رسائل ماجيستير
English
2021
Carbapenemases, Their Inhibitors, And Persister Cells In Mdr And Xdr Gramnegative Bacteria At Aubmc
Antoine Abou Fayad, Ghassan Matar , Hanin Sami Banna
كلية الطب-الجامعة الأمريكية في بيروت · لبنان
Antimicrobial resistance is posing an increasing risk to public health dueto the overuse, abuse and miss-use of the available agents, especially with multi-drugresistant (MDR) and extensive-drug resistant (XDR) Gram-negative bacilli. Accordingto the World Health Organization (WHO), Pseudomonas aeruginosa and Klebsiellapneumoniae fall in the critical priority group’s target of new antibiotics, consideringtheir increased resistance to carbapenems, one of the last resort drugs available. TheseGram-negative bacilli mainly develop resistance to carbapenems by hydrolyticcarbapenemase enzymes, downregulation of porin channels, and upregulation of effluxpumps or modification of carbapenem’s molecular target (penicillin-binding protein;PBP). Moreover, resistance in P. aeruginosa and K. pneumoniae is currently extendingto affect carbapenemase inhibitors too. Furthermore, P. aeruginosa and K. pneumoniaepopulations might survive under stressful environments due to the presence of persistercells which are genetically identical to the susceptible cells but metabolically dormant.Aim: Carbapenems resistance in K. pneumoniae and P. aeruginosa occurs throughtarget modification, expression of carbapenemases or overexpression of efflux pumps.The aim of this study was to assess at the phenotypic and genotypic levels carbapenemresistance in K. pneumoniae and P. aeruginosa and to determine the prevalence ofpersister cells in K. pneumonia.Methods: Sixty-one clinical P. aeruginosa isolates were screened for their susceptibilityagainst meropenem, imipenem, colistin and ceftolozane/tazobactam, and 55 of theseisolates were screened against meropenem with EDTA, meropenem with avibactam andmeropenem with PaβN. Furthermore, induction of resistance was carried out for K.pneumoniae ATCC 13883, P. aeruginosa ATCC 10145 & 27583, and PAN14 strainsagainst piperacillin/tazobactam, ceftolozane/tazobactam and meropenem as well asavibactam against K. pneumoniae ATCC 13883 only. Each strain was subculturedseparately on agar plates supplemented with increasing concentration of each antibioticalone. Antibiotic susceptibility testing was then performed on the highest targetedconcentration of each strain against several antibiotics to determine the minimalinhibitory concentration towards each antibiotic. In addition, the presence of persistercells among K. pneumoniae ATCC 13883 and K. pneumoniae ATCC 13883 inducedagainst 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. aeruginosaisolatesshowed that 58 isolates were resistant to imipenem, 52 to meropenem, 44 to3ceftolozane/tazobactam, and all the isolates were susceptible to colistin. Moreover,induction of resistance in P. aeruginosa ATCC isolates, reflected that P. aeruginosa wasable to develop resistance to carbapenems and piperacillin/tazobactm faster thanceftolozane/tazobactam by induction against increasing concentrations reaching thebreakpoint, 4-fold the breakpoint and 16-fold the breakpoint respectively. On the otherhand, induction of resistance on K. pneumoniae ATCC 13883, showed that K.pneumoniae was capable of developing resistance against piperacillin/tazobactm andceftolozane/tazobactam by induction against increasing concentrations reaching thebreakpoint and 6-fold the breakpoint respectively. Moreover, exposure of K.pneumoniae to increasing concentrations of meropenem induces the formation ofpersister 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 theoverexpression of efflux pumps, and detection of blaOXA, blaVIM, and blaIMPcarbapenemases encoding genes. Moreover,K. pneumoniae showed its ability to resistcarbapenemsthrough the formation of persister cells.