Colistin has been used for last-line treatment of multidrug-resistant (MDR) Gram-negative bacterial infection . Recently, the colistin-resistant Salmonella enterica isolates have been found globally, emerging as serious threats to human and veterinary health . In South Korea, S. enterica serotype Enteritidis (S. Enteritidis) has been one of the dominant serotypes involved in Salmonella outbreaks . However, lack of genomic information of colistin-resistant S. Enteritidis isolates from South Korea has become an issue in the field. In this study, we report a complete genome sequence of colistinresistant S. Enteritidis strain CRSE-01 isolated from a poultry carcass sample in South Korea.
S. Enteritidis strain CRSE-01 was isolated in 2018 from a poultry carcass at a slaughterhouse located in Gyeonggi-do. A single colony of CRSE-01 was inoculated into Luria-Bertani (Becton Dickinson and Company, USA) and incubated at 37℃ with aeration and shaking (220 rpm) for 18 h. The bacterial genomic DNA was extracted using HiYield genomic DNA mini kit (Real Biotech Corporation, Taiwan) and subjected to wholegenome sequencing analysis. DNA library was prepared with SMRTbell template prep kit (PacBio, USA) and sequenced using PacBio RS II platform (PacBio). Total 142,654 subreads were generated (Total subread bases, 1,473,548,582 bases; mean subread length, 10,329 bases; N50, 14,311 bases) and assembled using HGAP3 version 3.0 software . To correct errors in the assembled contigs, the gDNA was also subjected to Illumina sequencing. The sequencing library was prepared with TruSeq Nano DNA library prep kit (Illumina, USA) and sequenced using Illumina HiSeq X ten platform (Illumina). A total 9,836,008 reads were generated (Total read bases, 1,485,237,208 bases; G+C content, 52.11%; Q30, 98.5%) and mapped against assembled genomes using Pilon version 1.21 software  to construct contigs more accurately. Gene annotation was performed using Prokka version 1.12b software . The serotype and O:H antigen were determined using SeqSero software (https://cge.cbs.dtu.dk/services/SeqSero/) from Center for Genomic Epidemiology (CGE). In addition, sequence type (ST) was analyzed by Multilocus sequence typing (MLST) (https://cge.cbs.dtu.dk/services/MLST/) from CGE. The chromosomal point mutations and acquired genes related to antimicrobial resistance (AMR) were identified by using ResFinder software (https://cge.cbs.dtu.dk/services/ResFinder/) from CGE. To determine AMR profile of CRSE-01 strain, we performed minimal inhibitory concentration (MIC) analysis using KRNV5F Sensititre (TREK Diagnostic Systems, USA) . Complete genome sequence of CRSE-01 was submitted to the NCBI GeneBank database under accession numbers CP126166 and CP126167.
The assembled genome of CRSE-01 strain yielded two circular contigs (Fig. 1A and B). Contig 1 comprised a single chromosome (4,783,907 bp, 52% G+C contents, 192× depth) which includes 4487 coding sequences (CDSs), 86 tRNA, and 22 rRNA (Table 1). Contig 2 comprised a single plasmid (59,372 bp, 51.95% G+C contents, 92× depth) containing 75 CDSs. The CRSE-01 strain was identified as S. Enteritidis belonging to ST11 with 9:g, m:-antigen profile (O antigen: 9, H1 antigen: g, m, H2 antigen: -). MIC analysis showed that CRSE-01 strain is resistant to ampicillin, streptomycin, nalidixic acid, sulfisoxazole, tetracycline, and colistin. Corresponding to its AMR phenotypes, the CRSE-01 genome harbors various acquired AMR genes (sul2, tet(A), blaTEM-1B, aph(6)-Id, aph(3’’)-Ib) and single point mutation in gyrA (Asp87Asn). However, the CRSE-01 genome does not harbor chromosomal mutations or plasmid-encoded genes required for colistin resistance, suggesting that its colistin resistance is independent of genetic alteration.
Figure 1.A complete genome of Salmonella enterica serotype Enteritidis CRSE-01 strain.
Circular maps of contig 1 (A) and contig 2 (B). Each circular map was drawn by applying the contig annotation information. Marked characteristics are shown from outside to the center; coding sequence (CDS) on forward stand, CDS on reverse stand, tRNA, rRNA, G+C content and GC skew.