Molecular epidemiology of the nosocomial Salmonella Typhimurium ST328 clone based on whole-genome sequencing data

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Introduction

Salmonella enterica subsp. enterica remains one of the leading causes of acute bacterial gastroenteritis worldwide. Over the past decades, the global epidemiology of salmonellosis has been characterized by the predominance of two main serovars: S. Enteritidis and S. Typhimurium. In Russia and Belarus, the incidence of salmonellosis remains consistently high, at 13.7–55.0 cases per 100,000 population¹,².

A particular concern is the emergence and dissemination of high-risk clones with multidrug resistance and the ability to persist for prolonged periods in human populations [1, 2].

The first evidence of the circulation of third-generation cephalosporin-resistant strains of S. Typhimurium in Belarus dates back to the early 2000s. In 2001–2002, a series of outbreaks of nosocomial salmonellosis among young children was recorded in the Gomel region. The isolates exhibited a resistance phenotype unusual for that period, including resistance to extended-spectrum cephalosporins while remaining susceptible to carbapenems. Molecular genetic analysis revealed that the mechanism of resistance was the production of CTX-M-5 β-lactamase, and all strains, according to pulsed-field gel electrophoresis (PFGE) typing, belonged to a single genetic variant [3].

Subsequent studies have shown that the problem of CTX-M-5-producing strains of S. Typhimurium is not limited to Belarus. Molecular characterization of isolates using PFGE and multiple-locus variable-number-tandem-repeat analysis (MLVA) confirmed the clonal nature of the pathogen responsible for multiple geographically separated outbreaks. Parallel studies in Russia and Kazakhstan revealed the circulation of genetically related strains in a wide area. Systematic analysis of 88 cefotaxime-resistant isolates collected in 10 regions of 3 countries between 1996 and 2009 demonstrated the unprecedented long-term circulation of a single clone [4].

Typing using MLVA and multilocus sequence typing (MLST) showed that all 88 isolates belonged to ST328, a single-locus variant of ST19, the globally predominant sequence type. MLVA identified 17 types differing in 1–2 loci and indicated that the earliest isolates from St. Petersburg in 1996 could have been the progenitor strain, whose descendants spread across three countries. All isolates produced CTX-M-5 β-lactamase, associated with the mobile element ISEcp1 (insertion sequence Escherichia coli promoter 1) and localized on highly homologous plasmids of the pCTXM5 family. An important feature of the clone was its hypermutable phenotype, which led to a 10⁴-fold increase in the frequency of spontaneous resistance mutations and the independent acquisition of resistance to fluoroquinolones (FQ) in 43.2% of isolates through point mutations in the gyrA gene [5, 6].

The study of the S. Typhimurium ST328 clone reflects the evolution of methodological approaches in molecular epidemiology. Early research (2001–2004) was based on PFGE typing and PCR detection of resistance genes. The introduction of MLVA (2007–2012) increased discriminatory power, and MLST allowed local data to be integrated into a global context. However, all previous studies analyzed limited sets of genomic markers. The present study marks a transition to a fundamentally new level—whole-genome sequencing (WGS) with data integration into the specialized bioinformatics system EnteroBase [7].

EnteroBase³ is a global platform for storing and analyzing enterobacterial genomes, including more than 700,000 Salmonella genomes. The platform automatically performs genome assembly, annotation, serovar prediction and genotyping, identification of resistance determinants, MLST, ribosomal MLST (based on 51 ribosomal protein genes), and phylogenetic tree construction based on single nucleotide polymorphisms (SNPs) [8].

The application of WGS and EnteroBase to the ST328 clone allows us to refine the intraclonal phylogenetic structure, identify cryptic subgroups using the rMLST method, trace the global spread of the clone by comparing it with hundreds of thousands of genomes, establish the specificity of the association of bla_CTX-M-5 with the ST328 lineage across the entire S. enterica species population, and reconstruct the evolutionary history of the acquisition of resistance to various classes of antibiotics. Thus, this work represents a new stage in the study of the high-risk international clone S. Typhimurium ST328, based on modern genomic technologies and global bioinformatics resources.

