Complete Genome Sequence of Providencia stuartii CMC-4104, Isolated from a Human Splenic Abscess, Containing Multiple Copies of NDM-1 and PER-1 Carbapenem Resistance Genes

ABSTRACT We report the complete genome sequence of a clinical isolate of Providencia stuartii strain CMC-4104, isolated from a splenic abscess. Oxford Nanopore Technologies (ONT) and Illumina sequencing reads were assembled using Geneious to generate a 4,504,925-bp circular chromosome containing multiple copies of the NDM-1 and PER-1 genes in a genomic resistance island.

P. stuartii CMC-4104, isolated from a patient with splenic abscess aspirate, was inoculated onto sheep blood agar and MacConkey agar plates and incubated at 35°C aerobically for 24 to 72 h. Antibiotic susceptibility testing for 17 antimicrobial agents based on the CLSI M100 standards (7) was carried out by Quest Diagnostics at the Carilion Clinic (Table 1).
A single colony of P. stuartii CMC-4104 was grown in 25 mL lysogeny broth at 37°C and 200 rpm for 18 h. The cell pellet was used for genomic DNA isolation with a Genomic-tip 20/G kit (8,9). The unsheared DNA was sequenced without size selection using the ONT 9.4.1 MinION flow cell with the SQK-LSK109 ligation sequencing kit. MiSeq Illumina paired-end sequencing (350-bp insert size) was performed using the TruSeq DNA PCR-free library prep kit.
The ONT sequencing generated 19,109 reads with a maximum length of 170,418 bp and an average length of 15,700 bp for a total of 306 Mbp. Base calling was performed using Guppy 4.4.1, and Porechop 0.2.4 (10) was used for adaptor trimming. The resulting fastq files were initially assembled using Flye 2.8 (11) in Geneious Prime 2022.0.2 to produce a large, closed 4.59-Mbp draft genome with ;65Â coverage. The Illumina sequencing generated 2,290,766 paired-end reads 150 bp long for a total of 344 Mbp. The "Map to Reference" tool in Geneious Prime was used (with low sensitivity settings) to align all of the Illumina reads to the draft genome sequence and correct ONT sequencing errors. Variations in the Illumina read coverage were used to identify three circular plasmid sequences and a highly repeated set of MDR gene cassettes in the main chromosome. Finally, Geneious Prime was used to align the ONT reads to the plasmid and genome sequences (with medium sensitivity settings) to confirm closure of the circular plasmid and main chromosome sequences, as well as the repetition of the MDR gene cassettes. Default parameters were used for all software unless otherwise specified.
Final assembly of the P. stuartii CMC-4104 genome resulted in a main chromosome (GenBank accession number CP095443) of 4,504,925 bp with 41.4% GC content and an average Illumina coverage (AIC) of 65Â; a large, low-copy, circular plasmid (CP095444) of 278,489 bp with 47.3% GC content and an AIC of 85Â; a small, high-copy, circular plasmid (CP095445) of 2,683 bp with 41.8% GC content and an AIC of 4,616Â; and a phage-like circular sequence (CP095442) of 51,458 bp with 41.7% GC content and an AIC of 105Â. In the main chromosome (CP095443), the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) 6.1 (12) was used to identify 4,066 protein coding genes, 79 tRNAs, 22 rRNAs, and 4 CRISPR arrays. Of particular note was a large 215-kbp genomic resistance island (PsGRI) integrated with a site-specific integrase into the 39 end of a tRNA modification GTPase gene (mnmE), like the Salmonella genomic island (13,14). Remarkably, the PsGRI contained 15 copies of the MDR gene cassettes containing bla NDM-1 and 2 copies of bla PER-1 shown in Fig. 1. The large plasmid (CP095444) also contained one complete copy each of the bla NDM-1 and bla PER-1 MDR cassettes and an additional bla OXA-10 MDR cassette (14)(15)(16)(17)(18). The small, highcopy plasmid (CP095445) also contained a fluoroquinolone resistance gene, qnrD (19). The assembly revealed a circular phage-like sequence (CP095442) that is identical to a prophage in the main chromosome and is very similar to a second prophage integrated into a CRISPR locus (20).

ACKNOWLEDGMENTS
We acknowledge the faculty members of the Division of Infectious Disease, Carilion Clinic, and the Carilion clinical microbiology laboratory for their assistance in collecting patient isolates. Experiments were carried out at the Carilion Basic Science Research a MICs were determined (7) using the Vitek and Verigene testing systems at the Quest Diagnostics microbiology laboratory at Carilion. The carbapenemase-producing strain was confirmed by the Commonwealth of Virginia Consolidated Laboratory Services, VDH. b Susceptibility testing was performed using the E-test for ceftazidime1avibactam, imipenem1relebactam, and meropenem1vaborbactam. c Cefiderocol susceptibility testing was completed by Associated Regional and University Pathologists Laboratories in Salt Lake City, UT.
Laboratory, Carilion Roanoke Community Hospital. We thank the Carilion Clinic Healthcare Analytics Research Team for data storage and maintenance. We thank Sarah Cox, Radford University Carilion, for her valuable support in manuscript reading and suggestions. This study was supported by the Division of Infectious Diseases, Carilion Clinic (J.R.). The Oxford Nanopore sequencing was completed with the support of an FDA CVM Vet-LIRN Veterinary Diagnostic Laboratory Program Infrastructure Grant (K.K.L.), and the Illumina sequencing was performed at the Virginia Tech Genomics Resource Center with the support of the Fralin Life Science Institute (R.V.J.).