Comparison of Phenotype and Genotype Virulence and Antimicrobial Factors of Salmonella Typhimurium Isolated from Human Milk

Salmonella is a common foodborne infection. Many serovars belonging to Salmonella enterica subsp. enterica are present in the gut of various animal species. They can cause infection in human infants via breast milk or cross-contamination with powdered milk. In the present study, Salmonella BO was isolated from human milk in accordance with ISO 6579-1:2017 standards and sequenced using whole-genome sequencing (WGS), followed by serosequencing and genotyping. The results also allowed its pathogenicity to be predicted. The WGS results were compared with the bacterial phenotype. The isolated strain was found to be Salmonella enterica subsp. enterica serovar Typhimurium 4:i:1,2_69M (S. Typhimurium 69M); it showed a very close similarity to S. enterica subsp. enterica serovar Typhimurium LT2. Bioinformatics sequence analysis detected eleven SPIs (SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14, C63PI, CS54_island). Significant changes in gene sequences were noted, causing frameshift mutations in yeiG, rfbP, fumA, yeaL, ybeU (insertion) and lpfD, avrA, ratB, yacH (deletion). The sequences of several proteins were significantly different from those coded in the reference genome; their three-dimensional structure was predicted and compared with reference proteins. Our findings indicate the presence of a number of antimicrobial resistance genes that do not directly imply an antibiotic resistance phenotype.


Introduction
Salmonella enterica subsp. enterica serovar Typhimurium is a major cause of gastroenteritis and bacteraemia in humans [1,2] and has been included in the group of invasive non-typhoidal Salmonella (iNTS), frequently associated with human and animal diseases [3]. Infection with iNTS can be acquired through contaminated food and water and contact with animals, especially reptiles and amphibians. After infection, the incubation period is short, and gastrointestinal disease often develops within hours or days, during which S. Typhimurium develops alongside intestinal inflammation and has been found to survive and multiply in macrophages.
The invasiveness, virulence, and pathogenicity of Salmonella spp. have been attributed to genes located on Salmonella pathogenicity islands (SPI) [4]. In Salmonella, pathogenic islands such as SPI1 and SPI 2 include genes that are responsible for host-cell invasion, phagocytic cell inactivation, apoptosis, and alteration of intracellular transport pathways [5]. , navy for the contigs, green for the CDS on the forward strand, violet for the CDS on the reverse strand, turquoise RNA genes, red for the CDS with homology to known antimicrobial resistance genes, orange for the CDS with homology to know virulence factors, blue for the transporters genes, black for the drugs target genes, black line on orange background GC content, and black on purple GC skew (done with PATRIC-BV-BRC, https://www.bv-brc.org, accessed on 17 November 2022).
The annotation included 529 hypothetical proteins and 4235 proteins with functional assignments. Those with functional assignments included 1283 with Enzyme Commission (EC) numbers [26], 1048 with Gene Ontology (GO) assignments [27], and 900 proteins that were mapped to KEGG pathways [28]. PATRIC analysis indicated that the genome has 4647 proteins belonging to genus-specific protein families (PLFams) and 4673 belonging to cross-genus protein families (PGFams) [29].

Figure 1.
A circular graphical display of the distribution of the genome annotations of Salmonella enterica subsp. enterica Typhimurium 69M. The colors of the descriptive of the CDS elements of the Salmonella enterica subsp. enterica Typhimurium 69M were changed for the transparency of the view: grey for position label (MBP), navy for the contigs, green for the CDS on the forward strand, violet for the CDS on the reverse strand, turquoise RNA genes, red for the CDS with homology to known antimicrobial resistance genes, orange for the CDS with homology to know virulence factors, blue for the transporters genes, black for the drugs target genes, black line on orange background GC content, and black on purple GC skew (done with PATRIC-BV-BRC, https://www.bv-brc.org, accessed on 17 November 2022).

Figure 2.
A circular graphic of the IncFIB(S) plasmid found in the Salmonella Typhimurium 69M sequence. The length of the plasmid is 93926 bp, and the virulence genes carried by the IncFIB(S) are marked with maroon arrows (created in SnapGene 6.0.3 (www.snapgene.com on 2 February 2023)).

