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. 2025 Jul 31;14(9):e00221-25. doi: 10.1128/mra.00221-25

Identification of Acinetobacter radioresistens in wastewater effluent from a First Nation reserve in Manitoba, Canada

Rudra Patel 1, Dawn White 1, Ayush Kumar 1, Miguel Uyaguari 1,✉
Editor: Irene L G Newton2
PMCID: PMC12424402  PMID: 40741752

ABSTRACT

Acinetobacter radioresistens was identified in UV-treated wastewater effluent from a First Nation reserve in Manitoba, Canada, by whole-genome sequencing (3,195,655 bp; 41.7% GC) and digital DNA-DNA hybridization with A. radioresistens DSM 6976 (average contig nucleotide identity: 90.6%). Draft genome analysis revealed the presence of the clinically relevant carbapenem-resistance gene, blaOXA-23.

KEYWORDS: Acinetobacter radioresistens, UV light, effluent, wastewater treatment, First Nation community, whole-genome sequencing

ANNOUNCEMENT

Acinetobacter radioresistens was first isolated from irradiated cotton in 1988 (1) and is the original source of a mobile, clinically relevant, carbapenem-resistance gene bla0XA-23 (2). Most commonly found as a commensal microbe on healthy human skin (3), it is increasingly being identified in human infections (4). Here, we isolated A. radioresistens from wastewater treatment plant effluent at a First Nation reserve located 2 h away from Winnipeg, MB, Canada.

Wastewater (1 L) post-UV treatment was collected in a sterile bottle following standard procedures (5) on 9 September 2022. A 100 µL sample was spread on a Chromocult coliform agar plate and incubated at 37°C for 24 h. A small, white colony was isolated and re-streaked (×3) onto a fresh coliform agar plate and incubated at 37°C for 24 h to ensure colony purity. Antibiotic MIC testing revealed resistance against imipenem (64 µg/mL) following CLSI procedures and breakpoints (6). Genomic DNA was isolated from an overnight culture grown in lysogeny broth using the MasterPure Complete DNA and RNA Purification Kit (LGC BioSearch Technologies). No DNA shearing or size selection was performed. A DNA library was prepared using the Oxford Nanopore Technologies Native Barcoding kit (V14) (SQK-NBD114-24) following the manufacturer’s instructions. The library was sequenced using the MinION with a R10.4.1 flow cell (FLO-MINI114) using the Fast Model base-calling algorithm (400 bps) with a minimum read length of 200 bp without barcode trimming, mid-read filtering, modified base calling, and adapter trimming. All sequence analyses used default parameters unless noted otherwise.

From 7.65 million raw reads (N50: 2.05 kb), 73,803 sequences were assembled using default settings of Flye (v2.9.1-b1780) (7), yielding 47× mean coverage of 3 contigs totaling 3,195,655 bp and GC content of 41.7% (N50: 3.11 Mbp): contig 1 comprised 3,114,505 bp, 41.8% GC; contig 2 comprised 64,022 bp, 37.9% GC; and contig 3 comprised 17,128 bp, 37.0% GC (Fig. 1). Alignment of the sequenced genome using rMLST (8) and MiGA (v1.3.21.3) (9) identified our isolate as A. radioresistens; alignment using TYGS (v.391) (10) more specifically paired with A. radioresistens DSM 6976 and LH6. NCBI MSA alignment of our isolate and LH6 DNA sequences for 16S rRNA (99.5% identity) and rpoB (99.7% identity) further strengthened the identity of our isolate. The predicted antibiotic resistance-associated carbapenem-resistance gene, blaOXA-23 was also present (100% identity). The draft genome was estimated to be 93.4% complete using FastAAI (v0.1.17) (11). Annotation using RASTtk (v1.073) (12) revealed 3,502 CDS in contig 1, 83 CDS in contig 2, and 23 CDS in contig 3, where 3,409 were protein-coding and 93 were RNA genes (18 rRNAs, 75 tRNAs, and 0 ncRNAs).

Fig 1.

Circular genome maps of three contigs highlighting coding sequences, GC content, GC skew, tRNA, rRNA, and resistance genes OXA-23, qacJ, and adeF, with contig lengths of 2,757,930 bp, 64,022 bp, and 17,128 bp.

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Circular contig maps generated using Proksee (13). (A) Contig 1, (B) contig 2, and (C) contig 3. From the inner ring to the outer ring: GC skew (positive GC skew, mustard; negative GC skew, magenta); GC content (dark blue); Comprehensive Antibiotic Resistance Database (CARD) results (red, annotated); coding DNA sequences (CDS) (green); tRNA (bright blue); and rRNA (turquoise). Analysis of contigs 2 and 3 did not yield tRNA, rRNA, or CARD results.

Resistance-associated genes were found through the Comprehensive Antibiotic Resistance Database (v3.3.0) (14) using RGI main; perfect and strict cut-offs revealed blaOXA-23 and two antibiotic efflux pump genes (Table 1). Antimicrobial-resistant A. radioresistens is an emerging burden on the healthcare system (4). Increased surveillance of wastewaters—a documented source of antibiotic-resistant genes (15)—may alert us to potential new AMR bacterial threats.

TABLE 1.

Antibiotic-resistance related genes of A. radioresistens isolate identified using the Comprehensive Antibiotic Resistance Database

Antibiotic resistance related gene CARD accession Cut-off Gene family Percent length of reference sequence
blaOXA-23 ARO:3001418 Perfect OXA betalactamase 100
adeF ARO:300777 Strict Resistance nodulation- division efflux pump 99
qacJ ARO:3007014 Strict Small multidrug resistance efflux pump 102
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ACKNOWLEDGMENTS

We thank the members of the First Nation reserve for their valuable research partnership; without their help, this study would not have been possible.

This work was funded by a Discovery Grant from the Natural Science and Engineering Research Council of Canada and a Research Manitoba New Investigator Operating Grant awarded to MU. Additional support came from a Discovery Grant from the Natural Science and Engineering Research Council of Canada awarded to AK. RP was supported by a University of Manitoba Summer Research Award and the Indigenous CREATE program at the University of Manitoba for undergraduate students.

The authors acknowledge that the University of Manitoba campuses are located on original lands of Anishinaabeg, Ininewuk, Anisininewuk, Dakota Oyate, and Denesuline, and on the National Homeland of the Red River Métis.

Contributor Information

Miguel Uyaguari, Email: Miguel.Uyaguari@umanitoba.ca.

Irene L. G. Newton, Indiana University, Bloomington, Bloomington, Indiana, USA

DATA AVAILABILITY

The BioSample, BioProject, and SRA numbers for this A. radioresistens isolate (SEPT_EFF1) are SAMN42123106, PRJNA1129067, and SRR32145570, respectively.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The BioSample, BioProject, and SRA numbers for this A. radioresistens isolate (SEPT_EFF1) are SAMN42123106, PRJNA1129067, and SRR32145570, respectively.

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