Abstract:
BACKGROUND : Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.
METHODS : One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.
RESULTS : We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.
CONCLUSIONS : Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.
Description:
DATA AVAILABILITY : All raw sequencing data have been deposited in GenBank under the BioProject accession number PRJNA549322 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA549322). The custom code used in this study is freely available at GitHub [67].
ADDITIONAL FILE 1: Supplementary Table S1-S7. TABLE S1. The information of CG147 strains in this study. TABLE S2. Primers used in this study. TABLE S3. Plasmids and strains used in this study. TABLE S4. Type I-E CRISPR-Cas spacer sequences. TABLE S5. Type IV-A3 CRISPR-Cas spacer sequences. TABLE S6. STs, regions, type IV-A3 CRISPR-Cas system and anti-CRISPR proteins of 646 non-redundant strains by carbapenemases and ESBL CTX-M in 646 non-redundant strains. TABLE S7. Logistic regression of KPC-2 and KPC-3 with STs, regions, type IV-A3 CRISPR-Cas system and anti-CRISPR proteins in 646 non-redundant strains.
ADDITIONAL FILE 2: Supplementary Fig. S1-S3. FIGURE S1. Distribution of NDM and KPC positive CG147 isolates between 2000 and 2020. Verdigris dot: NDM; red: KPC. FIGURE S2. Distribution of spacers of type I-E CRISPR-Cas arrays and type IV-A3 CRISPR-Cas arrays in CG147. FIGURE S3. Prophages in CG147 strains. Alignment of two major types of acrIE8.1 carrying prophages. The distribution of acrIE8.1 and the containing prophages in 1,012 CG147 strains with different STs and K locus.
ADDITIONAL FILE 3: Supplementary results. The supplementary description of the results.