Introduction

S. enterica is a major cause of foodborne infections worldwide, responsible for a significant disease burden, including diarrhea and severe multi-organ invasive diseases [1,2,3,4]. An analysis of the global burden of Non-typhoidal Salmonella (NTS) gastroenteritis estimates approximately 93.8 million cases annually, resulting in 155,000 deaths [5]. Alarmingly, the incidence of Salmonella infections has risen in recent years. For instance, the occurrence of NTS infections has shown an upward trend since 2012 in the Netherlands [6]. Similarly, in Zhejiang, China, the positive detection rate for Salmonella increased markedly from 1.69% in 2012 to 6.61% in 2021 [7].

Antibiotic resistance in S. enterica has become a critical challenge in clinical treatment. The widespread use of antibiotics has led to increased resistance among clinical isolates, particularly to third-generation cephalosporins and fluoroquinolones [8, 9], complicating treatment and increasing the risk of failure and mortality. According to the standard of Clinical and Laboratory Standards Institute (CLSI), Salmonella isolated from intestinal tract in medical institutions does not routinely carry out antimicrobial resistance test, and clinicians need to adopt treatment strategies according to the epidemiological situation in their areas. Therefore, understanding the prevalence, antimicrobial resistance patterns, and genetic characteristics of Salmonella strains is crucial for effective surveillance, treatment and control strategies.

In recent years, there has been increasing attention on the molecular epidemiology and mechanisms of drug resistance in clinical isolates of S. enterica [10,11,12]. However, there is a knowledge gap in antimicrobial resistance profile and molecular characteristics of Salmonella isolated from human in Zhejiang, China [7].

This study aims to address this gap by assessing the prevalence, distribution, and antimicrobial resistance profiles of Salmonella strains isolated in Hangzhou, China, during 2020–2021. Additionally, we analyzed the differences in antimicrobial resistance between adult and pediatric populations and investigated the genomic characteristics and resistance mechanisms, particularly focusing on cephalosporins and quinolones, key antibiotics used in treating Salmonella infections.

Materials and methods

Bacteria isolation

Between January 1st 2020 and December 31st 2021, a total of 285 Salmonella strains were collected from Affiliated Hangzhou First People’s Hospital. The source of patients included outpatient service (intestinal outpatient service, pediatric outpatient service and digestive medicine outpatient service) and ward. The stools of outpatients with diarrhea as the first symptom were cultured and Salmonella strains were preserved. Clinical samples of patients suffering from infectious disease in wards were sent for culture, and Salmonella strains were preserved. Strains isolated from the same patient within 7 days were defined as replicated strains and were excluded. Finally, 271 Salmonella strains were included in our study. A Vitek 2 system (BioMérieux, Marcy-l’Étoile, France) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) (Bruker, Bremen, Germany) were used for bacterial species identification.

Whole genome sequencing and analyses

Genomic DNA of 271 Salmonella strains were extracted using a QIAamp DNA minikit (Qiagen) and subjected to the Illumina HiSeq (Illumina, San Diego, USA) platform. Shovill was used for de novo assembly of raw reads. Short reads generated by Illumina sequencing were assembled by Shovill 0.9.0 (https://github.com/tseemann/shovill). Multi-locus sequence typing (MLST) was identified using mlst v2.19.0 (https://github.com/tseemann/mlst). Antibiotic resistance genes were identified using ABRicate v0.8.13 with the refinder database. Samonella Serotyping were conducted using Seqsero2 v1.1.0 (http://www.denglab.info/SeqSero2). Alignment of core genes from all 271 Salmonella isolates was performed with panaroo v1.2.7 (https://github.com/gtonkinhill/panaroo). IQ-TREE v2.1.2 was then used for building the maximum-likelihood phylogenetic tree with the best-fit model according to BIC (http://www.iqtree.org/). The phylogenetic tree was visualized using iTOL v6 (https://itol.embl.de/).

