A Novel Anti-CRISPR AcrIE9.2 Is Associated with Dissemination of bla KPC Plasmids in Klebsiella pneumoniae Sequence Type 15.

Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of bla KPC plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of bla KPC plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on bla KPC plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded bla KPC plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both bla KPC and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted bla KPC plasmid was increased from 3.96 × 10 -6 to 2.01 × 10 -4 compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of bla KPC in ST15 by repressing CRISPR-Cas activity.