Analysis for Virulence and Antibiotic Resistance Genes
The sequence of S. Typhimurium 69M was determined using SPIFinder 2.0, the online bioinformatics tool of the Center for Genomic Epidemiology. The results identified eleven SPIs (SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-12, SPI-13, SPI-14, C63PI, CS54_island) with very high similarity (i.e., 98.58% to 100%) to the reference genome AE006468.2; it also identified other close matches regarding genes and regions of Salmonella spp. given in the NCBI Nucleotide Database, including JN673272, Z95891, AJ000509, Y13864, and AJ576316 (Table S1) [32]. All identified parts of the SPIs and their genes, together with their position in the contig and their accession number, are presented in Table 1. The S. Typhimurium 69M sequences were compared to the reference strain Salmonella Typhimurium LT2 genome. The results of fragment of SPI 1 with the mutS gene are given in Figure 3, with 100% matches marked in green, and differences such as deletions, insertions, and SNPs in red. Ten single nucleotide insertions and single nucleotide polymorphisms (SNPs) were detected in the mutS gene ( Figure 3). Despite these differences, the protein sequences generated by the mutS gene of the test strain demonstrated 98% and 100% similarity with the reference strains U16303 and S. Typhimurium LT2 (ProgramBlast 2 sequences [33]). The entire SPIfinder2.0 analysis is presented in the Supplementary Materials as Table S1. given in Figure 3, with 100% matches marked in green, and differences such as deletions, insertions, and SNPs in red. Ten single nucleotide insertions and single nucleotide polymorphisms (SNPs) were detected in the mutS gene ( Figure 3). Despite these differences, the protein sequences generated by the mutS gene of the test strain demonstrated 98% and 100% similarity with the reference strains U16303 and S. Typhimurium LT2 (ProgramBlast 2 sequences [33]). The entire SPIfinder2.0 analysis is presented in the Supplementary Materials as Table S1. The gene sequences obtained from S. Typhimurium 69M, particularly those within the identified SPIs, were analyzed using DNAPlotter [34]. It was found that the strain contains 221 genes responsible for virulence factors and antimicrobial resistance. The genes are located within 11 Salmonella pathogenicity islands ( Figure 4). SPI-1 includes 42 genes; SPI-2, 33 genes; SPI-3, three genes; SPI-4, nine genes; SPI-5, 10 genes; SPI-9, five genes; SPI-12, eight genes; SPI-13, four genes; and SPI-14, two genes (fixB and fixA). In addition, the CS54 island includes seven genes, and C63-PI includes five genes. The The gene sequences obtained from S. Typhimurium 69M, particularly those within the identified SPIs, were analyzed using DNAPlotter [34]. It was found that the strain contains 221 genes responsible for virulence factors and antimicrobial resistance. The genes are located within 11 Salmonella pathogenicity islands ( Figure 4). SPI-1 includes 42 genes; SPI-2, 33 genes; SPI-3, three genes; SPI-4, nine genes; SPI-5, 10 genes; SPI-9, five genes; SPI-12, eight genes; SPI-13, four genes; and SPI-14, two genes (fixB and fixA). In addition, the CS54 island includes seven genes, and C63-PI includes five genes. The location of individual pathogenicity islands and their genes, along with their arrangement on the forward and the reverse strands, are given in Figure 4.
The WGS analysis showed differences in the arrangement of 14 genes. Most of these, including pipB (SPI-2 type III secretion system effector PipB) and sspH2 (SPI-2 type III secretion system effector E3 ubiquitin transferase SspH2), were transferred from SPI-1 to SPI-5 and SPI-2 to SPI-12 ( Figure 4). The relevant SPIs are presented in Table S2, together with their component genes, gene length, strand direction, locus, and a brief description of the product and function.
Insertion 720_721insG Arg240_Lys241fs in yeiG (S-formylglutathione hydrolase gene) also creates a new STOP codon and the formation of a truncated protein. In addition, insertion 139_140insG Ile47_Asp48fs adds a new STOP codon, resulting in the formation of truncated YbeU protein ( Figure S3). In Figure S3 protein model with premature STOP codon in yeaL, rfbP, yacH, avrA, and ratB were present.
Insertion 1631_1632insC, Gly544_Ala545fs, deletes the STOP codon in the fumA gene, leading to the formation of a longer protein than in Salmonella Typhimurium LT2 ( Figure  S4). Table S4. Secondary structure of protein with insertions and deletions in gene of Salmonella Typhimurium LT2 and Salmonella Typhimurium 69 were described in Table S4.
