Salmonella is a Gram-negative, facultatively anaerobic, rod-shaped (bacillus) bacterial genus belonging to the Enterobacteriaceae family. They are non-spore forming, predominantly motile enterobacteria with peritrichous flagella [1]. Salmonella serovars are listed by the World Health Organization (WHO) as “High Priority Pathogens” (priority 2) which urgently require new antibiotics as they pose a significant threat to human health [2].

Salmonella genus comprises two major pathogenic species, Salmonella enterica and Salmonella bongori (Supplementary Figure S1). While the former has 6 subspecies, the latter has one subspecies [3]. More than 2500 serovars (alternatively, serotypes) of Salmonella are found in nature. These species are distinguished based on the combination of their surface antigens, somatic O (47) and flagellar H [H1(56) and H2 (20)] [4], [5]. Among them, Salmonella enterica subspecies enterica is responsible for about 99% of typhoidal and non-typhoidal human and animal infections [3], which also has the highest number of subspecies (nearly 60%). The Kauffmann-White-Le Minor (KW) scheme is a system that is used to classify Salmonella into various serotypes having different antigenic formulae (viz., the combination of O, H1 and H2) [6]. For instance, O-, H1- and H2-antigens having the combination O7, k and 1,5 antigens gives rise to the serovar Salmonella Thompson (antigenic formula - O7:k:1,5), whereas a change in even a single antigen (for example, from O7 to O8) results in a new serotype Salmonella Haardt (antigenic formula - O8:k:1,5). Based on the 47 O-antigen types, 2500 serovars are classified into 47 serogroups. The O-antigen type is determined based on the oligosaccharides associated with the lipopolysaccharide (LPS) which are structurally very different [5]. The H1- and H2-antigens depend on the flagellar proteins FliC and FljB which are alternatively expressed (viz., either H1 or H2 are expressed although both the genes are simultaneously present in the genome) as they exhibit phase variation [7], [8].

The periodic outbreak of Salmonella species [https://www.cdc.gov/salmonella/outbreaks.html][https://www.fda.gov/food/outbreaks-foodborne-illness/investigations-foodborne-illness-outbreaks] and their resistance against antibiotics [9] necessitate their systematic surveillance across the globe. Indeed, Salmonella spp. is under the Global Antimicrobial Resistance and Use Surveillance System (GLASS) of WHO [10]. Traditionally, the serotype of Salmonella spp. is identified experimentally by agglutination with specific antisera for O- and H-antigens which can be time-consuming and laborious depending on the isolate being identified [11]. To enhance flagellar antigenic expression and motility, some isolates require multiple passes through solid media. The situation becomes more complicated when serotype antigens are not expressed in some isolates, thus, limiting the efficiency of traditional serotyping. Since Salmonella spp. has a great number of serotypes, antisera testing is usually available for the most commonly found strains. This makes it difficult to experimentally detect uncommon strains or newly occurring strains [11]. Since conventional serotyping is a time-consuming process, in silico serotyping has become an alternative. Such serotyping relies on the genotype-serotype specificity, which has successfully been utilized in the serotyping of E. coli [12], Klebsiella spp. [13], Acinetobacter baumannii [14] and even Salmonella spp. [3], [15], [16], [17]. In this investigation, the protein/gene sequences involved in O-antigen biosynthesis and H-antigen formation are utilized in serotyping.