those conferring resistance to inter alia macrolides-lincosamide-streptogramin (erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)), tetracycline (tet(M), tet(L), tet(S)), aminoglycosides (aac(6')-Ii, aac(6')-Iih, ant(6)-Ia, aph(3')-III, ant(9)-Ia), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat). The litter-amended soil harboured new ARB (particularly E. faecium) and ARGs (ant(6)-Ia, aac(6')-Ii, aph(3')-III), lnu(G), msr(C), and eat(A), efmA) that were not previously detected in the soil. The identified ARGs were associated with diverse mobile genetic elements (MGEs) such as insertion sequences (IS6, ISL3, IS256, IS30), transposons (Tn3 and Tn916) and plasmids (repUS43, repUS1, rep9b, and rep 22). Twenty-eight virulence genes encoding adherence/biofilm formation (ebpA, ebpB, ebpC), antiphagocytosis (elrA) and bacterial sex pheromones (Ccf10, cOB1, cad, and camE), were detected in the genomes of the isolates. Phylogenomic analysis revealed a close relationship between a few isolates from litter-amended soil and the chicken litter isolates. The differences in the ARG and ARB profiles in the soil before and after the litter amendment and their association with diverse MGEs indicate the mobilisation and transmission of ARGs and ARB from the litter to the soil.

Quantitative microbial risk assessment for waterborne pathogens in a wastewater treatment plant and its receiving surface water body.

BACKGROUND: Access to safe water for drinking and domestic activities remains a challenge in emerging economies like South Africa, forcing resource-limited communities to use microbiologically polluted river water for personal and household purposes, posing a public health risk. This study quantified bacterial contamination and the potential health hazards that wastewater treatment plant (WWTP) workers and communities may face after exposure to waterborne pathogenic bacteria in a WWTP and its associated surface water, respectively. RESULTS: Escherichia coli (Colilert®-18/ Quanti-Tray® 2000) and enterococci (Enterolert®/ Quanti-Tray® 2000) were quantified and definitively identified by real-time polymerase chain reaction targeting the uidA and tuf genes, respectively. An approximate beta-Poisson dose-response model was used to estimate the probability of infection (Pi) with pathogenic E. coli. Mean E. coli concentration ranged from 2.60E+ 02/100 mL to 4.84E+ 06/100 mL; enterococci ranged from 2.60E+ 02/100 mL to 3.19E+ 06/100 mL across all sampled sites. Of the 580 E. coli isolates obtained from this study, 89.1% were intestinal, and 7.6% were extraintestinal pathogenic E. coli. The 579 enterococci obtained were 50.4% E. faecalis (50.4%), 31.4% E. faecium, 3.5%, E. casseliflavus and 0.7% E. gallinarum. The community health risk stemming from the use of the water for recreational and domestic purposes revealed a greater health risk (Pi) from the ingestion of 1 mL of river water from upstream (range, 55.1-92.9%) than downstream (range, 26.8-65.3%) sites. The occupational risk of infection with pathogenic E. coli for workers resulting from a once-off unintentional consumption of 1 mL of water was 0% (effluent) and 23.8% (raw influent). Multiple weekly exposures of 1 mL over a year could result in a Pi of 1.2 and 100% for the effluent and influent, respectively. CONCLUSION: Our findings reveal that there is a potentially high risk of infection for WWTP workers and communities that use river water upstream and downstream of the investigated WWTP.

Multidrug-resistant Gram-negative bacterial colonization in patients, carriage by healthcare workers and contamination of hospital environments in Ghana.

BACKGROUND: Patients already colonized with multidrug-resistant (MDR) Gram-negative bacteria (GNB) on admission to critical care units may be an important source of transmission of these bacteria in hospitals. We sought to determine the prevalence of MDR GNB colonization in patients, staff and the ward environment and to assess the risk factors for colonization of patients in wards. METHODS: The study was conducted from April 2021 to July 2021 in a teaching hospital in Ghana. MDR GNB were isolated from rectal, and hand swabs were taken from patients on admission and after 48 h. Swabs from HCW's hands and the ward environment were also taken. Risk factors for colonization with MDR GNB were assessed using univariate and multivariate analysis. RESULTS: MDR GNB rectal colonization rate among patients was 50.62% on admission and 44.44% after 48 h. MDR GNB were isolated from 6 (5.26%) and 24 (11.54%) of HCW's hand swabs and environmental swabs, respectively. Previous hospitalization (p-value = 0.021, OR, 95% CI= 7.170 (1.345-38.214) was significantly associated with colonization by MDR GNB after 48 h of admission. Age (21-30 years) (p-value = 0.022, OR, 95% CI = 0.103 (0.015-0.716) was significantly identified as a protective factor associated with a reduced risk of rectal MDR GNB colonization. CONCLUSION: The high colonization of MDR GNB in patients, the carriage of MDR GNB on HCW's hands, and the contamination of hospital environments highlights the need for patient screening and stringent infection prevention and control practices to prevent the spread of MDR GNB in hospitals.

