Genotypic study of the association between CRISPR/Cas system and antimicrobial resistance in Klebsiella pneumoniae (RSPM2.1)

Abstract

ABSTRACT CRISPR-Cas system is a recently discovered genomic engineering tool with the potential for knocking-out and knocking-in sequence-specific DNA targets. CRISPR is an abbreviation for Clustered regularly interspaced short palindromic repeat while Cas stands for CRISPR-associated enzymes. CRISPR/Cas has been originally known as a bacterial adaptive immune system utilized by host microbes for fighting foreign DNA invaders such as phages and plasmids. It has also been linked to higher susceptibility to antimicrobial agents in Enterococcus species as well as in Escherichia coli. Recent reports extending such findings to Klebsiella pneumoniae has been also published. This presents CRISPR/Cas as a potential solution for antimicrobial resistance. K. pneumoniae is a highly problematic species known for multidrug resistance and hypervirulence of some strains. The current study aimed at characterizing CRISPR/Cas systems in K. pneumoniae and their potential impact on acquisition of antimicrobial resistance. For this purpose, a collection of 46 K. pneumoniae isolates were tested for antimicrobial susceptibility as well as harboring CRISPR/Cas genes. Susceptibility profiles were analyzed using disk diffusion test and CRISPR/Cas genes were amplified using Polymerase Chain Reaction (PCR). The analysis revealed susceptibility to higher number of antimicrobials among the group of isolates carrying Cas genes compared to the Cas-negative isolates. As only five isolates were found to carry Cas genes, such findings were not considered as conclusive and a large scale bioinformatic analysis was done on a sample of K. pneumoniae genomes retrieved from the NCBI database. A total of 337 genomes were scanned for CRISPR/Cas genes which were identified in only 20% of the studied genomes. MLST analysis of the CRISPR/Cas positive isolates revealed that CRISPR/Cas systems were disseminated in different sequence types most commonly in ST23, a well-known multidrug resistant hypervirulent ST. Surprisingly, CRISPR/Cas-positive strains were found to carry larger number of resistance plasmids compared to others. In conclusion, CRISPR/Cas systems are disseminated with low prevalence in various sequence types and correlation to antimicrobial susceptibility is questionable. Further studies are required to investigate the role of CRISPR/Cas in acquisition of resistance by K. pneumoniae. A larger number of isolates should be phenotypically and genotypically analyzed.