Experience of using MALDI TOF minisequencing for studing TEM beta-lactamases in K. pneumoniae strains circulating in pediatric hospitals

Abstract

Study of the nature of K. pneumoniae antibiotic resistance and analysis of the structure of extended-spectrum β-lactamases (ESBL) are one of the most important aspects of modern clinical microbiology and epidemiology. The issue of studying the gene context of these enzymes in order to search for point mutations that lead to ESBL formation is urgent. Research objective : to analyze and search for SNPs in nucleotide sequences of genes encoding TEM β-lactamases in K. pneumoniae strains using MALDI TOF minisequencing. Materials and methods: bla TEM genes of 36 K. pneumoniae strains isolated during episodes of epidemic trouble in pediatric hospital settings were studied. The strains were identified using an Autoflex MALDI TOF mass spectrometer. DNA isolation was performed using a DNA-express kit, bla TEM genes were detected by PCR. The minisequencing reaction for SNP detection was performed using oligonucleotide probes described in the academic literature. The spectral data were taken using an Autoflex MALDI TOF mass spectrometer and FlexControl software in the manual mode. Results. In the course of the work it was found that SNPs in the bla TEM gene sequence at positions 104, 240 and 238 and those ones leading to the substitution of glutamic acid by lysine – Glu104Lys, Glu240Lys, as well as glycine substitution by serine – Gly238Ser were absent in all studied strains. In 67% strains, a mutation was found that led to alanine substitution by threonine at position 237 (Ala237Thr), which led to a change in the proteins substrate specificity and to formation of ESBLs resistant to inhibitors. Conclusions. In 67% strains, single nucleotide substitutions were found, leading to a change in the enzyme substrate specificity and to formation of ESBLs. This confirms the need to monitor the frequency of significant mutations in antibiotic resistance genes in hospital strains in order to ensure epidemic forecasting.