Ifediora, Afoma Chinwe2025-02-272025-02-272021-09Ifediora, A. C. (2021). Molecular characterization of methicillin resistant staphylococcus aureus from clinical specimens and antimicrobial activity of plant extracts against the isolates (Unpublished Doctoral Thesis). Federal University of Technology. Owerri, Nigeriahttps://repository.futo.edu.ng/handle/20.500.14562/1665The thesis is for the award of Doctor of Philosophy (Ph.D) in Medical MicrobiologyStaphylococcus aureus is a major bacterial pathogen that causes different community and hospital-acquired infections. S. aureus resistant to methicillin has become a big and expanding problem of concern in many developing countries. This study examined the molecular characterization and the effect of plant extracts on methicillin resistant Staphylococcus aureus (MRSA) from clinical specimens in Abia State, Nigeria using standard recommended procedures. Conventional cultural, morphological and biochemical methods were used to identify the isolates, while the antimicrobial susceptibility testing was performed by the disc diffusion method. Methicillin resistance was detected phenotypically using cefoxitin 30µg disc and oxacillin 1µg disc. Inducible clindamycin resistance was evaluated by the D-test. Polymerase chain reaction (PCR) was used to amplify genes for methicillin resistance (mecA), clindamycin resistance (ermB), beta-lactamase production (blaZ), Panton Valentine leukocidin (pvl) with 16SrRNA gene being the internal control. Sequencing was carried out on the amplified isolates. The Random Amplified Polymorphic DNA (RAPD) was implemented on the mecA strains isolated using three randomly selected oligonucleotide primers. Plant antimicrobial assay was done using the agar well technique and phytochemicals detected in the two plants tested. A total of 750 clinical specimens of blood, urine samples, wound, ear, nasal, high vaginal, urethral and ear swabs were collected from three major health facilities located in the three senatorial zones of Abia State, Nigeria. A total of 265 (35.3%) S. aureus isolates were recovered, out of which 126(47.5%) were from males and 139(52.5%) were from females, however there was no association between the prevalence and gender (p-value = 0.05) and also prevalence and age (p-value = 0.52). Phenotypic detection of MRSA using cefoxitin disc diffusion gave an MRSA prevalence of 164(61.9%) with 65(39.6%) being from urine, 3(23.1%) from wound, 31(18.9%) from high vaginal swab, 22(13.4%) from urethral swab, 4(2.4%) from ear swab, 3(1.8%) from nasal swab and 1(0.6%) from blood samples. All (100%) of the MRSA were susceptible to vancomycin, 120(73.2%) to clindamycin, 92 (56.1%) to gentamycin. All were resistant to ceftazidine, 157(95.7%) to cloxacillin, 146(89.0%) to augmentin, 136(82.9%) to ceftriaxone and 103(61.6%) to erythromycin. The MRSA strains showed much higher resistance rate than their methicillin susceptible Staphylococcus aureus (MSSA) counterparts to all tested antibiotic except clindamycin. Exactly 64(39.0%) of the MRSA were resistant to 4 classes of antibiotics indicating multi drug resistance (MDR). The overall prevalence of inducible clindamycin resistance among methicillin resistant isolates was 29(17.7%) while 66.5% xix produced beta-lactamase. Out of 40 cefoxitin positive isolates, 12 (30%) possessed mecA gene, 17.5% harboured the β-lactamase (blaZ) gene, 20% and 10% possessed the pvl gene. Dendogram analysis of RAPD-PCR amplification of mecA positive strains showed three different clones in circulation in the state. The plant extracts showed varied levels of antimicrobial activity against the MRSA isolates. The growth of the microorganisms used for the test was inhibited by the ethanolic extracts of the leaves of Alchornea cordifolia and Acalypha wilkesiana at concentrations of 50mg/ml to 200mg/ml. The inhibition zones ranged from 9.0mm to 21.0mm whereas the water extracts showed moderate activity against the isolates. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Alchornea cordifolia ranged from 12.5-50mg/ml and 25-100mg/ml for Acalypha wilkesiana. The results of the rates of kill revealed a gradual reduction in the total viable count of bacteria from 1hr to 24hrs in all the test isolates. The phytochemical screening of the ethanol extract revealed the presence of tannins, flavonoids, glycosides, resins and carbohydrates but in variable degrees. The percentage yields of phytochemical content of the leaves of the Alchornea cordifolia plants were as follows: alkaloids (1.85%), flavonoids (1.08%), Glycosides (1.05%), saponins (4.13%), and tannins (0.70%). The use of phenotypic and molecular methods in this study provided useful information on antibiotic resistance and genetic diversity of S. aureus isolates from clinical specimens in Abia State of Nigeria. The information provided could help in monitoring the evolution of S. aureus strains in Nigeria over time.enAttribution-NonCommercial-ShareAlike 4.0 InternationalStaphylococcus aureusMRSAD-testRAPDalchornea cordifoliaacalypha wilkesianaDepartment of MicrobiologyDoctoral Thesis