Complete Genome Sequence of a Colistin-Susceptible Salmonella enterica Serovar Minnesota Strain Harboring mcr-9 on an IncHI2/IncHI2A Plasmid, Isolated from Chicken Meat

ABSTRACT The recent emergence and dissemination of mobilized colistin resistance (mcr) genes have triggered extensive concerns globally. Here, we report the complete genome sequence of a colistin-susceptible Salmonella enterica serotype Minnesota strain (named SA18578), belonging to sequence type 548 (ST548) and carrying the mcr-9 gene on an IncHI2/IncHI2A plasmid, that was isolated from chicken meat in Saudi Arabia in 2020.

T he mobile colistin resistance (mcr-9) gene was first detected in Salmonella enterica serotype Typhimurium, which highlighted the need for rigorous monitoring of the potential spread of this new gene (1). Here, we determined the complete genome sequence of an mcr-9-carrying Salmonella enterica serotype Minnesota strain that was isolated from chicken meat.
This strain was isolated and confirmed to belong to Salmonella enterica serotype Minnesota based on the International Organization for Standardization guideline ISO 6579-1:2017 (2). Resistance to antibiotics was determined using standard broth microdilution and CLSI guidelines (3). MIC results showed that SA18578 was resistant to amoxicillinclavulanic acid, ampicillin, gentamicin, sulfisoxazole, and tetracycline; interestingly, SA18578 exhibited susceptibility to colistin (#1 mg/liter).
For DNA extraction, this strain was grown overnight in Oxoid nutrient agar at 37°C, and DNA was extracted using the QIAamp DNA minikit following the manufacturer's instructions (Qiagen). The short reads for this strain were generated by a MiSeq system (Illumina, CA) with a MiSeq v3 kit using 2 Â 300-bp paired-end chemistry. Raw data were demultiplexed using Illumina's bcl2fastq tool (version 2.20) and checked for quality using the FastQC tool (version 0.11.9) (4). The long reads for this strain were generated on a MinION sequencer (Oxford Nanopore Technologies) using the ligation sequencing kit SQK-LSK109. Default parameters were used for all software unless otherwise specified. The MinION raw data were base called using Guppy (version 4.3.4), which is integrated into MinKNOW (version 21.02.5), followed by filtering of reads with a minimum Q score of 7 and a minimum read length of 1,000 bp. A total of 595,286 short reads and 55,129 long reads were combined to obtain the complete genome sequence of the SA18578 isolate using the EToKi pipeline (Enterobase Tool Kit) (version 1.0) (5), generating 5 contigs with an average sequencing coverage depth of 40Â. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (version 5.2) (6) was used for genome annotation. Multilocus sequence typing (MLST), plasmid replicon typing, and determination of antibiotic resistance genes were performed with ABRicate (version 0.9.8) (https://github.com/ tseemann/abricate) using MLST (version 2.0.4) (7), PlasmidFinder (version 1.3) (8), and ResFinder (version 2.1) (9, 10). In silico MLST analysis indicated that the SA18578 strain belonged to sequence type 548 (ST548). Plasmid pSA18578_MCR-9_1 carried the colistin resistance mcr-9 gene and belonged to the incompatibility group IncHI2/IncHI2A. In addition to mcr-9, the resistance genes bla TEM-1B and aac(3)-IId were identified on this plasmid. Plasmid pSA18578_3 carried one resistance gene (qnrB19), and no resistance genes were identified on pSA18578_2 and pSA18578_4. Further resistance genes were detected on the chromosome, including sul2, tet(A), ant(30)-Ia, and bla CMY-2 , as well as a point mutation in parC (T57S).
Based on our results, it appears that the presence of the mcr-9 gene is actually not associated with colistin resistance. A few studies investigated the role of the mcr-9 gene in colistin resistance and concluded that this gene was neither inducible nor expressed under normal conditions (1,11,12). Therefore, further studies are necessary to determine the impact of the mcr-9 gene on colistin susceptibility.
Data availability.

ACKNOWLEDGMENTS
This work was funded and conducted by the Saudi Food and Drug Authority (SFDA) (Riyadh, Saudi Arabia). The long-read sequencing and colistin susceptibility testing were funded by and performed at the Infectious Diseases Research Department (IDRD), King Abdullah International Medical Research Center (KAIMRC) (Riyadh, Saudi Arabia).
The findings and conclusions of this report are those of the authors and do not necessarily represent the official views or policies of the SFDA.