Prevalence and Molecular Characterization of AmpC B-Lactamases Genes in Escherichia coli and Klebsiella pneumoniae from Clinical Isolates

Abstract

Plasmid mediated AmpC beta-lactamases (pAmpC) contribute significantly to confer resistance to broad-spectrum antibiotics among Gram-negative bacteria especially Enterobacteriaceae. Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are common pathogens causing nosocomial infections including blood stream and urinary tract infections. Enhanced production of drug resistance enzymes like, pAmpC and ESBL among these pathogens limit the efficacy of available therapeutic agents. The ultimate aim of the undertaken study was to determine the molecular prevalence, genetic characterization and diversity of pAmpC producing E. coli and K. pneumoniae collected during 2013 to 2015 from Tertiary Care Hospital Lahore. Pakistan. A total of 17500 clinical specimens were collected and processed for culture and biochemical identification. Out of 5125 isolates, 2013 (39.3%) were E. coli and K. pneumoniae. Among these 2013 isolates, E. co.li were 24% (n=1230) and K. pneumoniae were 15.3% (n=783). The antibiogram profile reflected that 1500 (74%) were resistant to cefotaxime and ceftazidime. Furthermore, 900 (60%) cefoxitin resistant isolates were investigated for pAmpC production. Out of these 293 isolates were pAmpC producers by phenyl boronic acid method. All phenotypically detected pAmpC E. coli isolates (n=75) exhibited resistance (100%) against ceftazidime, cefotaxime, cefuroxime and cefoxitin, followed by (n=74; 98.7%) cefixime, (n=73; 97.3%) co-amoxiclav and ceftriaxone each, moxifloxacin (n=69; 92%), gentamicin (n=66; 88%), ciprofloxacin (n=60; 80%) and levofloxacin (n=58; 77.3%). Only 53% (n=40) E. coli isolates were resistant to pipracillin-tazobactam. Relatively less resistance was observed against amikacin (n= 36; 48%), meropenem (n=33; 44%) and sulbactam-cefoperazone (n=26; 34.7%) whereas only 33.3% (n=25) isolates were resistant to cefepime and 26.7% (n=20) resistant to imipenem. No isolate displayed Summary 2 resistance against colistin of both E. coli and K. pneumoniae. In K. pneumoniae isolates, resistance (n=138; 100%) was observed against co-amoxiclav, ceftazidime, ceftriaxone, cefotaxime, cefuroxime and cefoxitin, followed by cefixime (n=132; 95.4%), gentamicin and levofloxacin (n=127; 91.7%) each, moxifloxacin (n=126; 90.8%) ciprofloxacin (n= 120; 86.7%) pipracillin-tazobactam 59.2% (n=82). cefepime (n= 81; 58.3%), amikacin (n=76; 55%), sulbactam-cefoperazone (n=74; 53.2%), meropenem (n=36; 47.4%) and imipenem (n=48; 34.4%). K. pneumoniae displayed relatively higher resistance than E. coli. Among pAmpC producing E. coli strains, detected by inhibitory based method, 88% (n=258) of the samples were obtained from the patients of age up to 15 years. Blood samples yielded relatively high number of pAmpC producing organisms (48%). Among all positive pAmpC samples, 42.7% were of male patients and 57.3% were of female patients. Among pAmpC producing K. pneumoniae confirmed by Inhibitory based method, 90.4% recovered from the patients of age group of up to 15 years, 45.9% recovered from blood samples. Among all pAmpC positive samples, 60.6% were of male patients and 39.4% were of female patients. Only153 were phenotypically confirmed ESBL producers among all 293 pAmpC positive isolates. Interestingly, high incidence of pAmpC beta-lactamases producing isolates was observed from June to September. Furthermore, some selective pAmpC producing isolates identified by phenotypic tests was further conformed by16S rRNA sequencing. Genotyping results revealed ACC, MOX and FOX were not detected in these samples. AmpC genes amplification results revealed that 63.70% isolates had active CIT, 14.70% isolates had active EBC, 4.70% isolates had active DHA; 6.30% isolates had active combined CIT and DHA, and 10.50% isolates had active combined CIT and EBC genes. Isolated pAmpC genes exhibited 99-100% homology with sequence of pAmpC genes already reported across globally. However, some mutations Summary 3 were observed in CIT against CMY-146 and CMY-42, in DHA against DHA-1 and DHA-2 and in EBC against MIR-1and MIR-4 genes indicating some possible amino acid substitutions in more than one position of these genes. Prevalence of pAmpC producing K. pneumoniae isolates 18% (n=138) is higher than pAmpC producing isolates of E. coli in Lahore. The undertaken study will significantly contribute to estimate the occurrence of pAmpC genes in bacteria recovered from different clinical specimens from patients in Pakistan. The data will also help the infection preventionists to take appropriate infection control measures to reduce the spread of such highly resistant bugs in the hospitals.