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Doctoral Degrees (Medical Microbiology)

Permanent URI for this collectionhttps://hdl.handle.net/10413/9619

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Browsing Doctoral Degrees (Medical Microbiology) by Author "Abia, Akebe Luther King."

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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.
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    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.