Antibacterial, Resistance Modulation, Anti-Biofilm Formation, and Efflux Pump Inhibition Properties of Loeseneriella africana (Willd.) N. Halle (Celastraceae) Stem Extract and Its Constituents.

This study investigated the antibacterial, resistance modulation, biofilm inhibition, and efflux pump inhibition potentials of Loeseneriella africana stem extract and its constituents. The antimicrobial activity was investigated by the high-throughput spot culture growth inhibition (HT-SPOTi) and broth microdilution assays. The resistance modulation activity was investigated using the anti-biofilm formation and efflux pump inhibition assays. Purification of the extract was carried out by chromatographic methods, and the isolated compounds were characterized based on nuclear magnetic resonance, Fourier transform infrared and mass spectrometry spectral data and comparison with published literature. The whole extract, methanol, ethyl acetate, and pet-ether fractions of L. africana all showed antibacterial activity against the test bacteria with MICs ranging from 62.5 to 500.0 µg/mL The whole extract demonstrated resistance modulation effect through strong biofilm inhibition and efflux pump inhibition activities against S. aureus ATCC 25923, E. coli ATCC 25922 and P. aeruginosa ATCC 27853. Chromatographic fractionation of the ethyl acetate fraction resulted in the isolation of a triterpenoid (4S,4αS,6αR,6ßS,8αS,12αS,12ßR,14αS,14ßR)-4,4α,6ß,8α,11,11,12ß,14α-Octamethyloctadecahydropicene-1,3(2H,4H)-dione) and a phytosterol (ß-sitosterol). These compounds showed antibacterial activity against susceptible bacteria at a MIC range of 31-125 µg/mL and potentiated the antibacterial activity of amoxicillin (at » MIC of compounds) against E. coli and P. aeruginosa with modulation factors of 32 and 10, respectively. These compounds also demonstrated good anti-biofilm formation effect at a concentration range of 3-100 µg/mL, and bacterial efflux pump inhibition activity at ½ MIC and » MIC against E. coli and P. aeruginosa. Loeseneriella africana stem bark extracts and constituents elicit considerable antibacterial, resistance modulation, and biofilm and efflux pump inhibition activities. The results justify the indigenous uses of L. africana for managing microbial infections.

SARS-CoV-2 genomic surveillance in wastewater as a model for monitoring evolution of endemic viruses.

As global SARS-CoV-2 burden and testing frequency have decreased, wastewater surveillance has emerged as a key tool to support clinical surveillance efforts. The aims of this study were to identify and characterize SARS-CoV-2 variants in wastewater samples collected from urban centers across South Africa. Here we show that wastewater sequencing analyses are temporally concordant with clinical genomic surveillance and reveal the presence of multiple lineages not detected by clinical surveillance. We show that wastewater genomics can support SARS-CoV-2 epidemiological investigations by reliably recovering the prevalence of local circulating variants, even when clinical samples are not available. Further, we find that analysis of mutations observed in wastewater can provide a signal of upcoming lineage transitions. Our study demonstrates the utility of wastewater genomics to monitor evolution and spread of endemic viruses.

Genomic epidemiology of SARS-CoV-2 during the first four waves in Mozambique.

Mozambique reported the first case of coronavirus disease 2019 (COVID-19) in March 2020 and it has since spread to all provinces in the country. To investigate the introductions and spread of SARS-CoV-2 in Mozambique, 1 142 whole genome sequences sampled within Mozambique were phylogenetically analyzed against a globally representative set, reflecting the first 25 months of the epidemic. The epidemic in the country was marked by four waves of infection, the first associated with B.1 ancestral lineages, while the Beta, Delta, and Omicron Variants of Concern (VOCs) were responsible for most infections and deaths during the second, third, and fourth waves. Large-scale viral exchanges occurred during the latter three waves and were largely attributed to southern African origins. Not only did the country remain vulnerable to the introductions of new variants but these variants continued to evolve within the borders of the country. Due to the Mozambican health system already under constraint, and paucity of data in Mozambique, there is a need to continue to strengthen and support genomic surveillance in the country as VOCs and Variants of interests (VOIs) are often reported from the southern African region.

