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Genetics

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    Genetic analyses of antimicrobial resistance and virulence genes in Enterococcus species isolated from livestock production systems in South Africa.
    (2021) Mnguni, Anele Buhle.; Zishiri, Oliver.
    Enterococcus species are widely dispersed in the environment this includes soil, water, plants, food and animals. Although Enterococcus constitute mostly as a commensal bacterium; over the past years the bacterium has evolved to cause nosocomial infections. The proliferation of this pathogen is attributed to its ability in successfully transferring antimicrobial and virulence genes using several channels such as mobile genetic elements. This study investigated the prevalence of Enterococcus spp. in small-scale commercial farms in rural South Africa. The dissemination of virulent E. faecium and E. faecalis isolates allied with livestock production in the Eastern Cape and KwaZulu-Natal provinces was investigated. A total of 276 samples randomly sampled from livestock and their associated environments (feed, soil and water) were screened for Enterococcus spp. using selective media and using DNA molecular methods. E. faecalis and E. faecium prevalence was confirmed by the amplification of the tuf and sodA genes. Sixty-one percent of total presumptive isolates were E. faecalis (n=61) and only 8% (n=8) were identified as E. faecium. The presence of virulence determining factors such as asa1, ccf, cylA, esp, gelE and hyl was screened in all samples that tested positive for Enterococcus species. Presumptive E.faecalis and E. faecium isolates were mostly recovered from Amandawe (KZN). E. faecalis isolates harboured the most virulence genes asa1 (25%; n=), ccf (84%; n=), esp(4%;n= ), gelE (69%; n=) and hyl (12%; n= ). Whilst E. faecium isolates only harboured of asa1(12.5%; n=1), ccf (100%; n=8), gelE (75%;n=6 ) and hyl (25%;n=2). The current study also evaluated the antibiotic resistance profiles and their associated genes in these two species. Antibiotic susceptibility profiles of E. faecium and E. faecalis were assessed using Kirby-Bauer disk-diffusion assay as per the CSLI guidelines. Erythromycin had the highest occurrence of resistant isolates in both species with 75% (n=6) and 54.1% (n=33) respectively. Isolates were least resistant to ampicillin, with 0.03% resistance in E. faecalis and 0% in E. faecium. E. faecalis had the highest prevalence of Multi Drug Resistance (MDR), exhibiting phenotypic resistance to macrolides, aminoglycoside, tetracyclines and fluoroquinolones. TET-CIP-ERY was the most observed antibiotic resistance pattern. Furthermore, the isolates were screened for vanA, vanB, vanC1, vanC2/3, aac(6”)-aph(2”) ,ermA and ermB. The resistance genes that amplified in E. faecalis included vanB (8%;n=5), vanC1 (37%;n=23), vanC2/3 (37%; n=23), ermB (96%;n=58), ermA (8%;n=5) and aac(6”)-aph(2”) (1.6%;n=1). The immense dissemination of E. faecalis that has potentially pathogenic virulent determinants is a cause for concern in livestock production systems. In addition, faecal contamination from livestock poses a threat to the dissemination of virulent strains. The study demonstrated that E. faecium and E. Faecalis isolated from livestock and their associated environment were predominantly resistant to macrolides, glycopeptides, tetracyclines and fluroquinolones. In addition to be the first study in South Africa to document the emergence of inducible vanC determinants in Vancomycin Resistant Enterococci isolates.
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    Identification of arthropods of forensic importance during cold and warm seasons in KwaZulu-Natal Province of South Africa.
    (2021) Tembe, Danisile.; Mukaratirwa, Samson.
    Forensic entomology is the study and use of insects and other arthropods in forensic investigations associated with death, abuse and neglect of both humans and animals. Although there has been an increased interest in forensic entomology and its application in predicting post-mortem interval (PMI) amongst other issues in many developed countries, the results cannot be extrapolated beyond the countries/regions of study since the arthropods species spectra may vary with region and geographical conditions. The present study aimed to determine the arthropod species of forensic importance found during different stages of decomposition of sheep (Ovis aries) and pig (Sus scrofa domesticus) carrion during the warm and cold season in KwaZulu-Natal province of South Africa. A scoping review was conducted to determine the state of knowledge of forensic entomology research and application in southern Africa. To determine the arthropod species associated with sheep and pig carcass during different stages of decomposition, two medium sized Large-White pigs and two medium sized Merino sheep were humanely killed and used for the cold and warm season. Adult arthropods found on and around the carcasses during different stages of decomposition were collected and identified using combined morphological identification keys and molecular technique based on the mitochondrial gene. The review showed that arthropod species that were found on a decomposing carcass could be useful in the estimation of PMI and provided clues in cases of criminal investigations. The review also confirmed the scarcity of forensic entomology research, and its application in southern Africa. Experimental results from this study showed that dipteran flies from the families Calliphoridae, Muscidae and Sarcophagidae were the first to colonize the sheep and pig carcasses during both warm and cold seasons. These include species of Chrysomya marginalis, Ch. putoria, Ch. albiceps, Ch. chloropyga, Lucilia cuprina, Musca domestica and Sarcophaga calcifera. On the sheep carcasses, Ch. marginalis, Ch. albiceps and M. domestica were the most dominant fly species, contributing 63.2 % of the collected flies in the warm season, and 68.9 % in the cold season. Colonization by coleopterans during the warm season started as early as the fresh stage with Dermestes maculatus, Thanatophilus micans and Onthophagus crassicollis. In the cold season these same beetle species were collected from the bloated stage of the sheep carcass. On the pig carcasses, Ch. marginalis (n = 111), Ch. albiceps (n = 99) and M. domestica (n = 131) were the most abundant species during the warm season. The same species were the most abundant species in the cold season (n = 55), (n = 34) and (n = 81) respectively, although in lower numbers than the warm season. Among the collected Coleoptera species, D. maculatus (n = 112) and N. rufipes (n=62) were the most abundant species found on the carcass during the warm season and the same species were the most abundant species in the cold season (n = 66) and (n = 48) respectively. In the warm season Dermestes maculatus was recorded on the pig carcass during the fresh stage and persisted on the carcass until the last of decomposition. However, in the cold season Dermestes maculatus was first recorded on the carcass during the active stage of decomposition. Molecular analyses confirmed the identification of twelve (12) arthropod taxa collected from both sheep and pig carcasses during the cold season. Results showed that 11/12 arthropod species were common in both sheep and pig carcasses, with exception to Onthophagus sp. and Atherigona soccata species which were unique to sheep and pig carcasses respectively. However, during the warm season, the sheep carcass attracted more (n=13) taxa as compared to the pig carcass. The variation in the arthropod was due to the presence of Onthophagus sp. which was also unique to the sheep carcass during this season. Furthermore, there was an addition of a beetle species Hycleus lunatus, which was collected from both sheep and pig carcasses but unique to the warm season. This study generated important information on the endemic arthropod species that are of forensic importance KwaZulu-Natal province. The arrival time and association of arthropods species with different stages of decomposition during the warm and cold season highlighted their value in estimating the PMI in forensic investigations in the locality of KwaZulu-Natal province. The studied arthropods can potentially be useful in the estimation of PMI and other cases of criminal investigations. The seasonal variations in abundance of both Diptera and Coleoptera in the two seasons seemed to indicate influence of seasons which subsequently influenced temperature. It is recommended that similar studies be conducted at other geographical locations of South Africa with a different ecological system to build a database of dipteran and coleopteran species of forensic importance which are endemic in these areas.