Detection of beta-lactamase genes and antibiotic susceptibility profiles of Staphylococcus aureus isolates in a hospital in Malaysia

• Main Author: Hamasalih, Bakhtiyar Mahmood
• Format: Thesis
• Language: English
• Published: 2017
• Online Access: http://psasir.upm.edu.my/id/eprint/71077/1/FPSK%28M%29%202017%2048%20-%20IR.pdf; http://psasir.upm.edu.my/id/eprint/71077/

Staphylococcus aureus is a versatile pathogen causing a variety of infections. Resistance to β-lactam antibiotics among S. aureus isolates has been attributed to the bla system detection. The bla system consists of three main components which are blaR1, a sensor signal transducer gene, blaZ, structural gene encoding β-Lactamase enzyme, and blaI, a repressor gene. The aim of this study is to detect the presence of blaR1gene and to determine its relationship with phenotypic resistance of S. aureus. Identification of 128 isolates of S. aureus was carried out based on colony morphology, Gram stain, catalase and coagulase tests. All isolates were subjected to susceptibility test of commonly used antibiotics by disc diffusion method. Resistant strains toward cefoxitin were considered as phenotypic methicillin resistant S. aureus(MRSA) and were subjected to further determination of vancomycin Minimum Inhibitory Concentration (MIC) by E-test. BlaR1 gene detection among all isolates was done by polymerase chain reaction (PCR). For phenotypic MRSA strains, resistance towards methicillin was further confirmed by mecA gene detection. All non urine isolates were sensitive to vancomycin and rifampin via disc diffusion method. Followed by trimethoprim/sulfamethoxazole (n=122, 98.4%). Whereas, all urine isolates were sensitive to vancomycin, trimethoprim/sulfamethoxazole, gentamicin and nitrofurantoin. Widespread of resistance against penicillin group (n=107, 83.6%) was detected, followed by cefoxitin (n=32, 25%) reported as phenotypic MRSA. PCR revealed all (n=107, 83.6%) penicillin group resistant isolates harboured blaR1 gene. Whereas, all (n=32, 100%) phenotypic MRSA strains harboured mecA gene. Five penicillin susceptible strains were positive for blaR1 gene, four had slightly larger amplicon size compared to blaR1 gene and one had similar amplicon size with the blaR1 gene. Penicillin and oxacillin E-test, nitrocefin disc test, bla system genes and mecA gene detection were conducted for these five strains. Sequencing analysis was performed for these five penicillin susceptible strains. Vancomycin E-test revealed two (n=2, 6.25%) MRSA strains were intermediate to vancomycin and one strain with reduced susceptibility (MIC 3 μg/mL). All five penicillin susceptible blaR1 positive strains were susceptible to penicillin and oxacillin by E-test and negative for nitrocefin test. Only one strain (A56) was positive for blaZand mecA gene. Sequencing and homology analysis of all five penicillin susceptible blaR1 positive strains did not have any similarity with any blaR1 gene sequence in NCBI website. However, four strains had 100% correspondence with S. aureus strain AUS0325 genome assembly. Conversely, sequencing result for blaZgene of strain A56 and blaR1 gene of representative penicillin resistant strains had 100% and 99% similarity with S. aureus strain N315 blaZ and blaR1 genes respectively. Statistical analysis found significance association of blaR1 gene with phenotypic resistant of S. aureus to beta lactam antibiotics. There was no significant association seen for other antibiotics. In conclusion, in this study Staphylococcus aureus strains were resistant to penicillin, followed by cefoxitin and erythromycin. All penicillin resistant strains harboured blaR1 gene, and all cefoxitin resistant strains harboured mecA gene. There may be a role for blaR1 gene detection among S. aureusto confirm beta lactam resistance, which would be useful to aid in managing infected patients efficiently.