- Title
- Phenotypic and molecular characterization of clinical isolates of s. Aureus and s. Epidermidis in the Eastern Cape province, South Africa : evaluation of plant-derived compounds
- Creator
- Mthethwa, Ntombeziningi Shirley
- Date
- 2015
- Type
- PhD Manuscript
- Identifier
- http://hdl.handle.net/11260/2030
- Identifier
- vital:40805
- Identifier
- Doctor Of Philosophy (Health Sciences) Medical Microbiology
- Description
- Staphylococcal species are known pathogens that are responsible for an extensive array of clinical manifestations in humans of diverse age groups in different settings and countries. They are responsible for cases of food poisoning, toxic shock syndrome, staphylococcal scalded skin syndrome, carbuncles, meningitis, among others. High level of antibiotic resistance patterns are common among isolates from different sources. The quest to discover alternative therapeutic regimes by use of medicinal plants are now topical and the subject of research activities. The aim of this study was to determine the prevalence and antibiotic resistant patterns of Staphylococcus aureus and Staphylococcus epidermidis isolated from patients attending different hospitals and clinics in rural communities around Mthatha; determine the activity of medicinal plants against S. aureus and S. epidermidis, including the characterisation of active compounds; ascertain phylogenetic relatedness of isolates; determine the genes coding for resistance in both pathogens and assess the role of human sera samples from different blood groups against S. aureus and S. epidermidis . In order to achieve these goals, the following aspects were explored and are hereby presented in chapters. Chapter one focused on the general introduction and literature review. The morphology, cultural characteristics, laboratory diagnosis, pathogenesis, antibiograms and clinical manifestations of Staphylococcus epidermidis and Staphylococcus aureus were reviewed. Furthermore the activities of medicinal plants and their various applications in the management of infections in different countries, including their possible active compounds and toxicity levels were also explored in an endeavour to provide suitable background for the study. iii The activity of human serum samples from different blood groups and their kinetics were also foregrounded. In Chapter 2, the antimicrobial susceptibility, genes coding for resistance and pathogenesis, and phylogenetic relatedness in S. aureus and S. epidermidis were investigated. Samples were collected from the Nelson Mandela Academic Hospital, Mthatha General Hospital and other samples were received from surrounding satellite hospitals. Samples were analysed using Kirby Bauer disk diffusion (antibiotic susceptibility testing), micro-broth dilution (MIC) and conventional PCR for the investigation of the genes (mecA, luks-lukf, msrA, ermA, ermC and icaA) responsible for resistance, virulence and pathogenesis of isolates. RAPD-PCR (Random Amplified Polymorphic DNA – Polymerase Chain Reaction) was used to determine phylogenetic relatedness amongst the S. aureus and S. epidermidis isolates. Results indicated that wound swabs and blood cultures were identified as the main sources of S. aureus and S. epidermidis and prevalence rates in children less than eleven years (25.7%) (S. aureus) and (7%) (S. epidermidis) were recorded. Vancomycin had the highest activity against both S. aureus and S. epidermidis, penicillin had the lowest activity.There were no significant differences in resistance patterns among different age groups and gender (p>0.01). Ninety three percent of S. aureus that were confirmed as MRSA with PCR method possessed mecA gene, whilst 16% of S. epidermidis had mecA and 33% possessed icaA gene. Fifty eight percent (58%) of S. aureus possessed luks-lukf PVL gene suggesting that it may have been acquired from the community. Forty two percent 42% of msrA was detected in S. epidermidis, 43% ermC and 34% ermA were detected in S. aureus isolates. Eleven percent 11% of S. aureus isolates were found to carry both ermA and ermC genes. mecA and luks-lufPV genes are implicated in iv methicillin resistant S. aureus that causes necrotizing pnemoniae and icaA is implicated in the formation of biofilm, another mechanism of resistance in S. epidermidis. RAPD profile resulting in DNA amplification fragments ranged in size from 300 to 1500 bp. The RAPD patterns obtained showed various trends among isolates meaning that S. aureus and S. epidermidis isolates investigated had high diversity among them. Dendograms constructed with each oligonucleotide showed that PCR patterns obtained with primer OPL11 (S. aureus) and ERIC1 (S. epidermidis) strains had genetic similarity indices ranging from 0.009 to 0.329. This wide range of similarity indices indicated a high level of DNA polymorphism among S. aureus and S. epidermidis isolates. Knowledge of the genes coding for resistance in this study will