Detection of mutations in gyra and parc by mama-pcr of quinolone-resistant isolates of escherichia coli from the komfo anokye teaching hospital, kumasi

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Antimicrobial resistance is a serious problem on the ascendency which has been documented in some parts of Africa and Ghana. By the World Health Organization’s assessment, infections caused by resistant microorganisms often fail to respond to conventional treatment, resulting in prolonged illness and greater risk of death (WHO, 2012). According to Namboodiri et al, 2011, data available on the prevalence of resistance to antimicrobial substances in Africa is limited to phenotypic resistance tests with little data on the molecular basis for resistance. Bacterial strains for the study were obtained from isolates of pathological samples of patients visiting the Komfo Anokye Teaching Hospital. Resistance to seven antimicrobials was tested using the disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin was also determined using the agar dilution technique in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Amplification of the quinolone-resistance determining regions was done by mismatch amplification mutation assay (MAMA-PCR) and visualization of PCR products by Agarose gel electrophoresis. The highest number of the isolates were from urine (n=138). A total of 139 of the 200 isolates obtained were from female patients while 55 were from male patients. The ages of patients from whom the isolates were obtained ranged from 2 days old to 88 years old. Out of the antibiotics tested, gentamicin showed the lowest percentage of resistance (47.5%) and ampicillin showed the highest percentage (94%). The number of susceptible isolates to the two quinolones tested was 21%. There were no isolates that were susceptible to nalidixic acid but resistant to ciprofloxacin. The most common phenotype in the study was the pan-resistant (30.5% of isolates) and 2.5% of isolates were susceptible to all antibiotics tested. Testing by MIC yielded 67.5% resistant, 2.5% intermediate and 30% susceptible isolates. MIC50 for ciprofloxacin in this study was >64μg/mL. A very good level of agreement was observed between the two methods used to determine resistance to ciprofloxacin (k=0.91). Five groups were defined by the results according to mutations in gyrA and parC. The group with the highest number of isolates (67%) was that with Ser-83 substitutions in gyrA. The group with the lowest number of isolates (1%) was that with substitutions in Ser-83 and Asp-87 in gyrA and Ser-80 in parC. Mutations in gyrA83 had the highest occurrence (149 isolates) and those in gyrA87 had the lowest occurrence (2 isolates). Isolates that had only a single mutation had minimum inhibitory concentration values ranging from 0.5μg/mL to >64μg/mL. With the exception of one isolate, all isolates that had minimum inhibitory concentration values less than 64 μg/ml (22 isolates) had only one mutation and no isolate that had two or more mutations (48 isolates) had a minimum inhibitory concentration value less than 64μg/mL. with the exception of one isolate, all isolates that had mutations in parC had MIC values that were greater than 64μg/mL. Mutations in the quinolone resistance development region were found to be associated with all quinolone resistant isolates in this study.
A thesis submitted to the Department of Clinical Microbiology, Kwame Nkrumah University of Science and Technology, in partial fulfillment of the requirements for the degree of Master of Science (Clinical Microbiology), 2013