School of Laboratory Medicine & Medical Sciences

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Molecular and genomic analysis of clinical multidrug-resistant coagulase-negative staphylococci from the uMgungundlovu District in the KwaZulu-Natal Province, South Africa.
(2020) Asante, Jonathan.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.; Abia, Akebe Luther King.
Coagulase-negative staphylococci (CoNS) are among the most commonly recovered bacteria in clinical specimens. They are usually colonisers (commensals) of the skin and nasal passages and considered contaminants of microbial cultures. However, they have been recognised as emerging pathogens, frequently causing opportunistic infections. The frequent use of indwelling medical devices and long-term hospitalisation present an increased risk of exposure to CoNS, resulting in infections usually caused by multidrug-resistant pathogens. Few studies focus on CoNS, including characterisation of their mechanisms of resistance, virulence, and persistence. Therefore, this study describes the molecular and genomic profiles of clinical CoNS from public sector hospitals in the uMgungundlovu District in KwaZulu-Natal, South Africa. Eighty-nine clinical CoNS isolates collected from three hospitals within the uMgungundlovu District between October 2019 and February 2020, constituted the sample. Isolates were speciated using the Vitek 2 system. Antibiotic susceptibility testing was done against a panel of 20 antibiotics according to Clinical and Laboratory Standards Institute (CLSI) guidelines using the Kirby-Bauer disk-diffusion method and minimum inhibitory concentration (MIC) was determined using the broth microdilution method for penicillin G, cefoxitin, ceftaroline, ciprofloxacin, moxifloxacin, azithromycin, erythromycin, gentamicin, amikacin, chloramphenicol, tetracycline, doxycycline, teicoplanin, tigecycline, linezolid, clindamycin, rifampicin, sulphamethoxazole/trimethoprim, nitrofurantoin and vancomycin. PCR was used to detect the presence of the mecA gene to confirm phenotypic methicillin resistance. Based on their resistance profiles, a sub-sample of isolates were subjected to wholegenome sequencing (Illumina MiSeq) to ascertain the resistome, virulome, mobilome, clonality and phylogenomic relationships using bioinformatic tools. The SPAdes software was used for the assembly of the raw reads. ResFinder 4.1 and CARD were used to identify antibiotic resistance genes in the isolates, while the virulence factor database (VFDB), Center for Genomic Epidemiology‘s MLST 2.0 server and MobileElementFinder v1.0.3 were used to identify virulence genes, sequence types and mobile genetic elements, respectively. Mutations in fluoroquinolone and rifampicin resistance genes were identified by manual curation using BLASTn alignment which was also used to determine the genetic environment of the resistance genes.S. epidermidis was the most abundant CoNS species isolated. Phenotypic methicillinresistance was detected in 76.4% (n=68) of isolates, 92.6% (n=63) of which were genotypically confirmed by PCR. Multidrug resistance (MDR) was observed in 76.4% (n=68) of isolates, with 51 antibiograms observed. The resistance genes mecA, blaZ, erm(A), erm(B), erm(C), msr(A), aac(6')-aph(2'') and fosB, among others, were detected and corroborated the observed phenotypes. Molecular mechanisms of resistance to tigecycline, teicoplanin, linezolid and nitrofurantoin were not detected even though some isolates were resistant to them. There was no association between ARG type and hospital/department. The ica operon known to facilitate biofilm formation was detected in 7/16 isolates sequenced. Known and putatively novel mutations in the gyrA, parC, parE and rpoB genes were also detected for fluoroquinolone- and rifampicin-resistant isolates. Prediction of isolates’ pathogenicity towards human hosts yielded a high average probability score (Pscore ≈ 0.936), which, together with the several virulence genes detected (including atl, ebh, clfA, ebp, icaA, icaB,icaC), support their pathogenic potential to humans. Seven MLST types were found, while the community-acquired SCCmec type IV was the most common SCCmec type detected. Mobile genetic elements (MGEs) haboured by isolates included plasmid replicon Rep10 and insertion sequence IS256. Defense systems such as arginine catabolic mobile element (type I and III), CRISPR system (16), and the restriction-modification system (type II) were detected. Genetic analysis showed that resistance genes were frequently bracketed by MGEs such as transposons (such as Tn554) and insertion sequences (such as IS257 and IS1182) that facilitated their mobility. Phylogenetic studies showed that the distribution of genes did not coincide with the phylogenetic clades. Despite the relatedness of isolates (clades A and B), there is still considerable variation within individual strains that can facilitate adaptation to local environments. The isolates exhibited several permutations and combinations of ARGs, virulence genes and MGEs, pointing to a complex milieu of mobilized antibiotic resistance and pathogenic characteristics in clonal and multiclonal strains. The study necessitates surveillance of CoNS as emerging pathogens.