The aim of the study was to characterize S. Typhimurium ST328 isolates based on genomic data, including determining its population structure, phylogeography, and mechanisms of antibiotic resistance in the context of the global S. Typhimurium population.

Materials and methods

The study included 11 non-duplicate isolates (no more than 1 isolate from 1 patient) of Salmonella enterica serovar Typhimurium recovered from clinical specimen (stool samples) of children aged 0–3 years hospitalized with acute gastroenteritis in 2002 in hospitals in Gomel, Belarus. The study is based on the analysis of archived bacterial isolates of Salmonella Typhimurium isolated in 2002 during routine diagnostic laboratory work. The study was conducted with the informed consent of the patients or their legal representatives. The research protocol was approved by the Ethics Committee of the Saint-Petersburg Pasteur Research Institute of Epidemiology and Microbiology (protocol No. 89, November 2, 2023).

Species identification of isolates was performed using commercial biochemical test systems, and serotyping was performed according to the White–Kauffman scheme using commercial agglutinating sera. The collection of isolates was stored at –70°C in broth with heart-brain extract and 30% glycerol until molecular genetic studies were performed. After recovery from frozen storage, the strains underwent no more than three passages.

Minimum inhibitory concentrations (MIC) of antibiotics were determined by the reference method of serial microdilutions in Mueller-Hinton broth, and susceptibility categories were interpreted in accordance with EUCAST v. 15 recommendations: S — susceptible (standard dosing regimen); I — susceptible with increased exposure; R — resistant⁴. Susceptibility to ceftazidime, meropenem, colistin, ciprofloxacin, and amikacin was determined.

Extended-spectrum β-lactamase (ESBL) production was detected using the double-disc method. A bacterial suspension (optical density 0.5 according to the McFarland standard) was spread on Mueller-Hinton agar. After 10 minutes, discs with amoxicillin/clavulanic acid (20/10 μg) were placed in the center and discs with ceftazidime and cefotaxime (30 μg each) were placed at a distance of 20 and 30 mm from the central disc. After incubation at 37°C for 18–20 hours, the presence of synergy between cephalosporins and the β-lactamase inhibitor was evaluated as phenotypic confirmation of ESBL production. In parallel with the analysis of the test cultures, control strains E. coli ATCC 25922 (ESBL-negative) and K. pneumoniae ATCC 700603 (ESBL-positive) were studied.

Genomic DNA was extracted using the QIAamp DNA Mini Kit (Qiagen). Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina). WGS was performed on the MiSeq platform (Illumina) with paired-end reads of 2 × 250 bp.

The raw paired-end reads were loaded into the Salmonella module⁵ of the EnteroBase database, where automated genome assembly (SPAdes), quality control, and annotation were performed. Serogenotyping was performed in silico using the SISTR (Salmonella In Silico Typing Resource) tool implemented in EnteroBase, which predicts Salmonella enterica serovars based on nucleotide sequences of antigenic and related genes, as well as cgMLST profiles [9].

Standard seven-gene MLST according to the Achtman scheme (aroC, dnaN, hemD, hisD, purE, sucA, thrA) was performed using the MLST module for sequence type (ST) determination built into EnteroBase [10]. According to the accepted MLST terminology, an isolate was considered a single-locus variant relative to another sequence type if their allele profiles differed in only one of the seven genes of the Achtman scheme. In this study, ST328 is considered a single-locus variant of the most common global genotype ST19 (difference in the aroC allele). Ribosomal MLST (rMLST) was also performed in EnteroBase based on the analysis of 51 ribosomal protein genes, which allowed ribosomal sequence types (rST) to be assigned to isolates and used to assess the intraclonal structure of ST328. rMLST provides higher discriminatory power compared to traditional 7-gene MLST and allows the identification of phylogenetic subgroups within a single ST [11, 12].

