Detection of a Novel Metallo-β-Lactamase, CAM-2, in a Metagenome-Assembled Genome from China

Abstract

C arbapenems are regarded as important antimicrobials for the treatment of serious bacterial infections caused by multidrug-resistant (MDR) Enterobacteriaceae. In 2019, Boyd and colleagues from Canada (1) described a novel class B carbapenemase, named CAM-1, in the chromosome of four closely related clinical Pseudomonas aerugi-nosa isolates collected from three patients. No subsequent isolates harboring this gene have been reported. Recent studies showed that this gene belongs to the molecular sub-class B1.4, a group dominated by Bacteroidetes and Firmicutes (2), suggesting that CAM-1 may have originated from a phylum other than Proteobacteria. Here, we report for the first time a novel metallo-b-lactamase, CAM-2, which is located on the chromosome of a metagenome-assembled genome (MAG) belonging to the family Pyrinomonadaceae (3). In July 2016, three estuary sediment samples were collected at Shenzhen Bay, China. We then performed total DNA extraction from three sediment samples using a DNeasy PowerSoil kit (Qiagen, Germany). Metagenomic sequence data were generated using Illumina HiSeq sequencing with 150-bp paired-end reads at Novogene Bioinformatics Technology Co., Ltd. (Tianjin, China). Raw metagenomic reads were dereplicated (100% identity over 100% length) and trimmed using sickle (https://github.com/najoshi/sickle). The remaining high-quality metagenomic reads were de novo metagenome assembled using MEGAHIT (4) with the parameters "-min-contig-len 1000-k-min 21-k-max 141-k-step 12-merge-level 20,0.95" and filtering out contigs shorter than 1 kb. We used the ABRicate tool (v1.0.1) (https://github.com/tseemann/abricate) to identify antibiotic resistance genes present in our metagenomic assemblies. The ResFinder (5) database was used as the reference database for ABRicate with the following settings: a minimum nucleotide identity of 70 % and a minimum DNA coverage of 80 %. The results showed that the assembled contig sequences of one of the samples contained a bla CAM-like gene designated bla CAM-2 , which was 741 bp long, 15 bases longer than the original bla CAM-1 gene (726 bp), and showed 91.21% nucleotide identity to bla CAM-1. In order to find out the bacterial host of the bla CAM-2 gene, genome binning of the assembled contigs was conducted using MetaBAT (6) with 12 sets of flags inducing different sensitivity and specificity combinations, and CheckM (7) was used to calculate the completeness and contamination of MAGs. Binning analysis produced a positive binning for the bla CAM-2 gene, consisting of 20 contigs (genome completeness, 94.84%; contamination, 3.42%). Finally, the bla CAM-2-carrying bin was selected for taxonomy classification using the GTDB-Tk package (8) and was taxonomically classified as the Pyrinomonadaceae family within the phylum Acidobacteria (3). The MAG consists of 5,719,470 bp. (The GC content is 46.4%.) The number of coding sequences (CDSs) is 5,465, as determined according to the Rapid Annotation Subsystem Technology (RAST) server (http://rast.nmpdr.org/). Except for bla CAM-2 , this MAG does not carry other antibiotic resistance genes or mobile genomic elements. Sequence analysis revealed that bla CAM-2 was located on a contig of 1,194,427 bp with Editor Cezar M. Khursigara, University of Guelph