Molecular characterization of antibiotic-resistant Staphylococcus aureus in an intensive pig production system in KwaZulu-Natal, South Africa.
(2021) Sineke, Ncomeka.; Amoako, Daniel Gyamfi.; Abia, Akebe Luther King.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.
The increase in antibiotic resistance in food animals and food of animal origin has been attributed to the extensive use of antibiotics during animal husbandry giving rise to multidrug-resistant bacteria. Staphylococcus aureus is a major threat in veterinary medicine, the agricultural sector and public health because of its zoonotic potential. Despite significant research on S. aureus in food animals in other parts of the world, in-depth studies outside healthcare facilities are limited in South Africa. This study characterized the molecular epidemiology of antibiotic resistant S. aureus from farm-to-fork in an intensive pig production chain in the uMgungundlovu district, Kwa-Zulu Natal, South Africa. A total of 333 samples collected along a pig production chain on the farm (faecal, litter and slurry samples) during transport (truck samples) and at the abattoir (caeca, carcass swabs, carcass rinsate and retail meat samples) were investigated for the presence S. aureus using selective media and biochemical tests. Confirmation was done by using PCR targeting the nucA gene. Antibiotic susceptibility patterns were investigated by the Kirby Bauer disk diffusion according to CLSI guidelines against the WHO-AGISAR recommended panel of antibiotics. Selected resistance and virulence genes were detected using PCR. REPPCR was used to evaluate the molecular relatedness of isolates across the pig production chain. Of the 333 samples, 141 (43%) yielded staphylococci isolates. After molecular confirmation, 97(69%) isolates were confirmed S. aureus and 44(31%) as other staphylococcal species. Isolates displayed resistance to erythromycin (85%), clindamycin (85%), penicillin-G (81%), tetracycline (79%), doxycycline (77%), vancomycin (69%), ampicillin (61%), trimethoprim/sulfamethoxazole (57%), rifampicin (57%), teicoplanin (52%), linezolid (51%), chloramphenicol (51%), nitrofurantoin (47%), moxifloxacin (33%), cefoxitin (20%), ciprofloxacin (15%), tigecycline (10%), levofloxacin (8%), gentamicin (8%), and amikacin (2%). Multidrug resistance (MDR) was recorded in 84% (80/97) of isolates with 56 different antibiograms. Resistance genes ermC, blaZ, tetK, tetM, msrA, aac’6, mecA were evident in 82%, 73%, 58%, 28%, 15%, 5%, and 53% respectively and not all resistance phenotypes were genotypically confirmed. The hla (39%), hld (23%), seb (3%), sed (2%), etb (1%), LukS/F-PV (30%) and tst (11%) virulence genes encoding hemolysin, cytotoxins, staphylococcal enterotoxins (sea and seb), exfoliative toxins, PVL pore-forming toxin and toxic shock syndrome toxin-1 were detected. Genetic fingerprinting revealed the diversity of MRSA isolates in the pig production chain with the major REP-types constituting isolates from different sources within the farm, suggesting transmission within the farm environment with no evidence of transmission across the production chain. This study highlights the phenotypic and genotypic diversity of the virulence and resistance profiles of S. aureus isolated across the pig production chain. Resistance to antibiotics used as growth promoters was evident and the high prevalence of MDR isolates with elevated MAR index values >0.2, specifically at farm level indicates exposure to environments of high antibiotic use, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.
Molecular epidemiology of antibiotic resistant Campylobacter spp. from farm-to-fork in an intensive pig production system in Kwazulu-Natal, South Africa.
(2021) Sithole, Viwe.; Amoako, Daniel Gyamfi.; Essack, Sabiha Yusuf.; Abia, Akebe Luther King.; Bester, Linda Antionette.
