Acinetobacter baumannii (A. baumannii) has developed several resistance mechanisms. The bacteria have been reported as origin of multiple outbreaks. This study aims to investigate the use of efflux pumps and quinolone resistance-associated genotypic mutations as mechanisms of resistance in A. baumannii isolates at a tertiary hospital. A total number of 103 A. baumannii isolates were investigated after identification and antimicrobial susceptibility testing by VITEK2 followed by PCR amplification of blaOXA-51. Conventional PCR amplification of the AdeABC efflux pump (adeB, adeS, and adeR) and quinolone (parC and gyrA) resistance genes were performed, followed by quantitative real-time PCR of AdeABC efflux pump genes. Phenotypic evaluation of efflux pump expression was performed by determining the difference between the MIC of tigecycline before and after exposure to an efflux pump inhibitor. The Sanger sequencing method was used to sequence the parC and gyrA amplicons. A phylogenetic tree was drawn using MEGA 4.0 to evaluate evolutionary relatedness of the strains. All the collected isolates were blaOXA-51-positive. High resistance to almost all the tested antibiotics was observed. Efflux pump was found in 75% of isolates as a mechanism of resistance. The study detected parC gene mutation in 60% and gyrA gene mutation in 85%, while 37% of isolates had mutations on both genes. A minimal evolutionary distance between the isolates was reported. The use of the AdeABC efflux pump system as an active mechanism of resistance combined with point mutation mainly in gyrA was shown to contribute to broaden the resistance spectrum of A. baumannii isolates.

No abstract

24Antimicrobial resistance is now globally recognised as the greatest threat to human 25 health. Acinetobacter baumanniis' (A. baumannii) clinical significance has been driven by its 26 ability to obtain and transmit antimicrobial resistance factors. In South Africa, A. baumannii 27 is a leading cause of healthcare associated infections (HAI). In this study, we investigated the 28 genetic determinants of multi-drug resistant A. baumannii (MDRAB) at a teaching hospital in 29 Pretoria, South Africa. 30One hundred non repetitive isolates of A. baumannii were collected for the study at Dr 31 George Mukhari Tertiary Laboratory (DGMTL). Antimicrobial susceptibility testing was 32 performed using the VITEK2 system (bioMerieux, France). The prevalence of common 33 resistance associated genes and AdeABC efflux pump system associated genes were 34 investigated using conventional PCR. Genetic relatedness of isolates was then determined 35 using rep-PCR. 36Seventy (70) of 100 isolates collected were confirmed to be multi-drug resistant and 37 were bla OXA51 positive. Phenotypically, the isolates where resistant to almost all tested 38 antibiotics. However, one isolate showed intermediate susceptibility to tigecycline while all 39 were susceptible to colistin. Oxacillinase encoding gene bla was the most detected at 40 99% and only 1% was positive for bla . The PCR results for metallo-betalactamase 41 (MBL) encoding genes showed that MBL bla VIM was the most frequently detected at 86% and 42 bla SIM-1 at 3% was the least detected. Out of 70 isolates, 56 isolates had the required gene 43 combination for an active efflux pump. The most prevalent clone was clone A at 69% of the 44 isolates. Regarding treatment; colistin and tigecycline are the most effective against strains 45 encountered at DGMTL as all tested carbapenems seem to have lost their effectiveness. 46The major genotypic determinants for drug resistances are oxacillinases: bla OXA-51 47 (100%) and bla (99%). The study reports for the first time, bla and bla SIM-1 48 detection in A. baumannii in South Africa. 49 50