Antimicrobial susceptibility testing

Antimicrobial susceptibility tests (ASTs) with cefoxitin (FOX), CAZ, FEP, CRO, CTX, piperacillin/tazabactam (PTZ), cefoperazone/sulbactam (F/S), ceftolozane/tazobactam (C/T), meropenem (MEM), imipenem (IMP), ertapenem (ETP), aztreonam (AZT), amikacin (AMI), LEV, CIP, ceftazidime/avibactam (CZA), fosfomycin (FOS), colistin (COL) and tigecycline (TGC) were performed using the broth microdilution method according to the CLSI guideline M100 [13], and the results were interpreted on the basis of the breakpoints from the CLSI except for tigecycline, which was determined according to the FDA 2019 guideline (https://www.fda.gov/drugs/development-resources/tigecycline-injection-products).

Results

Bacteria isolation

During the period from January 1st, 2020, to December 31st, 2021, a total of 271 non-replicated Salmonella strains were successfully isolated. Among these isolates, the majority (263 strains) were derived from fecal samples, while 7 strains were obtained from blood samples and 1 strain from pleural puncture fluid. Of the total isolates, 209 strains were recovered from adults, whereas 62 strains were obtained from children (below 18 years old). The monthly distribution of isolates is graphically represented in Fig. 1, highlighting a concentration of strains between May and October (summer and autumn) annually.

Fig. 1
figure 1

Temporal distribution characteristics of 271 Salmonella strains originating from adults and children

Full size image

Salmonella serotyping, sequence typing and phylogenetic analyse

The 271 Salmonella isolates were classified into 46 different serotypes, as depicted in Fig. 2. The most prevalent serotype was S. Enteritidis, accounting for 27.68% of the isolates (75/271), followed by S. 1,4,[5],12:i:- at 18.08% (49/271), S. Typhimurium at 9.59% (26/271), S. London at 7.38% (20/271), S. Thompson at 4.43% (12/271), S. Goldcoast at 3.69% (10/271), S. Agona at 2.58% (7/271), S. Give at 2.21% (6/271), and S. Rissen at 1.85% (5/271). Additional serotype and bacterial information can be found in Table S1. Among the 209 isolates from adults, 41 serotypes were identified, with S. Enteritidis being the most prevalent at 28.23% (59/209), followed by S. 1,4,[5],12:i:- at 13.88% (29/209), and S. Typhimurium at 10.45% (21/209). Among the 62 isolates from children, 18 serotypes were identified, with S. 1,4,[5],12:i:- being the most common at 32.26% (20/62), followed by S. Enteritidis at 25.81% (16/62), and S. Typhimurium at 8.06% (5/62). Salmonella serotypes including S. Indiana, S. Jangwani, S. 1,4,[5],12:i:-, S. Ohio, and S. Sereban were exclusively isolated from children.

Fig. 2
figure 2

Phylogenetic analysis and molecular characteristics of 271 Salmonella strains. Phylogenetic tree of ST11 CRKP isolates generated by panaroo v 1.2.7 and IQ-TREE 2.1.2, including information about isolate source, serotype, ST type, ESBL/AmpC genes and antibiotic resistance profile. The ESBL includes blaCTX-M-55, blaCTX-M-65, blaCTX-M-14, blaCTX-M-27, blaCTX-M-130, and blaCTX-M-199

Full size image

Phylogenetic analysis was conducted on the 271 Salmonella isolates, resulting in the identification of 54 sequence types (ST). The majority of the isolates belonged to ST11 at 27.68% (75/271), followed by ST34 at 18.08% (49/271), ST19 at 8.86% (24/271), ST155 at 7.38% (20/271), ST26 at 4.43% (12/271), ST358 at 2.95% (8/271), ST13 at 2.58% (7/271), ST516 at 2.21% (6/271), and ST469 at 1.85% (5/271). Notably, all S. Enteritidis strains belonged to ST11, S. 1,4,[5],12:i:- belonged to ST34, S. London belonged to ST155, S. Thompson belonged to ST26, S. Agona belonged to ST13, S. Give belonged to ST516, and S. Rissen belonged to ST469. S. 1,4,[5],12:i:- and S. Typhimurium exhibited a close genetic relationship, with the majority of S. Typhimurium isolates (92.31%, 24/26) belonging to ST19.

Plasmid characteristic of 271 isolates

Our findings reveal the presence of 12 distinct plasmid replicon types, including Col, IncA/C, IncFI, IncFII, IncHI, IncI, IncN, IncQ, IncR, IncX, IncY, and p0111.

