CARBAPENEMASES, THEIR INHIBITORS, AND PERSISTER CELLS IN MDR AND XDR GRAM-NEGATIVE BACTERIA AT AUBMC

Abstract

Background: Antimicrobial resistance is posing an increasing risk to public health due to the overuse, abuse and miss-use of the available agents, especially with multi-drug resistant (MDR) and extensive-drug resistant (XDR) Gram-negative bacilli. According to the World Health Organization (WHO), Pseudomonas aeruginosa and Klebsiella pneumoniae fall in the critical priority group’s target of new antibiotics, considering their increased resistance to carbapenems, one of the last resort drugs available. These Gram-negative bacilli mainly develop resistance to carbapenems by hydrolytic carbapenemase enzymes, downregulation of porin channels, and upregulation of efflux pumps or modification of carbapenem’s molecular target (penicillin-binding protein; PBP). Moreover, resistance in P. aeruginosa and K. pneumoniae is currently extending to affect carbapenemase inhibitors too. Furthermore, P. aeruginosa and K. pneumoniae populations might survive under stressful environments due to the presence of persister cells which are genetically identical to the susceptible cells but metabolically dormant. Aim: Carbapenems resistance in K. pneumoniae and P. aeruginosa occurs through target modification, expression of carbapenemases or overexpression of efflux pumps. The aim of this study was to assess at the phenotypic and genotypic levels carbapenem resistance in K. pneumoniae and P. aeruginosa and to determine the prevalence of persister cells in K. pneumonia. Methods: Sixty-one clinical P. aeruginosa isolates were screened for their susceptibility against meropenem, imipenem, colistin and ceftolozane/tazobactam, and 55 of these isolates were screened against meropenem with EDTA, meropenem with avibactam and meropenem with PaβN. Furthermore, induction of resistance was carried out for K. pneumoniae ATCC 13883, P. aeruginosa ATCC 10145 & 27583, and PAN14 strains against piperacillin/tazobactam, ceftolozane/tazobactam and meropenem as well as avibactam against K. pneumoniae ATCC 13883 only. Each strain was subcultured separately on agar plates supplemented with increasing concentration of each antibiotic alone. Antibiotic susceptibility testing was then performed on the highest targeted concentration of each strain against several antibiotics to determine the minimal inhibitory concentration towards each antibiotic. In addition, the presence of persister cells among K. pneumoniae ATCC 13883 and K. pneumoniae ATCC 13883 induced against 4 μg/mL meropenem was analyzed using concentration-dependent kill curves. Moreover, PCR was done on a panel of carabapenemase encoding genes. Results: Antimicrobial susceptibility testing results on the 61 clinical P. aeruginosa isolatesshowed that 58 isolates were resistant to imipenem, 52 to meropenem, 44 to 3 ceftolozane/tazobactam, and all the isolates were susceptible to colistin. Moreover, induction of resistance in P. aeruginosa ATCC isolates, reflected that P. aeruginosa was able to develop resistance to carbapenems and piperacillin/tazobactm faster than ceftolozane/tazobactam by induction against increasing concentrations reaching the breakpoint, 4-fold the breakpoint and 16-fold the breakpoint respectively. On the other hand, induction of resistance on K. pneumoniae ATCC 13883, showed that K. pneumoniae was capable of developing resistance against piperacillin/tazobactm and ceftolozane/tazobactam by induction against increasing concentrations reaching the breakpoint and 6-fold the breakpoint respectively. Moreover, exposure of K. pneumoniae to increasing concentrations of meropenem induces the formation of persister cells. PCR results revealed the presence of blaOXA, blaVIM, and blaIMP genes. blaNDM encoding gene was not detected. Conclusion: Resistance to carbapenems in P. aeruginosa is caused through the overexpression of efflux pumps, and detection of blaOXA, blaVIM, and blaIMP carbapenemases encoding genes. Moreover,K. pneumoniae showed its ability to resist carbapenemsthrough the formation of persister cells.