Determinants of antimicrobial resistance, including acquired resistance genes and chromosomal point mutations mediating resistance, were identified using the ResFinder 4.1 database [13]. To analyze the genomic context of the bla_CTX-M-5 gene in all assembled genomes of ST328 isolates, contigs containing this gene were additionally identified based on ResFinder results. These contigs were subjected to BLASTn analysis (NCBI nt) to determine the highest similarity to plasmid or chromosomal sequences of Salmonella and other enterobacteria. For each isolate, the presence of the flanking element ISEcp1, contig length, highest identity, and coverage were evaluated when aligned with known plasmids of the pCTXM5 family from previous studies.

A phylogenetic tree based on SNPs was constructed using EnteroBase tools. The analysis included 11 S. Typhimurium strains from the present study and 112 related S. Typhimurium ST328 strains represented in the EnteroBase database and recovered in various regions worldwide (Belarus, Armenia, Russia, Kazakhstan, Taiwan, Japan, Denmark, the United Kingdom, France, Canada, India, Ethiopia).

To construct the phylogenetic tree, the maximum likelihood algorithm with a nucleotide substitution model was used. The data were visualized using GrapeTree software integrated into EnteroBase. The tree displayed metadata for isolates, including country of origin, year of isolation, sequence types (ST and rST), presence of ESBL genes, and mutations associated with resistance to FQ.

Results

Phenotypic testing showed that 8 (72.7%) of 11 S. Typhimurium strains exhibited resistance or decreased susceptibility to third-generation cephalosporins (ceftazidime), with ceftazidime MICs ranging from 1 to 32 mg/L. ESBL production by the double disc method was detected in 7 (63.6%) of 11 strains. All isolates remained susceptibility to meropenem (MIC 0.016–0.060 mg/L) and colistin (MIC 0.06–0.25 mg/L).

Low-level fluoroquinolone resistance was detected in 1 (9.1%) of 11 strains: isolate No. 33 showed a ciprofloxacin MIC of 0.25 mg/L, which corresponds to the resistance category according to EUCAST criteria. The remaining 10 isolates had ciprofloxacin MICs of 0.03–0.06 mg/L, indicating continued susceptibility to this class of antibiotics (Table 1).

 

Table 1. MIC of antibiotics (mg/L), susceptibility categories, and production of ESBL by S. Typhimurium strains isolated during outbreaks of hospital-acquired salmonellosis in Gomel

Strain No.	Barcode (EnteroBase)	Year of isolation	Hospital	Ceftazidime	Meropenem	Colistin	Ciprofloxacin	Amikacin	ESBL
21	SAL_JC1502AA	2002	Hospital 1	4 (I)	0.016 (S)	0.125 (S)	0.06 (S)	4 (S)	+
22	SAL_JC1503AA	2002	Hospital 2	32 (R)	0.016 (S)	0.125 (S)	0.03 (S)	4 (S)	+
23	SAL_JC1504AA	2002	Hospital 3	4 (I)	0.03 (S)	0.25 (S)	0.03 (S)	8 (S)	+
25	SAL_JC1506AA	2002	Hospital 1	8 (R)	0.03 (S)	0.125 (S)	0.03 (S)	8 (S)	+
26	SAL_JC1507AA	2002	Hospital 1	1 (S)	0.03 (S)	0.125 (S)	0.03 (S)	8 (S)	–
27	SAL_JC1508AA	2002	Hospital 2	8 (R)	0.06 (S)	0.25 (S)	0.03 (S)	16 (R)	+
28	SAL_JC1509AA	2002	Hospital 1	4 (I)	0.03 (S)	0.25 (S)	0.03 (S)	4 (S)	–
29	SAL_JC1510AA	2002	Hospital 1	1 (S)	0.03 (S)	0.125 (S)	0.03 (S)	2 (S)	–
30	SAL_JC1511AA	2002	Hospital 1	4 (I)	0.03 (S)	0.125 (S)	0.06 (S)	4 (S)	+
31	SAL_JC1512AA	2002	Hospital 1	32 (R)	0.016 (S)	0.125 (S)	0.06 (S)	8 (S)	+
33	SAL_JC1514AA	2002	Hospital 1	1 (S)	0.016 (S)	0.06 (S)	0.25 (R)	4 (S)	–

 

WGS of all 11 isolates yielded high-quality genome assemblies with an average coverage of >50×. Serotyping by the SISTR method confirmed that all isolates belonged to serovar Typhimurium.