S. Enteritidis predominantly harbors the IncX type plasmid, found in 64 out of 75 isolates, followed by IncFII in 60, IncFI in 58, Col in 8, and IncI in 4. Notably, 44 isolates carry three plasmid replicons, and 9 carry four different types. Specifically, S. 1,4,[5],12:i:- exhibit a diverse plasmid profile, with 8 types identified, including Col in 19 out of 49, IncFI in 3, IncFII in 3, IncHI in 13, IncI in 5, IncQ in 27, IncR in 1, and p011 in 5. Seven of these isolates carry three plasmid replicon types. In the case of S. Typhimurium, the IncFII type is the most prevalent, found in 13 out of 26 isolates, closely followed by IncFI, also in 13. Other types present include Col in 4, IncA/C in 1, IncHI in 1, IncI in 1, and IncQ in 1. S. London isolates are characterized by the IncFI type plasmid in 12 out of 20, with Col, IncI, IncQ, IncR, and IncHI types also identified in varying frequencies. A detailed account of the plasmid profiles for other serotype isolates is provided in Table S2. It is important to highlight that 74 isolates lacked plasmid replicons, spanning a range of serotypes.

Antibiotic resistance profile

AST was conducted on 19 antibiotics, and 34 of the Salmonella strains were classified as MDR. The distribution of Minimum Inhibitory Concentrations (MIC) is presented in Table 1. Overall, the resistance rates of the 271 strains to the 19 antibiotics were below 25%. The resistance rate to FOX was 4.80%. Among cephalosporin antibiotics, the resistance rates to FEP, CAZ, CRO, and CTX were 7.38%, 9.23%, 15.87%, and 16.24%, respectively. Carbapenems exhibited high sensitivity, with sensitivity rates of 100%, 100% and 99.63% for MEM, IMP, and ETP, respectively. β-lactam-β-lactamase inhibitor combinations also demonstrated good sensitivity, with rates of 96.68% for PTZ, 90.77% for F/S, 100% for CZA, and 97.05% for C/T. The resistance rates to quinolones LEV and CIP were 8.49% and 19.19%, respectively, while the resistance rate to AMI was 0.74%. The resistance rates to AZT, FOS, COL, and TGC were 12.55%, 2.95%, 22.14%, and 0.37%, respectively.

Table 1 Distribution of MIC50 and MIC90 for 19 antibiotics of 271 Salmonella strains
Full size table

Differences in the antibiotic resistance profiles were observed between children and adult source isolates (Fig. 3a, b). Specifically, among the 19 antibiotics, strains isolated from children exhibited higher resistance rates, except for carbapenems, CZA, LEV, FOS, and TGC. The resistance rate of strains isolated from children to AZT was 27.42%. Notably, the resistance rates of strains isolated from children to CRO and CTX were both 30.65%.

Fig. 3
figure 3

a Resistance rates of 271 Salmonella strains to 19 antibiotics; b Comparative analysis of antibiotic resistance rates in Salmonella strains derived from children and adults; c Comparative analysis of antibiotic resistance rates in Salmonella strains derived from S. Enteritidis, S. 1, 4, [5], 12:i:-, S. Typhimurium, and S. London. Statistical analysis was performed with chi-square tests. *P < 0.05; **P < 0.01; ****P < 0.0001

Full size image

The comparative analysis of antibiotic resistance rates among various serotypes was conducted using a chi-square test, as depicted in Fig. 3c. For this comparison, we included serotypes with no fewer than 20 isolates: S. Enteritidis, S. 1, 4, [5], 12:i:-, S. Typhimurium, and S. London. These four predominant serotypes exhibited significantly distinct resistance profiles towards quinolones, cephalosporins, N-BIBIL, aztreonam, and colistin. Among them, strains of S. 1, 4, [5], 12:i:- showed the highest resistance rate to cephalosporins, at 28.57% (4/10), surpassing S. London with a 15% resistance rate (3/20), followed by S. Enteritidis at 8% (6/75), and S. Typhimurium at 7.69% (2/26). When it came to quinolone resistance, strains of S. London had the highest rate at 55% (11/20), followed by S. Typhimurium at 23.08% (6/26), S. 1, 4, [5], 12:i:- at 14.29% (7/49), and S. Enteritidis at 6.67% (5/ 75). Serotype S. 1, 4, [5], 12:i:- also presented the highest resistance rates towards both N-BIBIL (8.16%, 4/50) and aztreonam (24.49%, 12/50). In contrast, S. Enteritidis demonstrated the highest resistance rate towards colistin, with a striking 78.67% (59/75) of the isolates showing resistance. This detailed examination underscores the variability in antibiotic resistance among different serotypes of Salmonella, highlighting the importance of serotype-specific surveillance and antibiotic stewardship programs.

