Drug resistance phenotype analysis and mechanism investigation of extensively drug resistant Klebsiella pneumoniae / 中华检验医学杂志

ABSTRACT

Objective:This work aims to investigate the phenotype-characteristics of drug resistance and the possible mechanisms of extensively drug-resistance Klebsiella pneumoniae(XDRKP). Methods:Screened by the previous drug susceptibility results, 116 clinical Klebsiella pneumoniae isolates were collected from Shanxi Bethune Hospital from January 2018 to December 2020. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) rapid microbial identification system and VITEK-compact 2 were used. The modified carbapenem inactivation method (mCIM) combining with EDTA carbapenem inactivation method (eCIM) was used to identify the strains′ carbapenemase phenotypes, which were compared with subsequent qPCR results. The qPCR amplification combining with agarose gel electrophoresis were carried out to detect various drug-resistant related genes, including: carbapenemase genes: blaKPC, blaNDM, blaVIM, blaIMP, blaOXA; aminoglycosides resistance genes: ① 16S rRNA methylase genes: rmtA, rmtC, rmtD, rmtG, rmtH, armA, npmA, rmtB, rmtE, rmtF, ② variant of aminoglycosides acetyltransferase gene: aac(6′)-Ib-cr; quinolone resistance genes: DNA gyrase protection protein qnr family: qnrA, qnrB, qnrC, qnrD, qnrS, efflux pump protein gene: oqxAB, qepA, variant of aminoglycoside acetyltransferase gene: aac(6′)-Ib-cr; and tigecycline-resistant Tet protein genes: efflux pump protein gene: tet (A), tet (L), ribosome protection protein gene: tet (M), tigecycline modified enzyme gene: tet (X). Each isolate′s phenotype and resistance gene result were compared and analyzed correspondingly. Results:A total number of 116 XDRKP isolates were collected in 3 years, 115 of which are identified as carbapenem resistant. Both cephalosporins and quinolones resistant rate were 100%, while the resistant rate of aminoglycosides antibiotic gentamicin, tobramycin and amikacin was 95.69% (111/116), 94.83% (110/116), or 88.79% (103/116) respectively. Sulfonamide antibiotics and tigecycline showed a relatively lower resistant rate. Compared with PCR amplification results, mCIM combining with eCIM phenotype testing had a high conformity, up to 95.65% (110/115). Positive rate of each resistance related gene was: blaKPC 90.52% (105/116), blaNDM 10.34% (12/116), rmtB 81.90% (95/116), armA 2.59% (3/116), oqxAB 65.52% (76/116), qnrB 6.03% (7/116), qnrS 12.93% (15/116), aac(6′)-Ib-cr 7.76% (9/116), or tet(A) 21.55% (25/116), respectively. Other resistance related genes were not detected. Corresponding analysis between the resistant phenotypes and resistance related genes indicated that a total of 65 XDRKP didn′t have a matched pairs, i.e. bacteria′s resistance to specific antibiotic could not be interpreted by carrying some associated resistant genes.Conclusions:The wide distribution of resistant genes and multiple-antibiotic-inactivated trait of some genes(such as aac(6′)-Ib-cr and oqxAB) in XDRKP are potential causes of the generation of extensively drug resistant phenotype. Different XDRKP isolates may carry one or more resistant genes in responding to specific antibiotic. In addition, there are some bacteria with an unmatched phenotype-gene feature indicating that both resistance genes′ regulation and some other mechanisms also play a role in development of XDR.