MLST typing according to the Achtman scheme showed that 9 (81.8%) of the 11 strains belonged to ST328. Two strains (isolates No. 29 and No. 33) were assigned to ST19, which is a globally widespread sequence type of S. Typhimurium and represents a single-locus variant of ST328 (difference in the aroC gene allele).

Ribosomal MLST typing demonstrated greater discriminatory power and allowed differentiation of isolates within ST328. All 9 ST328 strains were assigned to rST60463. Two ST19 strains belonged to a different ribosomal profile, confirming their phylogenetic separation from the main ST328/rST60463 cluster (Table 2).

 

Table 2. Genetic determinants of antimicrobial resistance and typing characteristics of S. Typhimurium isolates (ST, rST, HC50)

Strain No.	ST	rST	HC50	ESBL	Aminoglycosides	Chloramphenicol	Tetracyclines	FQ	Sulfonamides	Trimethoprim
21	328	60463	41	blaOXA-1, blaCTX-M-5	aadA1, aph(3')-Ia, ant(2'')-Ia	catA1, catB3	tet(B)	–	sul1	dfrA19
22	328	60463	41	blaCTX-M-5, blaOXA-1	aadA1, aph(3')-Ia, ant(2'')-Ia	catA1, catB3	tet(B)	–	sul1
23	328	60463	41	blaOXA-1, blaCTX-M-5	aadA1, ant(2'')-Ia	catA1	–	–	sul1	dfrA19
25	328	60463	41	blaCTX-M-5	aph(3')-Ia, ant(2'')-Ia	–	–	–	sul1
26	328	60463	41	blaOXA-1	aadA1, aph(3')-Ia, ant(2'')-Ia	catA1, catB3	tet(B)	–	sul1	dfrA19
27	328	60463	41	blaOXA-1, blaCTX-M-5	aadA1, aph(3')-Ia, ant(2'')-Ia	catA1, catB3	tet(B)	–	sul1
28	328	60463	41	blaOXA-1	aadA1, aph(3')-Ia, ant(2'')-Ia	catA1, catB3	tet(B)	–	sul1	–
29	19	3484	395	–	aph(6)-Id, aph(3'')-Ib	catA1, catB3	tet(A)	–	sul2	dfrA19
30	328	60463	41	blaCTX-M-5	aph(3')-Ia	–	tet(B)	–	–	–
31	328	60463	41	blaCTX-M-5	aph(3')-Ia, ant(2'')-Ia	–	tet(B)	–	sul1	dfrA19
33	19	3484	305	–	aadA2	catA1, catB3	tet(G)	gyrA_D87N	sul1	–

 

To facilitate comparison with global EnteroBase data, the HierCC cluster numbers at the HC50 level are indicated for each genome in Table 2. All rST60463 isolates belong to the HC50_41 cluster.

Analysis using ResFinder 4.1 revealed a diverse spectrum of resistance genes in the studied strains (Table 2). The bla_CTX-M-5 ESBL gene was detected in 7 (77.8%) of 9 ST328 strains, which fully concordant with the ESBL production phenotype. The bla_OXA-1 gene encoding oxacillinase was detected in 6 ST328 strains.

Aminoglycoside resistance genes were widespread: aadA1 (adenylyltransferase, resistance to streptomycin and spectinomycin) was detected in 6 strains, aph(3')-Ia (phosphotransferase, resistance to kanamycin and neomycin) in 8 strains, ant(2'')-Ia (nucleotidyl transferase, resistance to gentamicin and tobramycin) in 7 strains. However, only one strain ST328 (isolate No. 27), carrying all three genes simultaneously, showed phenotypic resistance to amikacin (MIC 16 mg/L).

Resistance to chloramphenicol was mediated by the catA1 (chloramphenicol acetyltransferase) and catB3 genes, detected in 8 and 7 strains, respectively. The tet(B) gene, encoding an efflux pump for tetracyclines, was present in 7 ST328 strains. Resistance to sulfonamides was provided by the sul1 gene in 9 strains, and resistance to trimethoprim was mediated by the dfrA19 gene (dihydrofolate reductase) in 6 isolates.