Resistance mechanism of cephalosporins and quinolones

A total of 34 isolates were found to carry ESBL genes (Fig. 4), which included blaCTX-M-55, blaCTX-M-65, blaCTX-M-14, blaCTX-M-27, blaCTX-M-130, blaCTX-M-199. Among these, thirteen isolates carried blaCTX-M-65, with the majority of them belonging to S. 1,4,[5],12:i:- (8/13). Twelve isolates carried blaCTX-M-55, distributed among S. Goldcoast (3/12), S. Agona (2/12), S. Indiana (2/12), S. Enteritidis (1/12), S. 1,4,[5],12:i:- (1/12), S. Typhimurium (1/12), and S. Muenster (1/12). Six isolates carried blaCTX-M-14, specifically found in S. Enteritidis (4/6) and S. 1,4,[5],12:i:- (2/6). One S. 1,4,[5],12:i:- isolate carried blaCTX-M-130, while one S. Kentucky isolate carried blaCTX-M-199. Notably, one S. Saintpaul isolate carried both blaCTX-M-27 and blaCTX-M-55.

Fig. 4
figure 4

Distribution characteristics of ESBL genes in 34 Salmonella strains

Full size image

A total of forty-three isolates exhibited resistance to cephalosporins (Fig. 5a). Among them, twenty isolates demonstrated resistance to both third and fourth generation cephalosporins, and all of these isolates carried ESBL genes. The predominant ESBL gene was blaCTX-M-55 (12), followed by blaCTX-M-14 (4), blaCTX-M-65 (2), blaCTX-M-199 (1), blaCTX-M-130 (1), and one isolate carried both blaCTX-M-27 and blaCTX-M-55. Among the remaining twenty-three isolates that were only resistant to third generation cephalosporins, thirteen carried ESBL genes, with blaCTX-M-65 (11) being the most prevalent, followed by blaCTX-M-14 (2). Eight isolates carried AmpC genes, specifically blaCMY-2 (6) and blaDHA-1(2). One isolate sa824 carried blaTEM-1 and the gene encoding penicillin-binding protein 3 (PBP3) sal, presenting resistant profile towards third generation cephalosporins, CRO and CTX.

Fig. 5
figure 5

a Resistance mechanisms to cephalosporins in 43 Salmonella strains. The strains were categorized into five groups based on their resistance profiles: I, Resistant to both CRO and CTX (sa748-sa792). sa824 carried genes encoding PBP3sal. II, Resistant to both CTX and CAZ (sa784). III, Resistant to CRO, CTX, and CAZ (sa848-sa945). IV, Resistant to CRO, CTX, and FEP (sa949-sa1034). V, Resistant to CRO, CTX, FEP, and CAZ (sa759-sa1030); b Resistance mechanisms to quinolones in 53 Salmonella strains. The strains were classified into three groups based on their resistance profiles: I, Resistant to CIP (sa821-sa971), with sa971 carrying mutations in parC(p.T57S). II, Resistant to LEV (sa1036). III, Resistant to both CIP and LEV (sa758-sa908)

Full size image

Fifty-three isolates exhibited resistance to quinolones (Fig. 5b). Among the thirty isolates that were solely resistant to CIP, eleven isolates carried qnrS1 as the underlying mechanism, ten isolates carried qnrB6 and aac(6′)-Ib-cr and one isolate showed parC (p.T57S) as potential mechanism. One isolate that carried qnrS1 was solely resistant to LEV. Twenty-two isolates displayed resistance to both CIP and LEV, with the majority (10/22) attributed to qnrS1 and two isolates presented mutations in gyrA and parC.