Background: Campylobacter spp. are among the leading foodborne pathogens, causing Campylobacteriosis, a zoonotic infection that results in bacterial gastroenteritis and diarrhea disease in animals and humans. The emergence and transmission of antibiotic resistance and virulence in Campylobacter spp. is increasingly reported. We investigated the molecular epidemiology of antibiotic resistant Campylobacter spp. isolated across the farm-to-fork-continuum in an intensive pig production system in the uMgungundlovu District, Kwazulu-Natal, South Africa. Methodology: Following ethical approval, samples were collected over a period of 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 according to EUCAST and/or CLSI guidelines. Selected antibiotic resistance and virulence genes were identified using PCR and DNA sequencing. Clonal relatedness among the isolates was determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Results: Altogether, 378/520 (72.7%) samples were positive for Campylobacter spp. with C. coli as the most predominant (73.3%), followed by C. jejuni (17.7%) with 9.0% classified as “other”. Relatively high levels of resistance were 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. The lowest percentage resistance observed was for gentamicin (12%) for both C. coli and C. jejuni, and nalidixic acid (28% and 27%) for C. coli and C. jejuni respectively. Multi-drug resistance (MDR) was noted among 330/378 (87.3%) isolates. The antibiotic resistance genes observed were the tetO (74.6%), the blaOXA-61 (2.9%) and cmeB (11.1%) accounting for the resistance to tetracycline and ampicillin while the membrane efflux pump could confer resistance to ampicillin, tetracycline, ciprofloxacin, and erythromycin. All C. coli and C. jejuni isolates (21) with the gyrA gene exhibited mutation at the Thr-86-Ile region in the quinolone-resistancedetermining region (QRDR) and all C. coli and C. jejuni isolates (18) exhibiting erythromycin resistance showed common transitional mutations A2075G and A2074C in the 23S rRNA gene. 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. The ERIC-PCR banding patterns revealed that isolates along the continuum were highly diverse with isolates from the same sampling points belonging to the same major ERIC-types. Conclusion: We showed relatively high levels of resistance to antibiotics commonly used in intensive pig production in South Africa with some evidence, albeit minimal, of transmission across the farm-tofork continuum. This together with the virulence profiles present in Campylobacter spp. presents a challenge to food safety and a potential risk to human health. This is further exacerbated by the reduction in antibiotic treatment options necessitating routine surveillance and monitoring together with antibiotic stewardship, comprehensive biosecurity, and good animal husbandry in intensive pig production.
Molecular epidemiology of antibiotic resistant Escherichia coli from intensively-produced poultry in a farm-to-fork continuum in KwaZulu-Natal, South Africa.
(2020) McIver, Katherine Susan.; Essack, Sabiha Yusuf.; Bester, Linda Antionette.; Abia, Akebe Luther King.
The increased use of antibiotics in intensively produced food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. There is a risk of transfer of this resistance to humans and as such a public health risk. The aim of this study was to investigate the molecular epidemiology of antibiotic resistant Escherichia coli from intensively produced poultry in the uMgungundlovu district of Kwa-Zulu Natal, South Africa. This was a longitudinal descriptive study with the aim to determine the epidemiology of antibiotic resistance of E.coli from hatching through to the final retail product from an intensive poultry farm house. The farm reported the use of zinc bacitracin and Salinomycin included in the feed, but no therapeutic antibiotics used in this batch of chickens. However, the following antibiotics were used on the farm in the previous 12 months: Doxycycline, Sulfadiazine and Trimethoprim, Enrofloxacin, Ceva olaquindox 10%, Avilamycin, Tylosin 10% and Kitasamycin tartate. During the first five weeks, ten samples from litter and faeces were collected. During transfer from the house to abattoir ten swabs from transport trucks and transport crates were taken. At the abattoir ten samples from carcass wash were collected. After slaughter and dressing ten caecums, whole chickens, thighs and necks were collected. Again, during house washing, ten samples were collected. E.coli was putatively identified using Eosin Methylene Blue agar followed by Sorbitol MacConkey agar and confirmed by identification of the uidA gene by polymerase chain reaction. Susceptibility to a panel of