Chromosomal mutations in the gyrA gene, which determine resistance to FQ, were detected in only 1 ST19 strain (isolate No. 33), which had an Asp87Asn mutation in the quinolone-resistant determinant region. This mutation is associated with low-level resistance to ciprofloxacin (MIC 0.25 mg/L). All ST328/rST60463 strains had no mutations in the gyrA and parC genes and remained phenotypically sensitive to FQ.

Analysis of contigs containing bla_CTX-M-5 showed that in 6 of 7 ST328 strains carrying this gene, the corresponding contigs 7.5 thousand bp in length demonstrated > 99% identity and high coverage when aligned with the previously described plasmid pCTXM5-637 (S. Typhimurium ST328) from the studies by V.K. Kozyrev et al. [4, 5], indicating the plasmid localization of the ISEcp1::blaCTX-M-5 determinant. One strain (S. Typhimurium No. 23) was found to have a significantly longer contig carrying bla_CTX-M-5 together with ISEcp1. The size of the contig (length 502.1 kbp) containing the integrated ISEcp1::blaCTX-M-5 indicates chromosomal integration of the determinant in this strain. These data are consistent with the previously described model of coexistence of plasmid and chromosomal localization of bla_CTX-M-5 in the ST328 clonal lineage [5]. A phylogenetic tree constructed based on SNP analysis of 11 strains from this study showed a clear division into two main groups. Nine ST328/rST60463 strains formed a monophyletic cluster with minimal genetic differences (0–15 SNPs), indicating their clonal origin and recent epidemiological link. Two ST19 strains were located in a separate branch of the tree, demonstrating a phylogenetic distance of about 70 SNPs from the ST328 cluster. To assess the international spread of the ST328 clone, an extended phylogenetic analysis was performed, including 112 ST328 strains from the EnteroBase database, representing 10 countries. The analysis revealed the existence of two main phylogenetic subgroups within ST328, differing in their ribosomal profile: rST1344 and rST60463 (Fig. 1).

 

Fig. 1. A phylogenetic tree based on SNPs for 112 strains of S. Typhimurium ST328 represented in the EnteroBase database, showing rST assignment.

 

Subgroup rST1344 included 59 isolates from Taiwan, representing the dominant population in the Asia-Pacific region, as well as isolates from Japan (n = 6), Canada (n = 2), the United Kingdom (n = 2), Ethiopia (n = 2), and India (n = 1). Most rST60463 isolates belong to the HC50_41 cluster, while rST1344 isolates belong to the HC50_42541 cluster, consistent with their separation into two rST-defined groups.

None of the rST1344 strains had ESBL genes, indicating phenotypic sensitivity to extended-spectrum cephalosporins (Fig. 2).

 

Fig. 2. Presence of ESBL genes on the phylogenetic tree based on SNPs for 112 strains of S. Typhimurium ST328 represented in the EnteroBase database.

 

Subgroup rST60463 showed a more limited geographical distribution but was characterized by the presence of bla_CTX-M-5 ESBL-encoding genes. This subgroup included 8 strains from Belarus (including 7 isolates from the present study), isolates from Armenia collected between 1996 and 2016, strains from Denmark (2005–2007), and 1 isolate from France (2019).

Temporal analysis showed that the earliest ST328/rST60463 strains carrying the bla_CTX-M-5 gene were isolated in 1996 in Armenia (Fig. 3). Based on the available data, this observation suggests the Transcaucasian region as one of the places where this resistant lineage was first detected.

 

Fig. 3. Phylogenetic tree based on SNPs for 112 strains of S. Typhimurium ST328 represented in the EnteroBase database, indicating the years of strain isolation.

 

The Belarusian strains from 2002 are among the earliest representatives of the clone in Eastern Europe. The detection of the French isolate from 2019 indicates the continued circulation of the ST328/rST60463/ bla_CTX-M-5 lineage more than 20 years after its first detection. A search for bla_CTX-M-5 gene carriers among all 713,000 Salmonella strains represented in the EnteroBase database as of September 2025 revealed that this gene is detected exclusively in S. Typhimurium ST328/rST60463 strains. This indicates a specific association of bla_CTX-M-5 with this genetic lineage and the absence of widespread horizontal transfer of this gene between different serovars and genetic groups of Salmonella.