Clinical severity of SARS-CoV-2 Omicron BA.4 and BA.5 lineages compared to BA.1 and Delta in South Africa.

Omicron lineages BA.4 and BA.5 drove a fifth wave of COVID-19 cases in South Africa. Here, we use the presence/absence of the S-gene target as a proxy for SARS-CoV-2 variant/lineage for infections diagnosed using the TaqPath PCR assay between 1 October 2021 and 26 April 2022. We link national COVID-19 individual-level data including case, laboratory test and hospitalisation data. We assess severity using multivariable logistic regression comparing the risk of hospitalisation and risk of severe disease, once hospitalised, for Delta, BA.1, BA.2 and BA.4/BA.5 infections. After controlling for factors associated with hospitalisation and severe outcome respectively, BA.4/BA.5-infected individuals had a similar odds of hospitalisation (aOR 1.24, 95% CI 0.98-1.55) and severe outcome (aOR 0.72, 95% CI 0.41-1.26) compared to BA.1-infected individuals. Newly emerged Omicron lineages BA.4/BA.5 showed similar severity to the BA.1 lineage and continued to show reduced clinical severity compared to the Delta variant.

Longitudinal Surveillance of Antibiotic Resistance in Escherichia coli and Enterococcus spp. from a Wastewater Treatment Plant and Its Associated Waters in KwaZulu-Natal, South Africa.

We assessed the prevalence, distribution, and antibiotic resistance patterns of Escherichia coli and Enterococcus spp. isolated from raw and treated wastewater of a major wastewater treatment plant (WWTP) in KwaZulu-Natal, South Africa and the receiving river water upstream and downstream from the WWTP discharge point. Escherichia coli and enterococci were isolated and counted using the Colilert®-18 Quanti-Tray® 2000 and Enterolert®-18 Quanti-Tray 2000 systems, respectively. A total of 580 quantitative PCR-confirmed E. coli and 579 enterococci were randomly chosen from positive samples and tested for in vitro antibiotic susceptibility using the disk diffusion assay against 20 and 16 antibiotics, respectively. The removal success of the bacterial species through the treatment procedure at the WWTP was expressed as log removal values (LRVs). Most E. coli were susceptible to meropenem (94.8%) and piperacillin-tazobactam (92.9%), with most Enterococcus susceptible to ampicillin (97.8%) and vancomycin (96.7%). In total, 376 (64.8%) E. coli and 468 (80.8%) Enterococcus isolates showed multidrug resistance (MDR). A total of 42.4% (246/580) E. coli and 65.1% (377/579) enterococci isolates had multiple antibiotic resistance indices >0.2. The LRV for E. coli ranged from 2.97 to 3.99, and for enterococci the range was observed from 1.83 to 3.98. A high proportion of MDR E. coli and enterococci were present at all sampled sites, indicating insufficient removal during wastewater treatment. There is a need to appraise the public health risks associated with bacterial contamination of environmental waters arising from such WWTPs to protect the health of users of the receiving water bodies.

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.

Not All Street Food Is Bad: Low Prevalence of Antibiotic-Resistant Salmonella enterica in Ready-to-Eat (RTE) Meats in Ghana Is Associated with Good Vendors' Knowledge of Meat Safety.

Foodborne infections due to the consumption of meat is a significant threat to public health. However, good vendor and consumer knowledge of meat safety could prevent meat contamination with and transmission of foodborne pathogens like Salmonella. Thus, this study investigated the vendor and consumer perception, knowledge, and practices of meat safety regarding ready-to-eat (RTE) meat and how this affected the prevalence and antibiotic susceptibility of Salmonella enterica in RTE meats in the streets of Ghana. A semi-structured questionnaire was used to obtain the demographics, knowledge, and practices of meat safety data from RTE meat vendors (n = 300) and consumers (n = 382). Salmonella enterica detection was done according to the United State of America (USA)-Food and Drugs Administration (FDA) Bacteriological Analytical Manual. The disk diffusion method was used for antibiotic resistance testing. The results revealed that most of the respondents had heard of meat safety (98.3% vendors, 91.8% consumers) and knew that meat could be contaminated by poor handling (100.0% vendors, 88.9% consumers). The respondents knew that regular hand washing reduced the risk of meat contamination (100.0% vendors, 94.0% consumers). Responses to the practices of meat safety by vendors were generally better. A very low Salmonella enterica prevalence was observed in the samples, ranging between 0.0 and 4.0% for guinea fowl and beef, respectively. However, the six isolates obtained were resistant to five of the nine antibiotics tested, with all isolates displaying different resistance profiles. Overall, the good knowledge and practice of meat safety demonstrated by the respondents corroborated the negligible prevalence of Salmonella in this study, reiterating the importance of vendor meat safety knowledge. However, the presence of resistant Salmonella enterica in some of the meat samples, albeit in a very low prevalence, warrants stricter sanitary measures and greater meat safety awareness in the general population to prevent meat-borne infections and potential transmission of drug-resistant bacteria to humans.