A deletion was found in the gene encoding the long polar fimbrial operon protein (ipfD), i.e., 400_409delTTTGAGAATG, Phe134fs, which adds a new STOP codon. This results in the production of a truncated IpfD protein and loss of its transmembrane structure. Deletion 5807delT Met1936fs in ratB, encoding an outer membrane This was first thought to be the flipping of the O-antigen subunit on undecaprenylphosphate galactosephosphotransferase (EC 2.7.8.6) from the cytoplasm to the periplasmic space of the cytoplasmic membrane [37].
The detected insertion within the first half of the rfbP gene would result in a frameshift mutation leading to the creation of a STOP codon 25 codons below the frameshift. This would shorten the open reading frame to only 307 codons, compared to 476 codons in the reference-type rfbP gene.
Insertion 390_391insC, Val130_Arg131fs, creates a new STOP codon in the yeaL gene, resulting in the formation of a truncated Yeal multi-pass membrane protein and the disruption of its S4 and C-terminal parts. Similarly, insertion 67_68insT Ile23_Phe24fs causes a premature STOP codon in undecaprenyl-phosphate galactosephosphotransferase/Oantigen transfer protein gene rfbP; this results in the formation of a truncated RfbP protein and loss of transmembrane helix structure ( Figure 5).
Insertion 720_721insG Arg240_Lys241fs in yeiG (S-formylglutathione hydrolase gene) also creates a new STOP codon and the formation of a truncated protein. In addition, insertion 139_140insG Ile47_Asp48fs adds a new STOP codon, resulting in the formation of truncated YbeU protein ( Figure S3). In Figure S3 protein model with premature STOP codon in yeaL, rfbP, yacH, avrA, and ratB were present.
Insertion 1631_1632insC, Gly544_Ala545fs, deletes the STOP codon in the fumA gene, leading to the formation of a longer protein than in Salmonella Typhimurium LT2 ( Figure S4). Table S4. Secondary structure of protein with insertions and deletions in gene of Salmonella Typhimurium LT2 and Salmonella Typhimurium 69 were described in Table S4.
A deletion was found in the gene encoding the long polar fimbrial operon protein (ipfD), i.e., 400_409delTTTGAGAATG, Phe134fs, which adds a new STOP codon. This results in the production of a truncated IpfD protein and loss of its transmembrane structure. Deletion 5807delT Met1936fs in ratB, encoding an outer membrane protein/colonization factor, creates a new STOP codon and potentially leads to the synthesis of a truncated protein ( Figure 6). Deletion 850_857delGATCATCA Asp284fs creates a new STOP codon in the outer membrane gene (yacH) resulting in a truncated YacH protein sequence ( Figure 5). protein/colonization factor, creates a new STOP codon and potentially leads to the synthesis of a truncated protein ( Figure 6). Deletion 850_857delGATCATCA Asp284fs creates a new STOP codon in the outer membrane gene (yacH) resulting in a truncated YacH protein sequence ( Figure 5). Deletion 755delA Glu252fs in avrA again results in the creation of a premature STOP codon. This leads to the formation of a truncated Type III secretion injected virulence protein (YopP, YopJ), thought to be an inner-membrane protein ( Table 2).
The phenotype of S. Typhimurium was determined using VITEK2 software v 8.02, Biomerieux, Marcy-l'Étoile, France: β-lactams, wild; aminoglycosides, resistant to Ami Deletion 755delA Glu252fs in avrA again results in the creation of a premature STOP codon. This leads to the formation of a truncated Type III secretion injected virulence protein (YopP, YopJ), thought to be an inner-membrane protein ( Table 2).
In the Enterobacteriaceae, resistance to aminoglycoside (gentamycin) is associated with AAC(6 ), an aminoglycoside acetyltransferase encoded in the plasmid. The aac(6 )-I-cr variant gene can induce resistance against aminoglycoside and fluoroquinolone simultaneously. Our results also indicate the presence of the AcrAD-TolC efflux pump system, associated with aminoglycoside efflux. Although aminoglycoside resistance can also be caused by mutations within the gidB gene, causing changes in the structure of 16s rRNA, no such genetic changes were present in the investigated strain.
Other AMR mechanisms were also detected. These include AcrAB-TolC, a tripartite efflux system that confers resistance to tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin. This was accompanied by the AcrEF-TolC efflux pump system, involved in resistance to fluoroquinolones, and the MdtABC-TolC multidrug efflux system. The latter includes the macA gene, encoding the MacA membrane fusion protein, which forms an antibiotic efflux complex with MacB and TolC. Detailed functions of individual genes are provided in Tables S3 and S4.