Analysis of the distribution of mutations in the gyrA gene among 112 ST328 strains from the global database revealed a significant diversity of point mutations leading to amino acid substitutions at positions 83 and 87. Different types of mutations (Asp87Asn, Asp87Gly, Asp87Tyr, Ser83Phe) were distributed throughout the ST328 phylogenetic tree without forming monophyletic clusters (Fig. 4).

 

Fig. 4. Fluoroquinolone resistance-associated mutations on the phylogenetic tree based on SNPs for 112 strains of S. Typhimurium ST328 represented in the EnteroBase database.

 

This distribution indicates that resistance to FQ was acquired independently by multiple strains during the evolution of the ST328 genetic lineage, rather than as a result of a single mutation event followed by clonal spread. This pattern is consistent with the hypermutable phenotype of ST328 strains, which is characterized by an increased frequency of spontaneous mutations (four orders of magnitude higher than that observed in non-mutator strains) and contributes to rapid adaptation to fluoroquinolone antibiotic therapy.

Among the Belarusian isolates from this study, mutations in gyrA were not detected in ST328/rST60463 strains, which may be explained by the absence of selective pressure from FQ during the period of isolation of these strains (2002). However, the potential for the development of such resistance remains due to the mutator phenotype of the clone.

Discussion

This study presents the first characterization of the high-risk international clone S. Typhimurium ST328 in Belarus using modern WGS methods and the global EnteroBase database. The results demonstrate that most S. Typhimurium isolates from children with nosocomial salmonellosis in Gomel in 2002 belong to the ST328/rST60463 clone, characterized by CTX-M-5 ESBL production and multidrug resistance.

ST328 is a single-locus variant of the globally predominant ST19 genotype of S. Typhimurium, differing from it in the aroC gene allele. Despite its close phylogenetic relationship, ST328 exhibits unique phenotypic characteristics, including association with nosocomial outbreaks and a specific spectrum of antibiotic resistance. Previous studies have demonstrated the long-term circulation of the ST328 clone in Russia, Belarus, and Kazakhstan between 1996 and 2009. V.K. Kozyreva et al. showed that 88 isolates from multiple outbreaks and sporadic cases of nosocomial salmonellosis in various regions of the three countries belonged to ST328 and produced CTX-M-5 β-lactamase [5]. The results of this study confirm and expand on these data, demonstrating the circulation of the clone in the Gomel region of Belarus.

Use of the international EnteroBase database placed Belarusian isolates in a global phylogenetic and epidemiological context. Analysis of 112 ST328 strains from 10 countries revealed that the clone has an international distribution covering Europe, Asia, Africa, and North America. However, within ST328, there is a clear differentiation into two main subgroups (rST1344 and rST60463), which differ in geographical distribution, sources of isolation, and antibiotic resistance profiles.

One of the key findings of this study is the confirmation of a specific association between the bla_CTX-M-5 gene and the ST328/rST60463 subgroup. When searching among more than 713,000 Salmonella genomes in EnteroBase, all bla_CTX-M-5 carriers belonged exclusively to ST328/rST60463. This contrasts sharply with other CTX-M-β-lactamase variants, such as CTX-M-2, CTX-M-15, and CTX-M-65, which are widespread among various Salmonella serovars and demonstrate extensive horizontal transfer [14].

The limited spread of bla_CTX-M-5 may be explained by the peculiarities of the genetic structure carrying this gene. Previous studies have shown that the blaCTX-M-5 gene in ST328 strains is located on small (7.4–12.4 thousand bp) non-mobilizabed plasmids of the pCTXM5 type. These plasmids do not contain the tra operon and can only be transmitted in the presence of helper conjugative plasmids, which limits their spread [5]. At the same time, some ST328 strains show transposition of the ISEcp1::blaCTX-M-5 determinant into the chromosome, which ensures stable inheritance of resistance but excludes horizontal transfer [15].