Burden, Antibiotic Resistance, and Clonality of Shigella spp. Implicated in Community-Acquired Acute Diarrhoea in Lilongwe, Malawi.

Although numerous studies have investigated diarrhoea aetiology in many sub-Saharan African countries, recent data on Shigella species' involvement in community-acquired acute diarrhoea (CA-AD) in Malawi are scarce. This study investigated the incidence, antibiotic susceptibility profile, genotypic characteristics, and clonal relationships of Shigella flexneri among 243 patients presenting with acute diarrhoea at a District Hospital in Lilongwe, Malawi. Shigella spp. were isolated and identified using standard microbiological and serological methods and confirmed by identifying the ipaH gene using real-time polymerase chain reaction. The isolates' antibiotic susceptibility to 20 antibiotics was determined using the VITEK 2 system according to EUCAST guidelines. Genes conferring resistance to sulfamethoxazole (sul1, sul2 and sul3), trimethoprim (dfrA1, dfrA12 and dfrA17) and ampicillin (oxa-1 and oxa-2), and virulence genes (ipaBCD, sat, ial, virA, sen, set1A and set1B) were detected by real-time PCR. Clonal relatedness was assessed using ERIC-PCR. Thirty-four Shigella flexneri isolates were isolated (an overall incidence of 14.0%). All the isolates were fully resistant to sulfamethoxazole/trimethoprim (100%) and ampicillin (100%) but susceptible to the other antibiotics tested. The sul1 (79%), sul2 (79%), sul3 (47%), dfrA12 (71%) and dfrA17 (56%) sulfonamide and trimethoprim resistance genes were identified; Oxa-1, oxa-2 and dfrA1 were not detected. The virulence genes ipaBCD (85%), sat (85%), ial (82%), virA (76%), sen (71%), stx (71%), set1A (26%) and set1B (18%) were detected. ERIC-PCR profiling revealed that the Shigella isolates were genetically distinct and clonally unrelated, indicating the potential involvement of genetically distinct S. flexneri in CA-AD in Malawi. The high percentage resistance to ampicillin and sulfamethoxazole/trimethoprim and the presence of several virulence determinants in these isolates emphasises a need for continuous molecular surveillance studies to inform preventive measures and management of Shigella-associated diarrhoeal infections in Malawi.

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.

Prevalence, phylogenomic insights, and phenotypic characterization of Salmonella enterica isolated from meats in the Tamale metropolis of Ghana.

Characterization of foodborne pathogens including Salmonella species allows for the determination of their relationship and/or relatedness with others. This study characterized Salmonella enterica (S. enterica) isolated from five meat types (mutton, beef, chevon, guinea fowl, and local chicken) obtained from Tamale metropolis, Ghana. The S. enterica were characterized phenotypically (n = 44) based on their antibiotic resistance pattern with the disc diffusion method and genetically (n = 16) using whole-genome sequencing (WGS) as well as with bioinformatic analysis for the prediction of their clonal and phylogenomic relationship. Of the 225 meat samples examined, 107 (47.56%) were positive for S. enterica. Mutton was the most contaminated meat type and the least was local chicken. The 44 S. enterica isolates exhibited five different antibiotic patterns with multiple antibiotic resistance (MAR) index ranging from 0.13 to 0.63. Resistant to only erythromycin was most common and was exhibited by 34 isolates (77.27%). Four isolates were resistant to four different antibiotics (TeAmpSxtECro) with a percentage of 9.09%, while two isolates (4.55%) were resistant to none of the antibiotics. The sequenced S. enterica isolates consisted of 7 serovars and 8 clonal lineages with the S. enterica subsp. enterica serovar Hato (ST5308) being the predominate strain. Phylogenomic analysis showed that the isolates clustered according to their serovars and sequence types (clonal lineages). However, further metadata insights coupled with the phylogenomics revealed a complex intraspread of multiple S. enterica subsp. enterica serovars in diverse meat sources in areas in Tamale which is very worrying for infection management. In summary, our study provides useful insights into S. enterica in meat reservoirs obtained from Tamale metropolis, Ghana.