address the gaps in the mechanisms of resistance for effective intervention strategies. The infections caused by multi-resistant strains of Staphylococcal species represent an important problem that affects many health institutions. Due to the large number of patients infected with S. aureus and S. epidermidis and the diverse possibilities of contamination, it is always necessary to review the evolution of antimicrobial resistance and the therapeutic responses of the bacterial strains. This aspect of the study provides updated data on susceptibility patterns of local isolates of S. aureus and S. epidermidis for use in empiric management of patients including the various genes coding for resistance in the designated pathogens and their phylogenetic relatedness. Chapter 3 presents the antimicrobial and anti-HIV1 activities of selected medicinal plants from the Eastern Cape Province used for treating skin and respiratory tract infections. Anti-HIV activities were investigated because of the role of staphylococcal v species as opportunistic pathogens in HIV infections. Four medicinal plants were evaluated for antimicrobial activities and cytotoxicity on clinical isolates of S. aureus and S. epidermidis. Medicinal plants used in this study were Cassine transvaalensis, Croton gratissimus, Vangueria infausta and Vitex ferruginea. The Kirby-Bauer disk diffusion and 96 well microtiter plate methods were used in the antimicrobial activity studies but for the anti-HIV1 activity, MAGI assay was used. Microtetrazolium assay (MTT) was used for the evaluation of cytotoxicity of plants. Bio-autographic assay was used to locate the position of the active compound using Rf value; thin layer chromatography and column chromatography were used in the isolation of active compounds and NMR was used in the identification and structural elucidation of the active compounds. C. transvaalensis and V. infausta were found to possess high activity against S. aureus and S. epidermidis with the lowest MIC of 0.02. In the anti HIV1 MAGI assay, C. transvaalensis and C. gratissimus showed activity against HIV1. C. transvaalensis and C. grattisimus possessed high level of toxicity with CC50 reading of 0.2 while V. infausta and V. ferruginea had reading of 0.1. Two triterpenoids were successfully isolated in C. transvaalensis. Elucidation of the structure of active compounds of plant extracts of medicinal plants provides suitable templates for candidate drug designs and drug discovery. Such potential application will be contingent on the results of cytotoxicity testing and this underlines the significance of the test. Human serum is an important host defence mechanism against disease causing agents. Chapter four evaluates the bactericidal activity of different blood groups. Blood samples of different blood groups were screened for the presence of antibodies against S. aureus and S. epidermidis using agglutination test. Serum sensitivity assay vi was employed for susceptibility testing of S. aureus and S. epidermidis to blood samples. The Gunea-pig serum was used to determine the role of complement in serum bactericidal activity. The results showed that S. aureus and S. epidermidis were highly sensitive to normal human sera from blood group B with a percentage of 61% (S. aureus) and 83.3% (S. epidermidis). When the role of complement in bactericidal activity of the serum to staphylococci species was assessed it was shown that when both S. aureus and S. epidermidis were incubated with individual sera of blood groups and pooled serum of the same blood groups, the organisms were serum sensitive. The findings showed that different individual and pooled serum samples had a role to play in the defence mechanisms of individuals against S. aureus and S. epidermidis. In general the study provides a reference document on S. aureus and S. epidermidis in terms of their antibiograms, molecular characterization, sensitivity to human sera and susceptibility to medicinal plant extracts. Cytotoxicity profiles of the medicinal plants are also reported. Due to the on-going need to expand on the frontiers of knowledge, it is therefore recommended, as a way of building on this study, extensive investigations on the epidemiology of S. epidermidis, S. aureus and related pathogens to be conducted because studies of this nature have been limited by the fact that S. epidermidis strains are often considered to be contaminants. High-level surveillance of clinical samples of MRSA to define the instance and spread of CA-MRSA and HA-MRSA in hospital settings including health care workers and patients are also warranted in addition to further anti -cancer activities and cytotoxicity tests.
- Format
- Publisher
- Department Medical Microbiology
- Language
- English
- Rights
- Walter Sisulu University
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WSU Department of Laboratory medicine and Pathology
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