农业流域与奶牛粪便来源的多重耐药鲍曼不动杆菌基因组特征及公共卫生意义

《Microbiology Resource Announcements》：Draft genomes of multidrug-resistant Acinetobacter baumannii isolates from an agriculturally-dominated watershed and dairy cattle feces

本研究报道了从农业主导流域和奶牛粪便中分离的6株多重耐药鲍曼不动杆菌（A. baumannii）的基因组草图，揭示了其携带丰富的抗生素耐药性（AMR）和毒力因子基因，其中4株被鉴定为耐碳青霉烯类（CRAB）菌株，凸显了环境传播对公共卫生的威胁。

　　

ABSTRACT

Acinetobacter baumanniiis an opportunistic human pathogen that primarily affects individuals with compromised immune systems. Here, we report the draft genomes of six A. baumanniistrains isolated from animal fecal sources and mixed-use but primarily agricultural surface waters. These genomes contain a diverse array of antibiotic resistance and virulence-associated genes.

ANNOUNCEMENT

Acinetobacter baumanniiis a major opportunistic pathogen associated with severe (co)infections in immunocompromised individuals. The ability to acquire and express diverse antimicrobial resistance (AMR) determinants has driven the rise of multidrug-resistant and extensively drug-resistant strains. Its resilience in diverse environments supports persistence and facilitates dissemination into healthcare settings. Particularly concerning are carbapenem-resistant (CRAB) strains, which the World Health Organization designates as ‘critical’ priority pathogens due to limited treatment options and high clinical burden.

菌株分离与鉴定

Strains SNC-24, SNC-253, SNC-5, and SNC-9 were isolated from surface waters in an agriculturally dominated but mixed-use watershed, whereas CF0141 and CF0140 were recovered from dairy cattle feces in the South Nation River basin (45.170, -75.079), eastern Ontario, Canada. Collected samples were suspended in 1× phosphate-buffered saline and serially diluted 10-fold. Bacteria were cultured on Karmali agar supplemented with amphotericin B, cefoperazone, sodium pyruvate, and vancomycin, and plates incubated at 42°C under microaerophilic conditions (5% O₂, 85% N₂, and 10% CO₂) for 48 h. Single colonies were subcultured to ensure purity and stored at ?20°C for further analysis. Genomic DNA was extracted with the DNeasy UltraClean microbial kit. Identification was performed by 16S rRNA sequencing with primers pA-F (5’ – AGA GTT TGA TCC TGG CTC AG- 3’) and pH-R (5’ – AAG GAG GTG ATC CAG CCG C - 3’). Sequencing was performed on an ABI PRISM 3130XL Genetic Analyzer and BLAST analysis confirmed 100% identity to A. baumannii16S rRNA.

全基因组测序与分析

Whole-genome sequencing was performed using the Illumina NextSeq 500 platform (2×150 bp) using the Nextera DNA Flex Library Prep kit following manufacturer’s instructions. Reads were trimmed with Atria v3.1.2(first 10 bp removed), quality-checked with FastQC v0.12.1and MultiQC v1.19, and assembled de novowith MEGAHIT v1.2.9, discarding contigs <500 bp. Assembly quality was evaluated with CheckM v1.2.2, and taxonomy confirmed with GTDB-Tk v2.3.2. Scaffolding was performed using RagTag v2.1.0with A. baumanniiK09-14 as reference. Gene annotation was conducted with Prokka v1.14.6. Virulence factors were identified via BLASTp v2.12.0against the Virulence Factor Database, and AMR genes were detected using AMRFinderPlus v3.11. Default parameters were applied unless stated otherwise.

基因组特征与耐药毒力分析

High-quality draft genomes (3.71–3.98 Mbp; N50: 3.68–3.92 Mbp) were recovered from all isolates, each with >98% completeness, <1.3% contamination, and 72–140× coverage. Isolates were predicted to be resistant to spectinomycin/streptomycin and cephalosporins, with variable resistance to other antibiotics. SNC-5, SNC-24, CF0141, and CF0140 were identified as potential CRAB strains, based on the presence of carbapenem resistance genes. Virulence factors were predominantly associated with immune modulation (VFC0258), nutritional/metabolic factors (VFC0272), adherence (VFC0001), effector delivery systems (VFC0086), and biofilm formation (VFC0271).

ACKNOWLEDGMENTS

This research was supported by Agriculture and Agri-Food Canada (AAFC) through the Growing Forward 2 program (Project IDs: 1074 and 1236) and the Biological Collections Data Mobilization (BioMob) Initiative—Work Package 2, Molecular Characterization (Project ID J-001564). The first author received financial support from AAFC and the Canadian Poultry Research Council (CPRC) under SCAP-ASC-18-Poultry Cluster Activity 5A (Project ID J-003360).

DATA AVAILABILITY

The data generated in this study have been deposited in NCBI under BioProject accession no. PRJNA1271542.