Discussion

In this study, we found that S. enterica isolated from clinical settings in the Hangzhou area were predominantly S. Enteritidis, with the emergence of MDR strains. The resistance rates to cephalosporins, commonly used clinical antibiotics, were 7.38% to 16.24%, and the resistance rates to quinolones were 8.49% to 19.19%. Notably, the resistance rate to cephalosporins was significantly higher in strains isolated from children compared to adults. The resistance of S. enterica to cephalosporins was mainly due to the carriage of ESBL genes, and the resistance to quinolones was mainly due to the carriage of the qnrS1 gene, which has important value for guiding usage of clinical antimicrobials and public health management in the local area.

We observed that the isolation rate of S. enterica strains in the Hangzhou area from 2020 to 2021 increased significantly between May and October each year. Similarly, a 10-year epidemiological investigation by the Zhejiang Provincial Center for Disease Prevention and Control (CDC) from 2012 to 2021 found that the isolation of Salmonella has a higher incidence in summer and the isolation rate increased year by year [7]. The possible reason for this analysis may be related to the opening of gastroenterology clinics during this period each year.

S. Enteritidis and S. Typhimurium serotypes are the main serotypes of Salmonella infections worldwide [14,15,16] and are prone to invasive infections [17]. S. 1,4,[5],12:i:- is a monophasic variant of S. Typhimurium [18], and was observed in our study, presenting a close genetic relationship with S. Typhimurium through phylogenetic analysis. In our study, the most prevalent serotype was S. Enteritidis, accounting for 27.68% of the isolates, followed by S. 1,4,[5],12:i:- at 18.08% and S. Typhimurium at 9.59%. Similarly, data from the China National Foodborne Disease Surveillance Network from 2010 to 2019 showed that S. Enteritidis is the most common serotype, followed by S. Typhimurium, and is prone to outbreaks [19]. Interestingly, we observed a difference in the prevalence of Salmonella serotypes between adults and children. In adults, S. Enteritidis was most common, followed by S. 1,4,[5],12:i:- and S. Typhimurium. Conversely, in children, S. 1,4,[5],12:i:- was the most prevalent, followed by S. Enteritidis and S. Typhimurium. S. Typhimurium was observed to be the predominant serotype in children [20, 21]. Serotyping of clinical strains is conducive to a better understanding of the local Salmonella epidemiology and is crucial for the prevention and control of infections. At the same time, for clinicians, understanding the Salmonella serotype can help them make informed decisions about disease diagnosis, progression, and prognosis.

With the widespread use of antibiotics, the resistance of Salmonella has increased year by year. Epidemiological investigations around the world have shown that the prevalence of MDR strains and even extensively drug-resistant (XDR) strains has emerged in some regions [22,23,24]. Salmonella is a leading cause of foodborne bacterial illnesses, primarily transmitted to humans through animal-derived foods [25]. In China, Salmonella is a widespread foodborne pathogen, posing a significant risk to food safety and public health [26]. Studies have revealed a high prevalence of multidrug-resistant (MDR) Salmonella strains in various food samples, along with numerous virulence factors [27]. Since 2016, there has been a growing concern about the emergence of Salmonella strains carrying the mcr-1 gene, which confers resistance to colistin, a last-resort antibiotic [28,29,30]. In this study, the resistance rates of 271 strains of S. enterica to third-generation cephalosporins (CRO and CTX) were 15.87% and 16.24%, respectively, and the resistance rates to quinolones (LEV and CIP) were 8.49% and 19.19%, respectively. Among the 271 S. enterica, 34 strains were MDR. Third-generation cephalosporins and quinolones are the first-line antibiotics for the clinical treatment of Salmonella infections [31]. Once invasive infections related to antimicrobial resistance occur, the use of effective antibiotics will be restricted. Fortunately, S. enterica in this region showed high susceptibility to β-lactam-β-lactamase inhibitor combinations and carbapenems.

