Non-typhoidal Salmonella (NTS) has emerged as a leading cause of infectious diarrhea globally, underscoring its significance as both a foodborne and zoonotic pathogen. Reports indicated an alarming prevalence of NTS invasive disease, with an estimated 535,000 cases and 77,500 deaths attributed to this pathogen in 2017 alone, which indicated a mean all-age case fatality rate of approximately 14.5% (GBD 2017 Non-Typhoidal Salmonella Invasive Disease Collaborators, 2019). In the United States, the annual incidence was staggering, with around 1.35 million cases of NTS reported, resulting in approximately 26,500 hospitalizations and 420 fatalities (Lewis et al., 2023). In recent years, certain high-income countries like the United States, Australia, and Sweden have reported outbreaks of foodborne diseases (Ford et al., 2023; Kerr et al., 2022; Jansson Mörk et al., 2022). Additionally, Europe ranked NTS as the second most common zoonosis (European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2023), while data from the Chinese Center for Disease Control and Prevention (CDC) revealed that up to 80% of bacterial foodborne illnesses stem from Salmonella, highlighting its pervasive role in public health crises across various regions (Hu et al., 2022; Wang et al., 2021). In Guizhou province, food poisoning incidents attributable to Salmonella are common, underscoring the pathogen’s status as the leading cause of foodborne illness and infectious diarrhea in the region (Wei et al., 2023; Zhou et al., 2024). Studies have identified Salmonella as a prevalent etiological agent in diarrheal diseases across Guizhou province, revealing a significant public health threat that necessitates urgent attention and intervention.

The clinical manifestations of salmonellosis generally include diarrhea, fever, abdominal cramps, and vomiting, illustrating that while the disease is often self-limiting, certain populations—particularly those with compromised immune systems—are susceptible to severe complications (Wang Z. et al., 2024). While antimicrobial treatment was frequently unnecessary in most cases, its importance escalates in severe infections, especially amongvulnerable populations. Alarmingly, the global rise of antimicrobial resistance (AMR) poses a significant threat, with XDR Salmonella isolates emerging across multiple countries, including China, Australia, Italy, and Egypt (Chen et al., 2022; Liu B. et al., 2024; Cummins et al., 2020; Russo et al., 2022; Algammal et al., 2023; Sallam et al., 2024). The prevalence of XDR Salmonella, drastically limited treatment options and exacerbated clinical outcomes, emphasizing an urgent need for comprehensive understanding of the molecular mechanisms that underpin antibiotic resistance. In Guizhou province, China, the situation is particularly critical, as evidenced by a multidrug resistance rate of 86.7% among clinical Salmonella isolates from 2013 to 2017, with 4.4% categorized as extensively drug-resistant (Wei et al., 2023). Recent study on food revealed that a significant proportion of Salmonella isolates from retail food sources exhibited resistance to multiple antibiotic classes (Zhou et al., 2024). These data highlights the importance of understanding the underlying resistance mechanisms that facilitate the survival and proliferation of Salmonella. However, the available literature was lacking in comprehensive analyses of the molecular epidemiological characteristics and prevalence of resistance genes among clinical XDR Salmonella isolates in Guizhou. The gap in information limited the development of effective intervention strategies.

This study aimed to fill this critical gap by systematically investigating the molecular epidemiology and resistance patterns of XDR Salmonella isolates from clinical samples in Guizhou province. By exploring the incidence rates of resistance genes in clinical isolates, the findings served as a pivotal reference for public health strategies to prevent and manage Salmonella infections. Furthermore, this research informed the judicious use of antimicrobial therapies, contributing to efforts to mitigate the burgeoning public health challenge of antimicrobial resistance.

A tremendous increase in antimicrobial resistance among Salmonella isolates poses a significant global concern (Algammal et al., 2023; Chen et al., 2022; Cummins et al., 2020; Karampatakis, 2024; Liu B. et al., 2024; Sallam et al., 2024). In Guizhou province, surveillance data revealed a concerning escalation in XDR Salmonella prevalence, with detection rates climbing from 4.4% during 2013–2018 to 5.5% in the subsequent 2019–2023 period (Wei et al., 2023). Notably, our study revealed an accelerated upward trajectory, showing the detection rate of XDR Salmonella isolates nearly doubling from 3.8% in 2019 to 7.4% by 2023 within the same province. Due to their resistance to multiple antibiotic agents, the effective treatment of Salmonella infections was threatened, thus increasing the morbidity and mortality of patients. However, in 2022, the number of XDR isolates decreased, which may be attributed to environmental hygiene measures, less eating out, less travel, and less turnover during the pandemic of COVID-19. These changes highlighted the critical role of human mobility in the spread of resistant pathogens. In this study, we focused on XDR Salmonella isolates from humans because of the lack of data on this particular Salmonella phenotypes and resistance genes in Guizhou province and due to its severity in cases of outbreaks and surveillance. Limited data are available on genomic resistance characterization. Therefore, we investigated 51 XDR human Salmonella isolates from Guizhou province between 2019 and 2023 regarding genomic characterization, phylogenetic relationships, and resistance genes on chromosomes and plasmids. Understanding the underlying drug resistance mechanisms of these pathogens helps us finding solutions for the long-standing antibiotic resistance problem. This is the first study on genomic epidemiology of XDR human Salmonella isolates in Guizhou province, China.

