Draft genomes of meropenem-resistant Pseudomonads from the cloacae of turtles in Lake Opinicon, Canada

TL;DR

This study reports the draft genomes of seven meropenem-resistant bacteria found in turtles from Lake Opinicon, Canada, shedding light on antibiotic resistance in wild animals.

Contribution

The paper provides new draft genome sequences of meropenem-resistant Pseudomonads from wild turtles.

Findings

Seven meropenem-resistant bacterial isolates were identified in turtle cloacae.

The isolates were collected from painted and musk turtles in Lake Opinicon, Canada.

Abstract

We report the draft genome sequences of seven meropenem-resistant bacterial isolates from the cloacae of painted and musk turtles at Lake Opinicon, Canada. This study improves our understanding of the spread of meropenem-resistant bacteria in the wild.

Tables

TABLE 1. Assembly and genome characteristics for the seven isolates.a

Isolate	Location	Closest ANI neighbor	ANI neighbor % similarity	GC content (%)	No. of contigs	Contig N50	Total length (bp)	Number of raw reads
PT6	Fishcamp Bay	Pseudomonas fluorescens AU11706	91.4	62.3	53	619,162	6,718,404	2,982,833
MT1	Fishcamp Bay	Pseudomonas fluorescens AU11706	91.37	62.3	43	812,339	6,880,211	4,618,451
MT15	Stinkpot Bay	Pseudomonas sp. MWU12_3153	91.34	62.3	55	647,030	6,795,780	3,394,872
MT16	Stinkpot Bay	Pseudomonas sp. MWU12_3153	91.32	62.2	77	667,054	6,655,658	4,061,292
MT23	Brooks Bay	Pseudomonas sp. MWU12-2534b	97.92	63.2	89	345,326	6,966,590	3,989,925
MT19	Stinkpot Bay	Pseudomonas sp. MWU 341	95.99	62	125	188,314	5,721,847	3,332,021
MT21	Stinkpot Bay	Pseudomonas sp. MWU 341	99.22	62	114	238,284	6,233,820	3,632,238

Full text

ANNOUNCEMENT

Carbapenems are beta-lactam antibiotics used to treat infections caused by multidrug-resistant Gram-negative bacteria. Carbapenems bind to penicillin-binding proteins, disrupting cell wall synthesis and ultimately resulting in cell death. The primary mechanism of clinical carbapenem resistance is the production of carbapenemases, which hydrolyze the amide bond of the beta-lactam ring (1, 2).

Environmental transmission of antimicrobial resistance (AMR) genes and resistant organisms is an increasing concern (3). As a step toward understanding the distribution of AMR in freshwater systems, cloacal swabs were performed on Musk and Painted turtles from three different locations at Lake Opinicon (44°33′30″N 76°19′44″W, ON, Canada): Fishcamp Bay, Stinkpot Bay, and Brooke’s Bay. Swabs were streaked onto lysogeny broth (LB) agar (10 g/L tryptone, 5 g/L yeast extract, and 0.5 g/L NaCl) supplemented with 4 µg/mL meropenem, and then single colonies were streaked onto Uriselect agar (Bio-Rad) for the selection of Gram-negative bacteria based on the ability to ferment lactose. Single colonies were then restreaked onto LB agar supplemented with meropenem. Single colonies were inoculated into LB and grown overnight in a shaking incubator (125 rpm) at 37°C. Genomic DNA was extracted using the One-4-All Genomic DNA Mini-prep Kit (Bio Basic) as described in the manufacturer’s protocol. Whole-genome sequencing was carried out at the Microbial Genome Sequencing Center (Pittsburgh, PA, USA). Sample libraries were prepared using an Illumina DNA Prep kit and IDT 10 bp UDI indices and sequenced on an Illumina NextSeq 2000 producing 2 × 151 bp reads. Reads were trimmed using Trimmomatic 0.36 with sliding window 4:20 and minimum length 36 [Bolger et al., 2014 (4)], and quality control was performed using Fastqc 0.12.1 [Andrews, 2010 (5)]. Genomes were assembled using Unicycler 0.4.4 (6), and the assembly was assessed using Qualimap 2.2.2a (7). Coverage was assessed by mapping reads to the assembly using bowtie2 v2.4.5 (8) and calculating average depth across all positions with the samtools v1.14 depth function (9). Contigs of <200 bp were removed using the BBMAP v38.18 reformat function (10). Default parameters were used except where otherwise noted. The genomes were submitted to NCBI Prokaryotic Genome Annotation Pipeline v6.6 for annotation (11). Resistance genes were identified using ResFinder v4.1.5 (12), and taxonomic classification was performed by the identification of genomic closest average nucleotide identity (ANI) neighbors using LINbase (13).

All seven isolates were classified as members of the genus Pseudomonas (Table 1), with nearest neighbors belonging to unnamed species.

No known resistance genes were detected by ResFinder in any of the isolates (Table 1). The genomes varied in size between 5,721,847 bp (MT19) and 6,966,590 bp (MT23) in length, and the GC content varied between 62% and 63.2% (Table 1).

These genomes will support further investigations into the spread of carbapenem-resistant bacteria among wild animals. The absence of known carbapenem resistance genes in these meropenem-resistant isolates suggests novel resistance mechanisms. This study also contributes to our understanding of the distribution of Pseudomonads in freshwater lakes and their animal inhabitants.