Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Klebsiella spp. Isolated at the National Reference Laboratory, Kathmandu, Nepal

Abstract

Klebsiella spp. are associated with several nosocomial and opportunistic infections. Increasing antimicrobial resistance of Klebsiella species is aggravated by a number of intrinsic and extrinsic factors. The main aim of this study is to determine antimicrobial resistance due to production of β-lactamase enzymes, extended spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC and Klebsiella pneumoniae carbapenemase (KPC) and biofilm formation in Klebsiella isolates. A total of 2197 non-duplicate specimens of urine, sputum and pus were obtained from the National Public Health Laboratory (NPHL), Kathmandu, Nepal, between February and August 2019. Klebsiella species were isolated, identified and screened for antimicrobial susceptibility testing with the disk diffusion method. Phenotypic detection of ESBL, MBL, KPC and AmpC production was observed and biofilm production was detected by the microtiter plate method. Out of a total of 2197 clinical specimens, bacterial growth was detected in 8% (175/2197) of the specimens. Of the total isolates, 86.3% (151/175) were Gram-negative bacteria and 37.7% (57/151) were Klebsiella spp. Of the total Klebsiella spp., 56% (32/57) were multi drug resistant (MDR), 16% (9/57) were ESBL, 26% (15/57) were MBL, 4% (2/57) were KPC (class A carbapenemase), 16% (9/57) were AmpC producers and 95% (54/57) were biofilm producers. Gentamicin was the most effective antibiotic, followed by cotrimoxazole, as 68% (39/57) and 47% (27/57) of the Klebsiella isolates were susceptible towards these drugs, respectively. The study results show evidence of β-lactamase production, high prevalence of MDR and biofilm producing Klebsiella species. Integrating the test parameters for phenotypic confirmation of ESBL, MBL, AmpC β lactamase and KPC in routine diagnostic procedures can help in the early detection and management of these resistant strains.

Detection of NDM Variants (blaNDM-1, blaNDM-2, blaNDM-3) from Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae: First Report from Nepal

“…More specifically, Enterobacterales like E. coli and K. pneumoniae are the chief agents isolated from infections mediated by GNB. 39 Although recognized for irreplaceable choice in bacterial infections, antibiotics are losing their efficacy day by day, leading the clinical world to the fear of “no antibiotic era”. 40 Next to irrational practices of prescription and use of antibiotics, acquisition and transfer of resistant genes (responsible for expression of resistant enzymes) from multiple reservoirs of pathogenic bacteria, environment, and animals are recommended as the chief cause of an unabated spread of AMR worldwide.…”

“… 75 In general, production of MBL in Enterobacteriaceae isolates currently follows an increasing prevalence pattern and the prevalence rate may vary greatly within and between geographical locations. The increasing prevalence of ESBL-producing pathogens in Nepal 4 , 39 , 44 , 51 , 76 , 77 coupled with a higher frequency of prescription and use of carbapenem antibiotics may be the driving factors behind the increasing incidence of carbapenemase-producing organisms.…”

Antibiotic Susceptibility, Biofilm Production, and Detection of mecA Gene among Staphylococcus aureus Isolates from Different Clinical Specimens

“…Screening of biofilm-producing S. aureus was done using tissue culture plate method (TCP). This quantitative test was performed as described elsewhere [ 22 , 23 , 24 ]. Briefly, a loopful of test organism was isolated from a fresh agar plate and inoculated in 2 mL of tryptic soya broth (TSB).…”

Beta-lactamase determinants and molecular typing of carbapenem-resistant classic and hypervirulent Klebsiella pneumoniae clinical isolates from southwest of Iran

“…Subsequently, the inoculated plates were incubated for 24 h at 37°C. The isolates were confirmed as AmpC producers when the zone diameter of cefoxitin/PBA increased by ≥5 mm compared to cefoxitin alone ( Kuinkel et al, 2021 ).…”

“…The cefoxitin disk and cefoxitin/PBA disk were dispensed onto an MHA plate that had already been inoculated with the test isolate (equivalent to 0.5 McFarland) by the standard disk diffusion method. Subsequently, the inoculated plates were incubated for 24 h at 37 • C. The isolates were confirmed as AmpC producers when the zone diameter of cefoxitin/PBA increased by ≥5 mm compared to cefoxitin alone (Kuinkel et al, 2021).…”