Discussion
Human milk from the Women's Milk Bank is not available to private individuals: it is illegal to purchase breast milk or give it to a child outside the hospital premises. The milk collected by milk banks is intended primarily for premature babies or for sick infants as part of nutritional therapy. Therefore, it is very important that potential donors are screened for pathogens, including Salmonella enterica subsp. enterica serotype Typhimurium [15].
The investigated Salmonella strain from human milk could not be serotyped with classical methods according to the White-Kauffmann-Le Minor scheme; this approach is time-consuming and complicated, as it requires above one hundred and fifty specific antisera and well-trained personnel to interpret the results.
For the genus Salmonella, 47 O serogroups and 114 H antigens have been described according to the Kauffmann-White-Le Minor scheme [38,39]. The O antigen (polysaccharide O) is part of the lipopolysaccharide (LPS) component of the outer membrane. It is necessary for the survival of the bacteria and plays a role in the virulence of Salmonella spp. [39].
Often, precise identification is not possible due to the lack of well-expressed flagellar antigens, as was the case in our study. This problem is becoming increasingly common [1,22,40,41], and as such, a number of European countries have included WGS in their screening protocols, such as the UK Standards for Microbiology Investigations [42].
In the present study, the biochemical tests and serotyping of isolated pure colonies allowed only the strain to be identified to a subspecies, i.e., Salmonella enterica subsp. enterica. Subsequent PCR analysis indicated that the strain belonged to the Typhimurium serovar. However, the WGS data confirmed the presence of genes encoding the lipopolysaccharide (O antigen; encoded by rfb genes) and flagellar antigens (phases 1 and 2 of H antigen, encoded by fliC and fljB). The detected antigenic profile was characteristic for the strain of S. Typhimurium (4:i:1,2): O antigen 4, H1 antigen I, and H2 antigen 1,2, despite Salmonella Typhimurium 69M not revealing any flagellar antigens when tested with H sera.
RfbP belongs to a large family of bacterial membrane proteins required for initiation of O antigen synthesis, and which catalyze the transfer of galactose-1-phosphate to undecaprenyl phosphate (Und-P) [43]. The rfpB gene is involved in lipopolysaccharide biosynthesis; it encodes galactosyl transferase, which catalyzes the transfer of galactose to undecaprenol phosphate, which is involved in the initial step in O-polysaccharide synthesis. As noted by Kong et al. (2011), Salmonella with mutated wbaP (rfbP) were significantly attenuated compared to wild-type strains when administered orally to BALB/c mice and were less invasive in host tissues; the mutants also demonstrated substantially reduced bacterial motility [44]. Wand et al. reported the presence of a secondary translation starting within the rfbP gene, resulting in the synthesis of a polypeptide with GT activity [37]. These results indicate that the N-and C-terminal parts of RfbP are the T and GT functional domains, respectively.
The genes affected by the changes within the tested Salmonella Typhimurium 69M strain are believed to be responsible for the chemotaxis and motility of the bacterial cell, as well as its adhesion and colonization factors. One good example is the bapA gene; this encodes the BapA protein, which forms a biofilm together with cellulose, fimbriae, and the lpfD colonization factor, fimbrial gene lpfD, encoding the adhesin at the tip of the Lpf fimbriae [45].
The fumA gene (fumarase A) is responsible for switching flagellar rotation from one direction to another and is hence an essential part of bacterial chemotaxis. In cytoplasmfree bacterial envelopes containing CheY, fumarate has been shown to restore the ability of flagella to switch directions; it also increases the probability of reversal in intact cells. Fumarate acts as a switching factor, presumably by lowering the activation energy of switching; thus, fumarate modulates bacterial flagellar rotation during chemotaxis and plays a role in bacteria metabolism [46]. The encoded protein, FumA (fumarase A), plays an essential role in bacterial chemotaxis through switching the direction of flagellar rotation. Fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behavior [46].
The outer membrane protein yacH-CpxR is a conserved sensing system, i.e., regulated by genes associated with virulence; they are known to contribute to the resistance of E. coli to cationic antimicrobial peptide stress. In CpxR, extracellular protein transcription is reduced upon exposure to a sublethal dose of the cationic antimicrobial insect peptide cecropin A. Single-deletion strains (∆yacH) demonstrated better survival than wild-type strains after protamine challenge, suggesting that these target genes contribute to resistance to protamine in E. coli [47,48]. CpxRA is a two-component system that monitors envelope perturbations and responds by altering the gene expression profile to allow Salmonella to survive under harmful conditions. Therefore, CpxRA activation is likely to contribute to Salmonella gut infection. However, the role of the CpxRA-mediated envelope stress response in Salmonella-induced diarrhea is unclear. In S. enterica subsp. enterica serovar Typhimurium, it has been found that CpxRA is not needed for the induction of colitis, but is required for gut colonization [49].