Studies have shown that the antimicrobial resistance of Salmonella isolated from humans is related to the use of antibiotics in animal husbandry and food contamination [32]. The MDR patterns of Salmonella isolated from food are resistance to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole [33, 34]. According to the results of a global survey of foodborne diseases [35], blaCTX-M-14, fosA3, tet(A), and blaTEM-1 are common resistance genes in China, while qnrS1 is uncommon. Most of the Salmonella isolated from a slaughterhouse in China possess blaTEM-1 and/or blaCTX-M genes that are resistant to β-lactams, and the vast majority of quinolone-resistant strains have at least one mutation in gyrA or parC [36]. Similarly, we observed that the strains resistant to cephalosporins mainly carried ESBL genes predominantly blaCTX-M-65 and blaCTX-M-14. It is worth noting that one strain of S. Saintpaul carried blaCTX-M-27 and blaCTX-M-55 at the same time. Moreover, one of the isolates demonstrated resistance to CRO and CTX but did not possess the ESBL or AmpC genes. Upon analysis, it was noted that this isolate carried the PBP3sal gene, which is reportedly characterized by low affinity towards β-lactams [37, 38]. However, strains resistant to quinolones mainly carried the qnrS1 gene (27/53 strains). Most (10/22) of the strains that were resistant to both CIP and LEV were attributed to the carriage of qnrS1, and two strains that did not carry common resistance genes had gyrA and parC mutations. Among the 30 strains that were only resistant to CIP, it was shown that the carriage of qnrS1 (11/30), qnrB6 and aac(6')-Ib-cr (10/30) were the potential mechanisms, and one strain did not carry common quinolone resistance genes and had a parC (p.T57S) mutation to cause resistance (Fig. 5b). A recent report has shown that S. Worthington ST592, isolated from raw milk in Brazil, carries a conserved Col(pHAD28) plasmid, which contains the antimicrobial resistance determinant parC (p.T57S) that can confer resistance to CIP [39]. Our strain is S. London ST155, which also has a parC (p.T57S) mutation associated with CIP resistance. However, whether the parC (p.T57S) substitution contributes to quinolone resistance remains controversial [40]. We will conduct further analysis in future studies to identify the root cause. Studies have shown that the IncHI2 plasmid of Salmonella contains two class I integrons, which can simultaneously carry blaCTX-M-15 and blaOXA-1 resistance genes [41]. The presence of such a set of resistance genes on a mobile genetic element may increase the rapid spread of resistant pathogens in the environment. Other studies have shown that S. enterica carries blaCTX-M and qnrS genes through specific IncY plasmids to form XDR strains [42], which are resistant to both CRO and quinolones and lead to global dissemination, posing a major challenge to public health. We identified specific resistance genes and mutations associated with resistance to these antibiotics, revealing the genetic basis of antimicrobial resistance in S. enterica strains isolated clinically in the Hangzhou region. Understanding these resistance mechanisms is essential for selecting appropriate antibiotics and developing effective treatment strategies.

We have investigated the molecular basis of high colistin resistance among the 271 isolates, with a particular focus on the 60 isolates exhibiting resistance. Our findings indicate that none of the isolates carried the acquired colistin resistance mcr genes, nor were there any colistin-associated chromosomal point mutations detected. Among the colistin-resistant isolates, 59 were identified as S. Enteritidis, and one as S. Typhimurium. It is noteworthy that Group D Salmonella, including S. Enteritidis and S. Typhimurium, are known to exhibit a higher natural tolerance to colistin, as supported by the literatures [43, 44]. This observation is further corroborated by a recent report from the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC), which highlighted a significant association between colistin resistance and the prevalence of S. Enteritidis [45]. In S. Enteritidis and S. Typhimurium, the O antigens are structurally similar. Ricci et al. have reported that the O-antigen epitope in these Group D Salmonella species is a likely factor attributing to their colistin resistance [44]. This insight provides a plausible explanation for the high natural tolerance observed in our study.

This study also has some limitations. First, the study was conducted in a specific geographical area and may not fully represent the diversity and resistance patterns of Salmonella strains in other regions. Second, larger-scale studies are needed to more fully understand the epidemiology and resistance status of Salmonella nationwide or globally.

In general, our study investigated the epidemiology of S. enterica isolated clinically in a tertiary hospital in Hangzhou for two consecutive years and analyzed the characteristics of antimicrobial resistance. The results of the study revealed the molecular epidemiological characteristics of clinical S. enterica infections in this region and provided a theoretical basis for the resistance mechanisms of cephalosporins and quinolones. This is of great significance for clinical doctors to better use antibiotics empirically and for public health interventions and policy formulation. Further research is needed to delve into the underlying molecular mechanisms of antibiotic resistance in Salmonella to provide a more comprehensive theoretical basis for the prevention and treatment of Salmonella infections.