pmrCAB Recombination Events among Colistin-Susceptible and -Resistant Acinetobacter baumannii Clinical Isolates Belonging to International Clone 7

Abstract

Acinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB. In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance. IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales, the presence of mcr-type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB. We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.

Keywords: Gram-negative bacilli; colistin resistance; insertion sequences; mobile genetic elements; polymyxins.

FIG 1

(A to D) Protein sequence alignment of PmrC (A), PmrA (B), and PmrB (C) and SplitsTree-based neighbor-net of a 23.6-kb genomic region encompassing pmrCAB (D) between isolates MC1 (IC7), 72554 (IC7), 71813 (IC4), 67659 (IC7), 67098 (IC5), AYE (IC1), and ACICU (IC2). Sequences belonging to isolate MC1 were used as references for sequence alignment. Amino acid differences are highlighted in colors (panels A to C).

FIG 1

(A to D) Protein sequence alignment of PmrC (A), PmrA (B), and PmrB (C) and SplitsTree-based neighbor-net of a 23.6-kb genomic region encompassing pmrCAB (D) between isolates MC1 (IC7), 72554 (IC7), 71813 (IC4), 67659 (IC7), 67098 (IC5), AYE (IC1), and ACICU (IC2). Sequences belonging to isolate MC1 were used as references for sequence alignment. Amino acid differences are highlighted in colors (panels A to C).

FIG 1

(A to D) Protein sequence alignment of PmrC (A), PmrA (B), and PmrB (C) and SplitsTree-based neighbor-net of a 23.6-kb genomic region encompassing pmrCAB (D) between isolates MC1 (IC7), 72554 (IC7), 71813 (IC4), 67659 (IC7), 67098 (IC5), AYE (IC1), and ACICU (IC2). Sequences belonging to isolate MC1 were used as references for sequence alignment. Amino acid differences are highlighted in colors (panels A to C).

FIG 1

(A to D) Protein sequence alignment of PmrC (A), PmrA (B), and PmrB (C) and SplitsTree-based neighbor-net of a 23.6-kb genomic region encompassing pmrCAB (D) between isolates MC1 (IC7), 72554 (IC7), 71813 (IC4), 67659 (IC7), 67098 (IC5), AYE (IC1), and ACICU (IC2). Sequences belonging to isolate MC1 were used as references for sequence alignment. Amino acid differences are highlighted in colors (panels A to C).

FIG 2

(A and B) Spatial k-mer sharing plots of a 23.6-kb genomic region encompassing pmrCAB and flanking genes of isolate 67659 against isolates MC1 (IC7, top) and 67098 (IC5, bottom) (A) and 72554 against MC1 (IC7, top) and 71813 (IC4, bottom) (B). The plots show spatial variations in the proportion of k-mers present in the genomes described on the x axis also present in the genome of the different references described on the y axis, calculated in sliding windows of 40 bases along the genome of the first isolate and for k = 19. Plots are based on k-mer counts computed with Cortex and a custom R visualization script. pmrCAB coding regions are highlighted in red, and flanking genes are indicated in green.