Genome sequencing, annotation and analysis of Salmonella enterica sub species salamae strain DMA-1

During a study on identifying bacterial diversity of mouse stool samples, strain DMA-1 was isolated on tryptone soya agar (TSA, HiMedia, Mumbai, India). The strain DMA-1 was subjected to polyphasic taxonomic studies to identify the exact taxonomic status. The polyphasic taxonomical studies involved phenotypic, biochemical characterization and 16S rRNA gene sequencing that identified it as Salmonella enteric a subspecies salamae. The genus Salmonella was first proposed in 1952 by Kauffmann and Edwards [1]. Later on, it was emended in 1987 by Le Minor and Popoff [2]. The members of the genus Salmonella are Gram-negative, rod shaped, facultative anaerobes capable of aerobic respiration producing ATP and fermentation in the absence of oxygen. At present, the genus Salmonella consists of three species and six sub species. The Salmonella enterica subspecies include: S. enterica subspecies enterica (subspecies I), S. enterica subspecies salamae (subspecies II), S. enterica subspecies arizonae (subspecies IIIa), S. enterica subspecies diarizonae (subspecies IIIb), S. enterica subspecies houtenae (subspecies IV) and S. enterica subspecies indica (subspecies V). S. enterica includes the majority of Salmonella strains isolated from humans and warm blooded animals. But Salmonella bongori is typically obtained from cold blooded animals [3]. Most of the rodents present in laboratories are susceptible to Salmonella infections. Rodent feces cause infections via fecal-oral transmission, when a susceptible rodent ingests the bacteria present in their feed or contaminated drinking water. Immunocompromised caretakers of these rodents may get serious illness, which in some cases develop into life threatening diseases. Therefore in laboratories incoming rodents are quarantined and health monitoring for any rodent colony involves routine intestinal culture [4]. Infection caused by Salmonella enterica species is a major public health issue inflicting mortality globally. Contagious diseases caused by S. enterica depend mainly on their secreted proteins and adhesion from fimbrial and non-fimbrial sources, which produce biofilm and build contact with the host cell [5]. S. enterica also contains type I fimbriae, representative of other enteric bacteria, which help in attaching themselves to epithelial cells [6]. They are shorter in size than flagella but have a peritrichous distribution. Fimbriae are made up of major and minor protein subunits that make a cylinder around a hollow core [7]. Not all strains of S. enterica express fimbriae, but the fimbriaeted strains are more virulent [8].

Salmonella species could be differentiated on the basis of three antigens present on the cell: H antigen which is a flagellar antigen occurring only in one phase (1 or 2) and can interchange itself [9, 10]; O antigen is a somatic antigen present on the outer membrane. The specificity of the O antigen is obtained by the character of the repeating units present on the outer O-polysaccharide chain [9]. The Vi antigen, virulence antigen which is a capsular polysaccharide that overlays the O antigen, but it is not possessed by all serovars. The capsule present in the Salmonella sp. is not essential for infection, yet it increases the infectivity by making it less visible to the body’s immune system [11].

S. enterica is also able to extract iron from host proteins by siderophores, which are formed by the cell when iron concentration is low, siderophores also contribute to virulence [12]. It also shows a mixed-acid heterofermentation of glucose to produce ATP. This process also produces CO₂ and H₂ with a variety of acid final -products, for example formate, acetate, lactate, and succinate [13]. S. enterica can nourish on maltose and maltodextrins with the help of type I ATP-binding cassette transporter. This system is present on a maltose regulon. In a maltose regulon the maltose enter into the cell by crossing the outer membrane through a homotrimeric maltoporin, after that it binds to a protein having pore-forming subunits that result in the translocation of an ATP-binding subunit across the inner-membrane where it is used by the cell [14]. S. enterica causes infectious diseases by entering the host cells that are present around the intestine, and attaching themselves to the cells of intestine, thereby causing ruffling response in the host cell membrane [5]. Ruffling is linked to a triggering response that results in macropinocytosis. The vesicles of macropinocytosis are large, and provide an efficient route for non-selective endocytosis of macromolecules [15]. The entry of the bacterial cells also harms the microvilli which are present on the cell surface, which causes disturbance in the white blood cells as they start to flood the mucosa thereby altering the homeostasis between absorption and secretion of the body [16].

S. enterica has a number of virulence factors like enterotoxin, endotoxin, and cytotoxin which are responsible for virulence. Enterotoxin causes diarrhea and vomiting in which the cells discharge huge amounts of fluid into the lumen. Endotoxin consists of toxic lipopolysaccharide which causes fever by inspiring host cells to discharge endogenous pyrogens. The cytotoxin interferes with protein synthesis in the host cell and leads to an efflux of calcium ions [15]. In some cases the infectious cells are transferred to the liver or spleen, where they multiply and return either to the host’s intestinal tract or get defecated [9].

Food contamination often occurs from infested feces that are present in polluted water, soil or other contaminated environments. S. enterica is responsible for causing many abdominal complications like gastroenteric disorders which involve stomach pain, cramps etc. When there is an imbalance between absorption and secretion, first signs include abdominal cramps, diarrhea, fever and nausea. Mostly the infection caused by food contamination cures within one week’s time, but more serious cases generally need fluoroquinolone or cephalosporin’s that eliminate S. enterica by interfering with their cell wall synthesis [17].

Presently only two species of the genus Salmonella have been sequenced for their whole genome: Salmonella bongori (two isolates), Salmonella enterica, forty three isolates including two isolates of Salmonella enterica subsp salamae, isolated from different sources, Salmonella enteric a subspecies salamae serovar 58:1, z13, z38:z6 strain 00-0163 [18] and Salmonella enterica subspecies salamae strain 3588/07 [Young et al. unpublished]. It is for the first time whole genome of strain DMA-1, isolated from mouse stool sample was sequenced by us. The whole genome sequencing was done in order to produce accurate reference genome which aids in identification and comparative genomic study. Moreover it also helps to analyze genes involved in virulence, disease and multidrug resistance.

It was for the first time, whole genome sequencing of the strain DMA-1, isolated from mouse stool sample was carried out by us and the strain was identified as Salmonella enterica sub species salamae. Further, the genome sequencing data was subjected to annotation and the analysis revealed the genes responsible for the pathogenesis, virulence, defence, metabolism and other genomic features. The whole genome of S. enterica sub species salamae comprised of 4,826,209 bp in size with a G + C content of 52.0 mol%, having 4,322 predicted CDSs and 69 RNAs. The final assembly contained 32 contigs of total size 4,826,209 bp with N₅₀ contig length of 4,24,412 bp; the largest contig assembled measured 6,24,758 bp.