The WGS data confirm that in most of the studied ST328 isolates, bla_CTX-M-5 retains its characteristic association with small non-mobilized pCTXM5 plasmids, while one of the strains shows signs of chromosomal integration of ISEcp1::blaCTX-M-5. This combination of plasmid and chromosomal localization of the gene, previously demonstrated by V.K. Kozyreva et al. for ST328 strains from Russia, Belarus, and Kazakhstan [4, 5], probably contributes to the long-term stability of bla_CTX-M-5 carriage while minimizing the risk of horizontal transfer outside the clonal lineage.

Interestingly, the Belarusian strains from 2002 are among the earliest isolates of ST328/rST60463/ bla_CTX-M-5, along with the Armenian strains from 1996. This suggests that Eastern Europe and the South Caucasus may have been the center of origin or early dissemination of this resistant lineage. The subsequent detection of strains in Denmark (2005–2007) and France (2019) indicates the gradual spread of the clone to Western Europe, probably through population migration or international trade.

The ST328 strains from this study demonstrated a wide range of resistance determinants beyond the production of ESBL. The presence of genes for resistance to aminoglycosides (aadA1, aph(3')-Ia, ant(2'')-Ia), chloramphenicol (catA1, catB3), tetracycline (tet(B)), sulfonamides (sul1), and trimethoprim (dfrA19) forms a multidrug resistance phenotype. Such multidrug resistance is characteristic of nosocomial Salmonella strains and reflects adaptation to the selective pressure of antibiotics in the hospital environment. Previous studies have shown that the determinants of resistance to non-β-lactam antibiotics in ST328 are located on large conjugative plasmids, which enable the co-transfer of multiple resistance genes.

Of particular concern is the ability of the ST328 clone to acquire resistance to fluoroquinolones, the first-line agents for invasive salmonellosis in adults. Although in the present study only one strain (ST19) carried mutations in gyrA, a global analysis showed that 42% of all ST328 strains in three countries (Russia, Belarus, Kazakhstan) showed resistance to nalidixic acid and low-level resistance to ciprofloxacin [16].

A key factor contributing to rapid adaptation to FQs is the hypermutable phenotype of ST328 strains. The frequency of spontaneous mutants resistant to nalidixic acid in ST328 is about 1 × 10^–5, which is 4 orders of magnitude higher than in normomutable strains of S. Typhimurium [16]. Hypermutability is caused by defects in DNA repair systems and is widespread among nosocomial pathogens, contributing to their survival under conditions of intensive antibiotic use.

The diversity of mutations in gyrA (Asp87Asn, Asp87Gly, Asp87Tyr, Ser83Phe) and their distribution throughout the ST328 phylogenetic tree confirm the independent multiple emergence of resistance to FQ, rather than the clonal spread of a single resistant variant. This poses serious challenges for controlling the spread of resistance, as selective pressure from FQ can lead to convergent evolution of resistance in different geographic regions [17, 18].

The application of the international EnteroBase database proved to be important for understanding the population structure, evolution, and global distribution of the ST328 clone. EnteroBase provides an integrated platform for automated genome assembly, multiple typing schemes (MLST, rMLST, cgMLST), serogenotyping, and phylogenetic analysis. The advantages of EnteroBase include scalability — the database contains more than 713,000 Salmonella genomes and is automatically updated daily by scanning public repositories; standardization — the use of unified algorithms ensures comparability of results between laboratories; open access — the ability to upload your own data and compare it with the global genome collection; hierarchical clustering — automatic identification of population groups at various taxonomic levels [19].

The application of rMLST has proven particularly valuable for identifying phylogenetic substructures within ST328. While standard 7-gene MLST combines all isolates into a single ST328, rMLST based on 51 ribosomal protein genes allowed the differentiation of two main subgroups (rST1344 and rST60463) with different phenotypic characteristics. The high concordance between MLST-based eBG (eBurst Groups) and rMLST-based reBG (Adjusted Rand index 0.992) confirms that these groupings correspond to natural evolutionary populations.

