Phenotypic and genetic screening of Klebsiella pneumoniae isolates from human UTI patients for beta-lactamases and their genetic diversity analysis by ERIC and REP PCRs

Abstract

Klebsiella pneumoniae is one of the major nosocomial pathogens responsible for pneumoniae, septicaemia, liver abscesses, and urinary tract infections. Coordinated efforts by antibiotic stewardship and clinicians are underway to curtail the emergence of antibiotic-resistant strains. The objective of the present study is to characterize K. pneumoniae strains through antibiotic resistance screening for production of beta-lactamases (β-lactamases) such as extended spectrum beta lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by phenotypic and genotypic methods and genetic fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and repetitive element palindromic PCR (REP-PCR). A total of 85 K. pneumoniae strains isolated from 504 human urinary tract infections (UTI) were used in this study. Only 76 isolates showed positive in phenotypic screening test (PST), while combination disc method (CDM) as phenotypic confirmatory test (PCT) confirmed 72 isolates as ESBL producers. One or more β-lactamase genes were detected by PCR in 66 isolates (91.66%, 66/72) with bla_TEM gene being the most predominant (75.75%, 50/66). AmpC genes could be detected in 21 isolates (31.8%, 21/66) with FOX gene being the predominant (24.24%, 16/66), whereas NDM-I was detected in a single strain (1.51%, 1/66). Genetic fingerprinting using ERIC-PCR and REP-PCR revealed wide heterogeneity among β-lactamase producing isolates with discriminatory power of 0.9995 and 1, respectively.

Keywords: AmpC β-lactamases; ERIC-PCR; ESBL; Klebsiella pneumonia; NDM-1; REP-PCR.

Fig. 1

A Phenotypic screening test for detection of ESBL production. B Phenotypic confirmation test for confirmation of ESBL production. C Ezy strip test for detection of metallo-β-lactamases

Fig. 2

A Molecular detection of ESBL genes (blaTEM, blaSHV, and blaOXA) in Klebsiella pneumoniae. B Molecular detection of ESBL genes (blaCTX-M1, bla-CTXM2, and blaCTX-M9) in Klebsiella pneumoniae. C Positive rate of ESBL (blaTEM, blaSHV, blaOXA genes and blaCTX-M group genes) genes in percentage. The number of isolates detected positive for ESBL genes by PCR are as follows: TEM — 36/66; CTX-M1 — 8/66; OXA — 7/66; TEM and OXA — 6/66; SHV — 5/66; TEM and SHV — 4/66; TEM, SHV, and OXA — 4/66; CTX-M2 — 2/66; SHV and OXA — 1/66

Fig. 3

A Molecular detection of AmpC genes (FOX, EBC, ACC, DHA, and CIT) in Klebsiella pneumoniae. B Molecular detection of VIM gene (390 bp) and NDM-1 gene (621 bp). C Positive rate of AmpC and carbapenemase genes in percentage. The number of isolates detected positive for AmpC beta lactamase genes by PCR are as follows: FOX and CIT — 11/66; FOX — 5/66; CIT — 4/66; DHA — 1/66; NDM-1 — 1/66

Fig. 4

DNA fingerprints of ESBL producing K. pneumoniae isolates generated by REP-PCR. Dendrogram resulting from analysis of REP-PCR profiles from 66 K. pneumoniae isolates. Neighbour joining (NJ) tree was constructed using PHYLIP version 3.6 program. Strains shown in same colour are isolates having similar band patterns and thus cluster together in phylogenetic tree

Fig. 5

DNA fingerprints of ESBL producing K. pneumoniae isolates generated by ERIC-PCR. Dendrogram resulting from analysis of ERIC-PCR profiles from 66 K. pneumoniae isolates. Neighbour joining (NJ) tree was constructed using PHYLIP version 3.6 program. Strains shown in same colour are isolates having similar band patterns and thus cluster together in phylogenetic tree