Detection and molecular characterization of multi-drug resistant nonfermentative Gram-negative bacteria from clinical isolates

Abstract

The increasing rate of carbapenem resistant nonfermentative Gram-negative bacteria is a serious global health threat. Almost all studied isolates of Pseudomonas aeruginosa and Acinetobacter baumannii showed resistance to carbapenem. Carbapenem is the last option for treating Multi-drug resistant (MDR) P. aeruginosa and A. Baumannii but resistance against this drug has been emerging. Two types of β-lactamases differentiated by their mode of target hydrolysis, serine β-lactamase and metallo-β-lactamase (MBL). These are major foci of the present study in nonfermentative Gram-negative isolates of P. aeruginosa and A. baumannii. When studying various virulence properties, a majority of both P. aeruginosa and A. baumannii isolates showed hemolytic activity. However, formation of capsule, invasiveness and cytotoxic activity were observed only in the case of A. baumannii. Among the 100 P. aeruginosa and A. baumannii isolates tested, 92% and 35% showed MBL positive, respectively. Verona Integron-encoded metallo-β-lactamase (VIM) is an important MBL which confers resistance to regular using antimicrobials and now a days carbapenems became resistant due to this enzyme. We obtained a total of 16 VIM encoded MBLs from 100 P. aeruginosa isolates, but none from a similar number of A. baumannii strains. The sequences of VIM gene from 16 strains together with 22 other similar sequences, retrieved from the NCBI GenBank database were used to construct the phylogenetic tree to understand the nearest neighbour of the study sequences. Genetic divergence and homogeneity of the sequences are apparent in the phylogenetic tree. For VIM type of MBL, P. aeruginosa phylogenetic analysis showed similarities with the strains of India, Thailand, Nepal, Egypt, Turkey, UK, USA and Tunisia (93-100%). These findings suggest that the blaVIM gene is similar to global circulating strains. From Bootstrap table it has been found that 90-100% nucleotide similarities exist among the 16 Bangladeshi strains. Comparable results of nucleotide and amino acid sequences of VIM showed differences with a reference strain retrieved from the NCBI for 4 Bangladeshi P. aeruginosa strains and among the changes, three were in important positions (72, 145 and 146) as these changes brought significant modification in amino acid sequences from polar/nonpolar to nonpolar/polar that might affect the enzymatic nature of VIM. We obtained 6 New Delhi Metallo-β-lactamase-1(NDM-1) positive isolates from 100 A. baumannii strains, but none from the same number of P. aeruginosa strains. Among these 6 blaNDM-1 genes, 5 sequences together with 17 other similar sequences retrieved from the NCBI GenBank database were used to construct the phylogenetic tree to understand the nearest neighbor of the study sequences. Genetic divergence and homogeneity of the sequences are apparent in the phylogenetic tree. Amongst the DNA sequences obtained in our study, the isolates MN226842, MN226843, MN226844 and MN226846 formed distinct lineages with the strains from India, while only isolate MN226845 shared similarity and was found to be closely related to the sequences with Iran, Egypt and Korea. From these findings, we can predict that the NDM-1 may have been transmitted in Bangladesh from India. Bootstrap table shows 100% nucleotide similarities among the 5 Bangladeshi strains. On the other hand, 10 strains from China showed 52% similarity. Comparable results of NDM-1 of A. baumannii showed no differences in nucleotide and amino acid sequences with a reference strain retrieved from the NCBI which suggest that these are clonal. The study reports on the presence of blaVIM and blaNDM-1 gene in nonfermentative Gram-negative bacteria in Bangladesh for the first time.