Genetic Characterization of blaSHV/VEB/PER Genes in ESBL-producing MDR Klebsiella Pneumonia Strains Isolated from Patients in Isfahan, Iran

Abstract

This study was conducted to detect three genetical variants of Extended-Spectrum Beta-Lactamase (ESBL, in which142 Klebsiella pneumoniae (K.pneumoniae) isolates were collected from sections of a teaching hospital in Isfahan and were detected using standard IMVIC biochemical tests and urease. These were confirmed by identification of the ureD gene. Antimicrobial susceptibility testing was performed using the standard Kirby-Bauer disk-diffusion method on Mueller-Hinton agar (Merck,Germany) .The performance and interpretation were based on the guidelines from the Clinical Laboratory Standards Institute (CLSI, 2013). Screening and phenotypic identification of ESBLs isolates were performed by DDST. The presence of genes responsible for ESBL resistance, such as SHV, PER and VEB type ESBL genes was identified by PCR and indicator isolates sequencing performed by Macrogen (Seoul, Korea). The nucleotide sequences were analysed using the BLAST program (http://www.ncbi.nlm.nih.gov/BLAST), the Lahey database, and CromasPro-2 and Mega-4 software to determine the subvarients of the three variants of ESBL (SHV, PER, VEB). These were compared with blaSHV-11 gene from K. pneumonia (accession.no.X98101), blaSHV-5 gene from K. pneumonia (accession no. X55640), blaPER-1 gene from P.aeruginosa transposed on Tn2345 (accession no AY866517.2) and blaVEB-1 gene from K. pneumonia (accession no. AF010416). A total of 120 isolates (84%) were recognized as MDR. The highest rate of resistance was recorded for piperacillin (80%), ceftazidime (76%), and cefotaxime (73%) and the lowest rate was for ertapenem (47.3%), meropenem (50.8%), and imipenem (58.7%) following detection of ESBL isolates of K.pneumoniae (101 isolates; 71%).The ward and the clinical specimen with the most prevalence were ICU with 55(38.7%) and urine with 61(42.9%). The lowest prevalence was related to the neurosurgery ward with 8(5.6%) samples and the clinical specimen with the lowest prevalence was cerebrospinal fluid (CSF) with 2 (1.4%) samples. PCR detection in ESBL-producing K. pneumoniae showed that, of the clinical isolates, 42.2% contained blaSHV (42/101), 2.9% contained blaVEB (3/101) and2% contained blaPER (2/101). Sequencing of 10 selected PCR products of SHV genes showed that 7/10isolates were similar to the strain SHV-11 and 3/7 isolates were similar to the strain SHV-5. The sequencing of two PCR products of the PER genes showed they were similar to the strainPER-1.Sequencing of three PCR products of the VEB genes showed they were similar to the strain VEB-1. The overall prevalence of ESBL-producers was found to vary greatly in different geographical areas; this may be the result of differences in the type and amount of antibiotics consumed and differences in the time of collection of isolates. The present study reflects anincrease in the prevalence of ESBL-producers in Iran. The most common ESBL type found in this study was SHVand that VEB and PER types were rare. In addition, sequence analysis results of our study show the rate of SHV-11, PER-1 and VEB-1was maximum.