ISOLATION AND IDENTIFICATION OF CLASS 1, 2, AND 3 INTEGRONS IN MULTI-DRUG RESISTANT UROPATHOGENIC Klebsiella pneumoniae FROM PATIENTS ATTENDING TERTIARY HOSPITALS IN SOUTH WEST, NIGERIA

Abstract

ABSTRACT Integrons play an outstandingrole in the evolution and dissemination of antibiotic resistant genes especially among uropathogenicKlebsiella pneumoniae. However, the prevalence of extended spectrum beta-lactamases (ESBL) or Metallo-beta-lactamases (MBL)-producing Klebsiella pneumoniae are increasingly becoming health care concern. This study aimed to characterize three classes of integrons identified among Multi-drug Resistant (MDR) uropathogenicKlebsiella pneumoniae isolated from patients visiting two tertiary hospitals in Southwest, Nigeria. Two hundred and fifty-three suspected Klebsiella pneumoniae isolated from the urine benches of University College Hospital (UCH), Ibadan (205) and Federal Medical Centre (FMC), Abeokuta (48) were examined. Isolates were identified using Analytical Profile Index 20E, and 16S rRNA gene sequencing. Susceptibility of isolates to 14 antibiotic discs under 8 classes was determined using disc diffusion method. MDR was based on resistance to ≥3 classes of antibiotics. 10 isolates with resistant to ≥3 classes of antibiotics were selected for molecular studies. The Clinical laboratory standard institute confirmation test was used for the evaluation of ESBL or MBL production. Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration were ascertained using microdilution technique. The DNA of the 10 MDR Klebsiella pneumoniae (MDRKP) was extracted using ZYMO research miniprep kit. Presence of blaIMPorblaCTX-M genes, class 1, 2, and 3 integrons, and the content of conserved segment (CS) were amplified using polymerase chain reaction. A total of 194 (95%) isolates from UCH and 10 (5%) from FMC were presumptively identified as Klebsiella pneumoniae by API 20E. Similarly, 88 (97.8%) from UCH, and 2 (2.2%) from FMC were putatively confirmed as Klebsiella pneumoniae by 16S rRNA gene sequencing. While 86.7% of the isolates were susceptible to colistin, 71.5% and 59.8% were sensitive to imipenem and cefoxitin respectively. High resistant phenotype was recorded against ampicillin (97.3%), amoxicillin clavulanate (82.4%), cephalothin (80.1%), cefotaxime (76.6%), sulfamethazine (73.4%) and ceftazidime (71.1%). The ESBL production was identified in 42.2% and MBL in 57.8% of the positive isolates in 57.8%. The MIC value for both colistin and imipenem ranged from 8-32 µg/mL, while cefotaxime value ranged from 4-64 µg/ml. All the isolates harboured blaIMPgroup while 87.5% carried blaCTX-M. Class 1, 2 integrons were detected in 87.5% strain while 25% harboured class 3 integrons. The CS of class 1, 2 and 3 integrons revealed 7 different arrays of antibiotic resistant genes (dfrA5, dfrA30; aadA1, dfrA1-sat1; dfrA1-sat1; dfrA5, dfrA30; aadA2; aadA2, dfrA12 and dfrA5, dfrA30, aadA2, aadA2, dfrA12). This study revealed high prevalence of class 1 and 2 integrons carrying gene cassettes and the presence of class 3 integrons among multi-drug resistant Klebsiella pneumoniae harbouring blaCTX-M and blaIMP genes in the studied tertiary hospitals in Nigeria.