In BALB/c mice, a strain of Salmonella Typhimurium with a deletion in the ratB gene was not able to colonize the cecum, but was still noted in Peyer's patches, the mesenteric lymph nodes, and spleen. In addition, mutations in shdA, ratB, and sivH resulted in a reduced ability to colonize intestinal tissues. The genes were encoded on the CS54 island and appear to be required for optimal colonization in the mouse cecum [50]. In contrast, despite large deletions and premature codon arrest, shdA from S. Typhi (shdA STy ) remained fully functional and was found to allow adherence and invasion in a fibronectin-producing epithelial cell line [51].
A significant change was found in yeiG, encoding S-formylglutathione hydrolase (esterase), which plays a role in L-glutamine production. The change resulted in alanine replacement in the protein YeiG, demonstrating that Ser145, Asp233, and His256 are essential for protein activity: the residues represent a serine hydrolase catalytic triad in the protein.
The enzyme also appears to contribute to the detoxification of formaldehyde, and may be involved in the degradation of methylglyoxal and/or other aldehydes [52,53].
The yeaL gene is responsible for the synthesis of the transmembrane protein in inter alia E. coli, Salmonella enterica subsp. Enterica, Klebsiella pneumoniae, and Yersinia enterocolitica [54]. The gene is also believed to involved in cell-wall biogenesis, which is required by S. Typhimurium to survive after desiccation [55].
Our findings indicate that 80.44% of Salmonella enterica subsp. Enterica showed aminoglycoside resistance facilitated by AAC(6 )-Ib-cr. The gene encodes an aminoglycoside acetyltransferase, which acetylates an amino group at position 6 in aminoglycoside. Despite the presence of a number of potential antibiotic resistant mechanisms, S. Typhimurium 69M was still sensitive to most antibiotics.
Moreover, one plasmid, IncFIB(S), carrying virulence genes spvB and spvC, associated with fimbriae (pefA, pefC, pefD, pefI) and vapB (type II toxin-antitoxin system VapB family antitoxin), was observed. The SpvB protein exhibits a cytotoxic effect on host cells and is required for delayed cell death by apoptosis following intracellular infection. The SpvC protein demonstrates phosphothreonine lyase activity and has been shown to inhibit MAP kinase signaling [56]. Interestingly, strains isolated from HIV positive patients, usually carry spv genes, strongly suggesting that CD4+ T lymphocytes are required to control disease caused by spv-positive Salmonella.
The IncFIB(S) plasmid belongs to the IncF family, which is widely distributed throughout the Enterobacteriaceae, in particular, Salmonella enterica subsp. Enterica. These plasmids carry a variety of virulence factors, such as AMR genes and adhesion factors [57]. In S. Typhimurium, resistance genes are carried predominantly on IncFII(S)/IncFIB(S)/IncQ1-type plasmids. The detected IncFIB(S) plasmid affects the virulence of this serovar but is not involved in antibiotic resistance because of the absence of the AMR gene (bla TEM , tet(A), dfrA15, sul1, catA1, strA/strB, addA1).
In the presence of antibiotic stress, Salmonella overexpresses the global activator protein MarA, which induces MDR efflux pump AcrAB, and downregulates the synthesis of the porin OmpF. In addition, S. Typhimurium 69M showed the presence of the marR gene encoding the MarR protein; this regulates the expression of marA, the activator of multidrug efflux pump AcrAB.
In the stress-response pathways, Salmonella Typhimurium alternates sensitivity to triclosan due to point mutations in the gyrA gene; similarly, point mutations in fabI used in lipid metabolism and fatty acid biosynthesis are disturbed by this biocide.
In addition to direct AMR mechanisms, the Salmonella genome expresses AcrAB-TolC, a tripartite efflux system that spans the cell membrane (AcrB) and the outer-membrane (TolC) and is linked together in the periplasm by AcrA. This efflux pump confers resistance to tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin. The cells also expressed AcrAD-TolC, associated with efflux of aminoglycosides, and the AcrEF-TolC efflux pump system, associated with resistance to fluoroquinolones.
The replacement of classical Salmonella spp. serotyping methods with molecular biology methods, especially WGS, has been extensively discussed in previous studies [58]. Our present analysis of the Salmonella enterica subsp. enterica serotype Typhimurium 4:i:1.2_69M sequence revealed the presence of numerous antimicrobial resistance genes. However, the strain turned out to be sensitive to all antibiotics, except for gentamicin, to which the Enterobacteriacea demonstrate natural resistance. This probably indicates that the strain was not subject to environmental pressure in terms of the presence of antibiotics. However, there is the genetic potential for activation of these genes. In the environment, such a strain may serve as an effective donor of antibiotic resistance genes for other bacteria.

