Detection, inhibition and molecular analysis of multidrug resistant aerobic gram-negative clinical isolates from a tertiary hospital in Nigeria

Abstract

The challenge of combating the ever emerging multi-drug resistant (MDR) clinical isolates in the face of a slow rate of discovery of new classes of antibiotics is a problem in antibiotic chemotherapy. This study was aimed at (i) linking phenotypic antibiotic drug-resistance characteristics detected in randomly-sampled clinical isolates with detectable genetic markers. (ii) screening a suspected efflux pump inhibitor (EPI) [1-(3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could be helpful in combating this challenge. Fifty-one isolates; 28 Klebsiella pneumonia, 3 E. Coli, 1 Enterobacter cloacae, 1 E. aerogenes, 5 Proteus mirabilis, 4 Providencia rettgeri, 1 P. stuartii, 1 Serratia liquefaciens, 6 S. odorifera, and 1 Acinetobacter baumannii obtained from infections of urinary tract, upper respiratory tract, gastrointestinal tract, ear swab, eye swab, and blood culture were screened for (i) antibiotic-susceptibility over a range of 11 classes of antibiotics, (ii) β-lactamase production, (iii) ESBL production and (iv) Efflux pump activity (EPA) in the presence and absence of 1-[3-(trifluoromethyl) benzyl]-piperazine (TFMBP). Molecular analysis was done using DNA extraction by boiling and the randomly-amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) procedure with 2% agarose gel electrophoresis stained with ethidium bromide at 10 μg/ml and visualized by UV trans-illumination. AmpC β-lactamase (4%) and K1 β-lactamase (5.8%) were detected with no carbapenemase producers. AcrA and AcrB marker genes were detected in 12% of the isolates while blaCTX-M (8%) and blaTEM (4%) were also detected. Antibiotic resistance due to EPA can be combated with a suitable EPI as demonstrated by TFMBP when combined with specific antibiotics.

Keywords: TFMBP, Efflux Pump Activity, ESBL, MDR, Carbapenemase