antibiotics recommended by the World Health Organization Advisory Group on the Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) was ascertained by the Kirby-Bauer disk diffusion method for 20 antibiotics according to CLSI guidelines. Realtime PCR was used to test for resistance genes tetA, tetB, qnrB, qnrS, aac(6)-lb-cr, sul1, sul2, sul3, blaSHV, blaCTX-M, blaTEM conferring resistance to tetracyclines, quinolones, sulphonamides and cephalosporin antibiotics. Clonal similarities were investigated using ERIC-PCR. A total of 266 E.coli isolates constituted the sample size with a non-susceptibility profile of ampicillin 48.1%, tetracycline 27.4%, nalidixic acid 20.3%, trimethoprim-sulphamethoxazole 13.9%, chloramphenicol 11.7%, cefalexin 4.5%, ciprofloxacin 4.1%, amoxycillin-clavulanic acid 3.4%, gentamicin 1.9%, cefoxitin 1.1%, cefepime 1.1%, cefotaxime 1.1%, amikacin 1.1%, ceftriaxone 0.8% and azithromycin 0.8%. Isolates were fully susceptible to ceftazidime, imipenem, meropenem and tigecycline. Of the 266 isolates 6.4% were multidrug resistant (resistant to one or more antibiotics in three or more distinct antibiotic classes). The most frequently observed resistance genes were blaCTX-M (100%), sul1(80%), tetA(77%), tetB(71%). Using ERIC-PCR the isolates were grouped into 27 clusters with a 75% similarity. Eight clusters comprised of isolates from only one sample. xiv There was an increase in MDR and resistance genes over the farm to fork continuum with lowest and highest levels seen in transport and waste-water samples respectively. ERIC-PCR did not indicate the transmission of clones across the farm-to-fork continuum. There instead appeared to be de novo or evolution of resistance genes or the introduction of plasmids over the time period. As the only antimicrobials used in this flock were salinomycin and zinc bacitracin it is postulated that the resistance observed could be attributed to the co-selection of resistance genes and/or horizontal gene transfer from the environment, insects, chicken food and workers. Overall resistance levels were low over the six weeks of the study, MDR and the prevalence of resistance genes increased over time. The diverse clonality shown by the ERIC PCR results did not support the transmission of clones across the farm-tofork continuum but indicated a de novo evolution of resistance genes and/or the loss or gain of plasmids over the time period.
Molecular epidemiology of antibiotic-resistant Escherichia coli and Enterococcus spp. from agricultural soil fertilized with chicken litter in uMgungundlovu district, KwaZulu-Natal Province, South Africa.
(2021) Fatoba, Dorcas Oladayo.; Abia, Akebe Luther King.; Essack, Sabiha Yusuf.; Amoako, Daniel Gyamfi.
The application of animal manure contaminated with antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) represents a major route by which antibiotic resistance is transmitted into the soil environment. The introduction and persistence of ARB in agricultural soil may pose a risk to public health via the consumption or handling of contaminated farm produce. Understanding the impact of animal manure application on the agricultural soil resistome and the risk it poses on public health is critical. However, such information is limited in South Africa as most antibiotic resistance research focuses on humans and food animals. This study, therefore, describes the prevalence and the genomic profiles of antibiotic-resistant Escherichia coli and Enterococcus spp. isolated from agricultural soil fertilized with chicken litter and the chicken litter. A total of 237 samples were examined and included soil before litter application, the litter-amended soil, and the chicken litter. Isolation and quantification of Escherichia coli and Enterococci were carried out using the Colilert® -18 / Quantiti-Tray® 2000 system and the Enterolert® -18® Quanti-Tray®/2000 system, respectively. The antibiotic susceptibility profiles of the isolates was determined using the Kirby-Bauer disk diffusion method. Whole-genome sequencing (WGS) and bioinformatics tools were used to determine the resistome, virulome, mobilome, clonal lineages, and phylogenies of the isolates circulating between the soil and the chicken litter. The application of chicken litter to the soil statistically significantly increased Enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil. A total of 835 enterococci (680 from soil and 155 from litter) isolates recovered from the samples was dominated by E. casseliflavus (56%), followed by E. faecalis (22%), E. faecium (8%), E. gallinarum (2%) and other Enterococcus spp 102 (12%). Overall, 55.8% (466/835) of the enterococci isolates were resistant to one or more antibiotics with the highest