Serological Testing
Serotyping was performed by slide agglutination according to the White-Kauffmann-Le Minor scheme [61]. Commercial H poly antisera were used to verify the genus of

Biochemical Strain Identification
The colonies demonstrating morphology typical of Salmonella spp. on selective agars were subjected to biochemical identification using VITEK2 COMPACT automated system for bacterial identification and VITEK ® 2 GN cards (Biomerieux, Marcy-l'Étoile, France). E. coli ATCC 25922, Salmonella Typhimurium ATCC 14028, Salmonella Enteritidis ATCC 13076, and P. aeruginosa ATCC 27853 were used as reference strains. Tests were performed according to the manufacturer's instructions.

Confirmation of Salmonella Identification with Molecular Biology Methods
DNA for Real-Time PCR was extracted from bacterial cells using a Kylt DNA Extraction-Mix II kit (Anicon, Emstek, Germany). A Kylt Salmonella spp. kit (Anicon, Emstek, Germany) was used to detect Salmonella spp., and a Spp-Se-St PCR kit (BioChek, Reeuwijk, The Netherlands) to detect Salmonella Enteritidis and Salmonella Typhimurium. Both Real-Time PCR tests were performed according to the manufacturer's instructions using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo, Waltham, MA, USA).
The general information about the assembly quality and gene content of Salmonella enterica subsp. enterica serovar Typhimurium 69M isolates and genomic components was obtained using the genomics tools of the Bacterial and Viral Bioinformatics Resource Center (BV-BRC, https://www.bv-brc.org accessed on 9 July 2022).
The serotypes of the isolated S. Typhimurium 69M strain were identified using a webbased tool: SeqSero 1.2 (https://cge.food.dtu.dk/services/SeqSero/ accessed on 9 July 2022) [30]. Bakta was used for rapid and standardized annotation of the bacterial genome and plasmids; this tool provides dbxref-rich, sORF-including, and taxon-independent annotations in machine-readable JSON and bioinformatics standard file formats for automated analysis of whole-genome sequences (Bakta version 1.4.2 was installed via BioConda with its native database publicly hosted at Zenodo [66]).

Antimicrobial Sensitivity Testing: Phenotypic Antibiotic Resistance
The antimicrobial susceptibility studies were performed as described previously [60] using a 96-well MICRONAUT plate in a VITEK2 Compact reader-incubator module, and AST-GN96 cards for gram-negative bacteria (BioMérieux, Marcy-l'Étoile, France). The AST card is a miniaturized and abbreviated version of the doubling dilution technique used to determine MICs by microdilution. The MICs were interpreted according to Clinical and Laboratory Standards Institute (CLSI) and FDA breakpoints (CLSI M100-ED28, 2018).

Conclusions
Salmonella serovars are typically classified based on serotyping according to the Kauffman and White classification scheme. However, some isolates require several passages through semi-solid media to enhance motility and flagellar antigenic expression. Some strains, like the S. Typhimurium 69M described herein, do not express serotype H antigens; S. Typhimurium 69M possesses a nine-nucleotide deletion (TTTGAGAATG, Phe134fs) in the ipfD gene (long polar fimbrial protein gene, part of the fimbrial operon), which creates a premature STOP codon. Pseudogene formation was also evident in a number of host adhesion factors. Two fimbrial genes including lpfD (encoding the tip adhesin of the Lpf fimbriae) and fimH (encoding the adhesin of the mannose-specific type 1 fimbriae) are inactive in the Sparrow MpSTM strain [45]. It was predicted that the mutated IpfD was truncated, i.e., with only 134 aa; this would result in the loss of its transmembrane structure, and thus potentially limit the value of traditional serotyping. In such cases, WGS Salmonella subtyping may ultimately prove to be more reliable and efficient. Although WGS serotyping requires higher technical and informational capacities, and remains expensive, it is clearly a very useful technique for both identifying Salmonella spp. strains and predicting the potential development of antibiotic resistance.