Genomic analysis of a multidrug-resistant clinical Providencia rettgeri (PR002) strain with the novel integron ln1483 and an A/C plasmid replicon.

Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624 bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harboring the gene blaOXA-2 , was identified, with other uncharacterized class 1 integrons harboring aacA4cr and dfrA1. Aac(3')-IIa and blaSCO-1 , as well as blaPER-7 , sul2, and tet(B), were found bracketed by composite Tn3 transposons, and IS91, IS91, and IS4 family insertion sequences, respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131, and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries. Multidrug resistance in P. rettgeri is rare, particularly in clinical settings, making this case an important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity, and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of these resistance genes.

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.

Molecular Epidemiology of Antibiotic-Resistant Escherichia coli from Farm-To-Fork in Intensive Poultry Production in KwaZulu-Natal, South Africa.

The increased use of antibiotics in food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. This study aimed to investigate the molecular epidemiology of antibiotic-resistant Escherichia coli from intensively produced poultry in the uMgungundlovu District, KwaZulu-Natal, South Africa. Samples were collected weekly between August and September 2017 from hatching to final retail products. E. coli was isolated on eosin methylene blue agar, identified biochemically, and confirmed using polymerase chain reaction (PCR). Susceptibility to 19 antibiotics was ascertained by the Kirby-Bauer disc diffusion method. PCR was used to test for resistance genes. The clonal similarity was investigated using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). In total, 266 E. coli isolates were obtained from all the samples, with 67.3% being non-susceptible to at least one antibiotic tested and 6.7% multidrug resistant. The highest non-susceptibility was to ampicillin (48.1%) and the lowest non-susceptibility to ceftriaxone and azithromycin (0.8%). Significant non-susceptibility was observed to tetracycline (27.4%), nalidixic acid (20.3%), trimethoprim-sulfamethoxazole (13.9%), and chloramphenicol (11.7%) which have homologues used in the poultry industry. The most frequently observed resistance genes were blaCTX-M (100%), sul1 (80%), tetA (77%), and tetB (71%). ERIC-PCR grouped isolates into 27 clusters suggesting the spread of diverse clones across the farm-to-fork continuum. This reiterates the role of intensive poultry farming as a reservoir and a potential vehicle for the transmission of antibiotic resistance, with potentially severe public health implications, thus, requiring prompt and careful mitigation measures to protect human and environmental health.

Genomic analysis of methicillin-resistant Staphylococcus aureus isolated from poultry and occupational farm workers in Umgungundlovu District, South Africa.

This study detected methicillin-resistant Staphylococcus aureus (MRSA) isolates circulating in poultry and farm workers at an intensive poultry production system in uMgungundlovu, South Africa and established the genetic relatedness and characteristics of the isolates using whole genome sequencing (WGS). A total of 145 S. aureus were isolated from poultry (120) and occupational workers (25) in the "farm to fork" continuum (farm, transport, slaughterhouse, and retail points). Twelve MRSA (12/145; 8.3%) isolates were found in the poultry food-chain. MRSA isolates were subjected to antibiotic susceptibility testing against a panel of 20 antibiotics using the broth dilution method and their whole genome was sequenced via the Illumina MiSeq. All the MRSA isolates were multi-drug resistant (MDR) and carried the mecA gene on the SCCmec mobile genetic element (MGE). The majority (11/12) of the MRSA isolates circulating between humans and animals in the continuum belonged to a human-associated clone, ST612-CC8-t1257-SCCmec_IVd (2B), previously reported in South Africa. Other MGEs present in the isolates included: plasmid replicons based on Rep 7 and 20, insertion sequences (IS1182), and prophages (phi2958PVL). Genomic analysis identified a distinct acquired antibiotic resistome in the clone, which accurately predicted the phenotypic antibiograms. Phylogenetic analysis clustered the isolates within the major cluster (I), suggesting the spread of the local dominant multidrug resistance MRSA clone ST612-CC8-t1257-SCCmec_IVd (2B) between humans and animals along the 'farm to fork' continuum. The findings of this study suggest the need to establish appropriate control measures to curb the spread of MDR-MRSA in the food chain.