rate in the litter-amended soil (68.9%, 321/466), followed by chicken litter (19.9%, 93/466) and the least in the soil samples collected before the litter amendment (11.2%; 52/466). The enterococci isolates were mostly resistant to tetracycline (33%), erythromycin (25%), and trimethoprim-sulfamethoxazole (23%), among others, intimating the high usage of these antibiotics in poultry farms in South Africa. Additionally, multidrug resistance (MDR) was recorded in 27.8% (130/466) of the enterococci isolates with MAR indices ranging from 0.13 (resistance to two antibiotics) to 0.44 (resistance to seven antibiotics). A total of 63 different resistance patterns were recorded in the MDR enterococci isolates. Notably, enterococci count and the number of antibiotic-resistant enterococci in the litter-amended soil were reduced to levels comparable to the unamended soil at 50 and 28 days after soil amendment respectively. The whole-genome analysis of the few selected enterococci isolates revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). Several resistance genes that confer resistance to aminoglycosides (aac(6’)-Ii, aac(6’)-Iih, ant(6)-Ia, aph(3’)-III, ant(9)-Ia), macrolide-lincosamide-streptogramin AB (MLSAB) [erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)], trimethoprim-sulfamethoxazole (dfrE, and dfrG), tetracycline (tet(M), tet(L), and tet(S)), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat) were detected in the isolates. The bioinformatics analysis further revealed that the chicken litter amendment increased the number and diversity of ARGs in the soil, resulting in increased detection of tetracycline resistance genes (tet(M), tet(L)), and the macrolide resistance gene erm(B) and appearance of some ARGs (ant(6)-Ia, aph(3’)-III, lnu(G), dfrG)) that were not detected in the unamended soil. ARGs were mostly associated with diverse insertion sequences (ISs) (IS982, ISL3, IS6, IS5, IS3, IS256, IS30) and/or transposons (Tn3, Tn916, Tn6009) on plasmids or chromosome. The tet(M) and erm(B) were also co-located on Tn916-like transposons (Tn644, Tn645, and Tn659) in the three sample groups. Some of the isolates also harboured virulence genes that encoded adherence/biofilm formation (ebpA, ebpB, ebpC), anti-phagocytosis (elrA), and bacterial sex pheromones (Ccf10, cOB1, cad, and camE). Phylogenomic analysis showed that few isolates from litter-amended soil clustered with the chicken litter isolates. The isolates from this study also clustered with clinical and animal isolates from South Africa (Pretoria, Pietermaritzburg), Angola, and Tunisia. There was also an increase (albeit statistically insignificant) in E. coli count and the number of antibiotic-resistant E. coli in the soil following chicken litter amendment. A total of 126 E. coli was recovered from the soil and chicken litter samples. In total, 76% (96/126) of the E. coli isolates displayed resistance to at least one antibiotic, with the highest prevalence in the litter-amended soil (71.9%, 69/96) and the least (1%, 1/96) in soil samples collected before the litter amendment. The E. coli isolates displayed a high percentage resistance to tetracycline (78.1%), chloramphenicol (63.5%), ampicillin (58.3%), trimethoprim-sulfamethoxazole (39.6%), cefotaxime (30.2%), ceftriaxone (26.0%), and cephalexin (20.8%). Lower percentages of XVI resistance to cefepime (11.5%), amoxicillin-clavulanic acid (11.5%), cefoxitin (10.4%), nalidixic acid (9.4%), amikacin (6.3%), ciprofloxacin (4.2%), imipenem (3.1%), tigecycline (3.1%), and gentamicin (3.1%) were also recorded in the isolates. All the isolates were completely susceptible to meropenem and ceftazidime. Approximately 54% (52/96) of the resistant isolates were MDR, and the MAR indices of the isolates ranged between 0.11 (resistance to two antibiotics) and 0.56 (resistance to ten antibiotics). Overall, 38.5% (37/96) of all the resistant isolates had a MARI > 0.2, with the highest rate (51.4%) in the litter-amended soil and the least in the soil before litter amendment (2.7%). Twenty-one multidrug resistance patterns were observed among the isolates. These results show that the soil resistome was augmented by chicken litter application. Agricultural soil and chicken litter are rich reservoirs of multidrug-resistant E. coli and Enterococcus spp. that could threaten public health through contamination of food products and the surrounding water bodies. There is therefore a need for urgent and stringent measures to mitigate the spread of antibiotic resistance in the environment via prudent use of antibiotics in food animal production and treatment of animal manure before its application onto agricultural soil.

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