Characterization of CRISPR-Cas Systems in Clinical Klebsiella pneumoniae Isolates Uncovers Its Potential Association With Antibiotic Susceptibility

Abstract

Prokaryotic CRISPR-Cas systems limit the acquisition of genetic elements and provide immunity against invasive bacteriophage. The characteristics of CRISPR-Cas systems in clinical <i>Klebsiella pneumoniae</i> isolates are still unknown. Here, 97 <i>K. pneumoniae</i> genomes retrieved from the Integrated Microbial Genomes & Microbiomes genome database and 176 clinical isolates obtained from patients with bloodstream (BSI, <i>n</i> = 87) or urinary tract infections (UTI, <i>n</i> = 89) in Taiwan, were used for analysis. Forty out of ninety-seven genomes (41.2%) had CRISPR-Cas systems identified by the combination of CRISPRFinder and <i>cas1</i> gene sequence alignment. The phylogenetic trees revealed that CRISPR-Cas systems in <i>K. pneumoniae</i> were divided into two types (type I-E, 23; subtype I-E^∗, 17) based on the sequences of Cas1 and Cas3 proteins and their location in the chromosome. The distribution of type I-E and I-E^∗ CRISPR-Cas systems was associated with the multilocus sequence typing and the pulsed-field gel electrophoresis results. Importantly, no CRISPR-Cas system was identified in published genomes of clonal complex 258 isolates (ST11 and ST258), which comprise the largest multi-drug resistant <i>K. pneumoniae</i> clonal group worldwide. PCR with <i>cas</i>-specific primers showed that 30.7% (54/176) of the clinical isolates had a CRISPR-Cas system. Among clinical isolates, more type I-E CRISPR-Cas systems were found in UTI isolates (BSI, 5.7%; UTI, 11.2%), and subtype I-E^∗ CRISPR-Cas systems were dominant in BSI isolates (BSI, 28.7%; UTI, 15.7%) (<i>p</i> = 0.042). Isolates which had subtype I-E^∗ CRISPR-Cas system were more susceptible to ampicillin-sulbactam (<i>p</i> = 0.009), cefazolin (<i>p</i> = 0.016), cefuroxime (<i>p</i> = 0.039), and gentamicin (<i>p</i> = 0.012), compared to the CRISPR-negative isolates. The strains containing subtype I-E^∗ CRISPR-Cas systems had decreased numbers of plasmids, prophage regions, and acquired antibiotic resistance genes in their published genomes. Here, we first revealed subtype I-E^∗ CRISPR-Cas system in <i>K. pneumoniae</i> potentially interfering with the acquisition of phages and plasmids harboring antibiotic resistance determinants, and thus maintained these isolates susceptible to antibiotics.