CRISPR-Cas System in Antibiotic Resistance Plasmids in Klebsiella pneumoniae.

Muhammad Kamruzzaman,Jonathan R Iredell

doi:10.3389/fmicb.2019.02934

Abstract

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is a microbial adaptive immune system involved in defense against different types of mobile genetic elements. CRISPR-Cas systems are usually found in bacterial and archaeal chromosomes but have also been reported in bacteriophage genomes and in a few mega-plasmids. Klebsiella pneumoniae is an important member of the Enterobacteriaceae with which they share a huge pool of antibiotic resistance genes, mostly via plasmids. CRISPR-Cas systems have been identified in K. pneumoniae chromosomes, but relatively little is known of CRISPR-Cas in the plasmids resident in this species. In this study, we searched for CRISPR-Cas system in 699 complete plasmid sequences (>50-kb) and 217 complete chromosomal sequences of K. pneumoniae from GenBank and analyzed the CRISPR-Cas systems and CRISPR spacers found in plasmids and chromosomes. We found a putative CRISPR-Cas system in the 44 plasmids from Klebsiella species and GenBank search also identified the identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmid and chromosome sequences. 45 of 47 plasmids with putative type IV CRISPR had IncFIB replicon and 36 of them had an additional IncHI1B replicon. All plasmids except two are very large (>200 kb) and half of them carried multiple antibiotic resistance genes including blaCTX–M, blaNDM, blaOXA. To our knowledge, this is the first report of multi drug resistance plasmids from Enterobacteriaceae with their own CRISPR-Cas system and it is possible that the plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence. Both chromosomal and plasmid CRISPRs target a large variety of plasmids from this species, further suggesting key roles in the epidemiology of large plasmids.

Highlights

Acquisition of genetic material including virulence, fitness and antibiotic resistance genes by horizontal gene transfer (HGT) is an essential process in bacterial adaptation to different environments (Frost et al, 2005)
In this study we have identified a putative type IV CRISPR-Cas system in the plasmids of K. pneumoniae
Type IV CRISPR-Cas was previously identified in a megaplasmid of Aromatoleum aromaticum species, an aromaticdegrading β-proteobacteria found in freshwater and soils

Summary

Introduction

Acquisition of genetic material including virulence, fitness and antibiotic resistance genes by horizontal gene transfer (HGT) is an essential process in bacterial adaptation to different environments (Frost et al, 2005). In an initial adaptation step foreign DNA fragments (protospacers) from infecting bacteriophages and plasmids are incorporated into the CRISPR array as new spacers. These spacers provide the sequence specific memory for a targeted defense against subsequent invasions by the same bacteriophage or plasmid. After expression of the array, mature crRNAs, aided by Cas proteins, identify specific targets and cleave the nucleic acid strands of corresponding viruses or plasmids (van der Oost et al, 2009; Garneau et al, 2010; Makarova et al, 2011b, 2015; Barrangou, 2015)

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