Phylogenetic analysis based on SNPs provided high resolution for assessing epidemiological links and reconstructing the pathways of international spread of the clone. The small number of SNP differences (0–15) between Belarusian isolates from 2002 indicates their recent common origin and possible nosocomial circulation within a single outbreak.

This study has a number of limitations. The small sample size (11 strains) from a single geographical region and limited time period does not allow for a full assessment of the microevolution of the clone in Belarus. The lack of available preserved epidemiological data on links between cases makes it difficult to reconstruct chains of transmission.

Conclusion

The study presents the first characterization of the high-risk international clone S. Typhimurium ST328/rST60463 in Belarus using WGS and the EnteroBase database. A total of 81.8% of isolates from children with nosocomial salmonellosis in Gomel in 2002 belong to the ST328/rST60463 clone, characterized by the production of CTX-M-5 β-lactamase and multiple drug resistance.

Analysis of the global phylogenetic context, including 112 ST328 strains from 10 countries, revealed a specific association of the bla_CTX-M-5 gene with the rST60463 subgroup, represented in Belarus, Armenia, Denmark, and France. A search of more than 713,000 Salmonella genomes in EnteroBase confirmed that bla_CTX-M-5 carriers are found exclusively among ST328/rST60463 strains, indicating that there is no widespread horizontal transfer of this gene between different genetic lineages. The Belarusian strains from 2002 are among the earliest isolates of ST328/rST60463/ bla_CTX-M-5, suggesting a possible role of Eastern Europe and the Caucasus in the early dissemination of this clone. The identification of French isolates in 2019 indicates the continued circulation of the lineage more than 20 years after its first detection.

Multiple drug resistance, a hypermutable phenotype, and the potential to acquire resistance to fluoroquinolones define ST328 as a clone of high epidemiological risk. Ribosomal MLST has proven to be a valuable tool for identifying phylogenetic substructures within sequence types defined by standard MLST.

The results emphasize the need to strengthen genomic surveillance of nosocomial infection pathogens using international databases for the timely detection of high-risk clones and the development of effective infection control strategies.

 

¹ On the state of sanitary and epidemiological well-being of the population in the Russian Federation in 2023: State report. Moscow; 2024. 368 p.

² Report "On the sanitary and epidemiological situation in the Republic of Belarus in 2024." Minsk; 2025. 178 p.

³ URL: https://enterobase.warwick.ac.uk

⁴ EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. Version 15.0;2025. URL: https://eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_15.0_Breakpoint_Tables.pdf

⁵ URL: https://enterobase.warwick.ac.uk/species/index/senterica

About the authors

Dmitry V. Tapalski

Institute of Physiology of the National Academy of Sciences of Belarus

Author for correspondence.
Email: tapalskiy@gmail.com
ORCID iD: 0000-0002-9484-7848

Dr. Sci. (Med.), Professor, Director

Belarus, Minsk

Elena V. Karpova

Institute of Physiology of the National Academy of Sciences of Belarus

Email: lenakarpova1108@gmail.com
ORCID iD: 0000-0002-3952-6187

Cand. Sci. (Med.), Associate Professor, Head, Center for medical microbiology and antibiotic resistance

Belarus, Minsk

Mariia A. Makarova

Saint-Petersburg Pasteur Research Institute of Epidemiology and Microbiology; North-Western State Medical University named after I. I. Mechnikov

Email: makmaria@mail.ru
ORCID iD: 0000-0003-3600-2377

D. Sci. (Med), Assistant Professor, senior researcher, Head, Laboratory of enteric infection, professor, Department of medical microbiology

Russian Federation, St. Petersburg; St. Petersburg

Lidiya A. Kaftyreva

Saint-Petersburg Pasteur Research Institute of Epidemiology and Microbiology; North-Western State Medical University named after I. I. Mechnikov

Email: kaflidia@mail.ru
ORCID iD: 0000-0003-0989-1404

D. Sci. (Med), Head, Microbiological department, Professor, Department of medical microbiology

Russian Federation, St. Petersburg; St. Petersburg

References

Supplementary files