Metagenomic analysis of Salmonella Enterica isolated from diarrhea patients living in Babylon and identification of some virulence genes

During this study, a total of 130 clinical stool samples were collected from patients of all ages and both sexes with watery diarrhoea or mucus, pus and a small amount of blood. Stool samples were collected from patients attending Imam Sadiq and Al-Hillah Surgical Teaching Hospital in Babil province between January and March 2023. The samples were then cultured on selective media and identified using bacteriological and biochemical tests and specific primers. Only 41 (31.5%) of 130 samples were identified as S. enterica isolates by culture and vitek 2 compact system. However, using the specific gene, a total of 34 (82.9%) isolates were positive for specific genes, including 24 (70.58%) S. typhimurium and 10 (29.41%) S. enteritidis isolates. Some virulence factors of Salmonella enterica isolates were analysed as hilA, stn genes and PCR was performed using specific primers on genomic DNA obtained from these isolates. In PCR amplification of these genes, it was determined that 33 (97.06%) and 32 (94.12%) isolates had hilA and stn genes, respectively. Antibiotic susceptibility test was performed with vitek 2 compact system to determine the microbial susceptibility and resistance of Salmonella enterica isolates. Accordingly, the isolates showed 100% susceptibility to Trimethoprim/Sulfamethoxazole, Imipenem and Tigecycline, 91.18% to Piperacillin/Tazobactam, Cefepime and Ertapenem, 82.35% to Ceftazidime and 82.35% to Ceftriaxone. However, the isolates showed moderate susceptibility to Ampicillin, Ciprofloxacin, and Nitrofurantoin (61.76%, 61.76% and 50%) respectively. Furthermore, the results revealed that some isolates were resistant to Cefazolin, Cefoxitin and Levofloxacin by 82.35%. In addition, the isolates were resistant to Amikacin and Gentamicin by 88.23%. At the genomic level, this study used metagenome sequencing to differentiate between the closely related isolates Salmonella enterica subsp. enterica serovar Typhimurium (5TMM) and Salmonella enterica subsp. enterica serovar Entretidis (13SE). Accordingly, it was determined that the chromosomes of S. enterica serovars 5TMM and 13SE differed slightly and the genomic size of 5TMM (4,902,206 bp, 52.3% GC before filtration, 52.28% GC after filtration) was larger than that of 13SE (5,032,992 bp, 52.2% GC before filtration, 52.23% GC after filtration). After comparative genome analysis, all S. enterica genomes iv examined (5TMM and 13SE) showed different evolutionary event patterns with each other or with the reference genome. In this study, whole genome sequencing with the next generation meptagenome sequencing technique produced crude paired-end reads. The GC content of Salmonella enterica subsp. enterica serovar Typhimurium 5TMM was 52.35% and the GC content of 13SE in Salmonella enterica subsp. enterica serovar Entretidis was 52.57%. The genome size was 4,583,358 bp for Salmonella enterica subsp. enterica serovar Typhimurium (5TMM) and 4,292,569 bp for Salmonella enterica subsp. enterica serovar Entretidis (13SE). Furthermore, the 5TMM and 13SE draft genomes contain 83 and 102 contigs. In addition, the sequence similarity of S. enterica serovars was analysed in comparison with the reference genome, and it was revealed that the sequence similarity in the 13SE strain was higher than that in the 5TMM strain, while 5TMM had more gaps than 13SE. In addition, in this study, 4,548 CDS, 71 tRNA, 3 rRNA and 428 hypothetical proteins were found in the 5TMM genome, while 4,213 CDS, 68 tRNA, 4 rRNA and 344 hypothetical proteins were found in the 13SE genome. According to the variant counts of the studied genomes, the 5TMM genome showed the presence of 99.09% (24203) SNPs, 0.46% (112) insertions and 0.45% (109) deletions (24424), while the 13SE genome had 99% (42027) SNPs, 0.49% (207) insertions and 0.51% (215) deletions (42449). Accordingly, the number of base changes in each SNP was calculated to determine the type of sequence variation. All genomes studied showed patterns of similarity variation, where the most common base substitution patterns were C↔️ T and G↔️ A substitutions. These patterns represent Transition substitution rather than Transversion substitution in all genomes studied after SNPs.