In the present study, we noted that the prevalence of XDR Salmonella varied significantly across different regions, with the highest proportion of XDR isolates observed in Zunyi city, reaching up to 35.3%. These variations might be attributed to a combination of factors, including the distinctive geographical characteristics, the diverse habits of food consumption among local populations, as well as the climatic conditions. The climatic conditions in each region, including temperature, humidity, and seasonal changes, can greatly affect the survival and proliferation of XDR Salmonella. Zunyi city, located in the northern region of Guizhou province, is warmer and more humid conditions, which may contribute to a longer survival period for pathogens outside their host, potentially accelerating their transmission rates. Serological identification of 51 XDR Salmonella isolates revealed that S. 1,4,[5],12:i:-, S. Enteritidis, and S. Kentucky were the most frequent serovars, with ST34, ST11, and ST198 being the predominant sequence type, which was consistent with previous reports in Guangdong, Jiangsu, and other European countries, indicating a widespread clonal (Sun et al., 2024; Liu B. et al., 2024; European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2019). These findings highlight the critical role of regional-specific environmental and behavioral factors in shaping the distribution and prevalence of XDR Salmonella.

The WGS analysis of 51 XDR Salmonella isolates revealed an extensive AMR landscape, with 105 ARGs conferring resistance to 15 antimicrobial classes. When examining genetic determinants contributing to resistance against FQs, we noted the presence of double mutations in the gyrA (D87 and S83) and one mutation in parC (S80I) in XDR isolates. These mutations were regarded as significant target-site alterations, which was consistent with findings from previous studies (Abd El-Aziz et al., 2021; Chen et al., 2024; Petrin et al., 2023). Furthermore, the PMQR genes determinants lead to decreased susceptibility to FQs and facilitate the selection of higher levels of FQ resistance (Chang et al., 2021; Vázquez et al., 2022). In the current study, eight PMQR genes were detected, with qnrS being the most prevalent in 47.1% of the isolates. Additionally, qnrS1 was identified among the examined isolates from various serovars, consistent with previous studies (Chen K. et al., 2020; Chen Z. et al. 2020). Meanwhile, it was important to note that qnr genes were found on multiple plasmids, which can disseminate across different regions and sources (She et al., 2023). In our study, 75.0% of the identified conjugative plasmid types were IncHI2/IncHI2A. Previous studies indicated that the IncHI2/IncHI2A plasmid was an essential lineage contributing to the spread of antibiotic resistance in Salmonella, suggesting that qnr genes could undergo horizontal gene transfer through conjugation between bacteria, thus promoting the spread of resistance genes (She et al., 2023).

In our study, the bla_TEM-1 gene conferring ampicillin resistance was the most common β-lactam resistance gene, consistent with its global dominance in Salmonella serovars (Chen et al., 2024). Meanwhile, we identified four subtypes of bla_CTX-M, a critical resistance gene family associated with third-generation cephalosporins resistance. Among these, bla_CTX-M-55 gene was predominant, mirroring its high prevalence in Salmonella isolates from Australia and in zoonotic and human isolates in China (Ingle et al., 2021; Sun et al., 2024; Zhou et al., 2023). The selective pressure from extensive clinical and veterinary use of third-generation cephalosporins has driven the global rise of CTX-M extended-spectrum β-lactamases (ESBLs), displaying traditional resistance determinants like TEM and SHV variants as the dominant ESBLs (Yu et al., 2024). This epidemiological shift was further exacerbated by the genetic mobility of bla_CTX-M genes, which were frequently plasmids-mediated and capable of horizontal tranfer between bacterial isolates through conjugation (Wei et al., 2024; Yu et al., 2024). The increasing prevalence of bla_CTX-M variants among Salmonella isolates poses a growing public health crisis, as these multidrug-resistant pathogens threatened the efficacy of frontline β-lactam therapies worldwide.

The production of AmpC β-lactamases has become a critical resistance mechanism in Gram-negative bacilli, conferring broad-spectrum resistance to β-lactam antibiotics. Of particular concern is the plasmid-mediated bla_CMY-2 gene, which encoded an AmpC-type enzyme capable of hydrolyzing extended-spectrum cephalosporins. While bla_CMY-2 has been extensively documented in food-producing animals and retail meat products across China, South Korea, and Spain (Na et al., 2020; Vázquez et al., 2021; Zhang et al., 2024b), its detection in human clinical isolates remains uncommon. Recent evidence from China, however, has confirmed the emergence of bla_CMY-2-harboring Salmonella in human infections (Zhang et al., 2024a), highlighting potential zoonotic transmission pathways. In this study, we identified one clinically derived S. Thompson isolate carrying bla_CMY-2 on a IncC-type plasmid. Notably, this isolate concurrently harbored a distinct plasmid bearing macrolide resistance determinants (mphA, msrE, mphE, and mrx), creating a multidrug-resistant genetic configuration. The dual-plasmid system provided compelling evidence for horizontal gene transfer between environmental and clinical reservoirs. The colocalization of β-lactamase and macrolide resistance genes on mobile genetic elements raised critical concerns about coselection pressures that may perpetuate resistance traits, even under targeted antimicrobial stewardship. These findings highlighted the urgent priorities that enhanced surveillance of plasmid-mediated AmpC dissemination across the food-animal-human interface and implementation of stricter antimicrobial usage policies to disrupt resistance transmission networks.

Additionally, we identified one S. Typhimurium isolate carrying 18 ARGs, including bla_NDM-1, oqxA/B, and mphA, on the IncHI2/IncHI2A plasmid for the first time. This isolate exhibited high resistance to third-generation cephalosporins (ceftazidime and cefotaxime), ciprofloxacin, azithromycin, and eleven other antimicrobials. The co-existence of ESBLs genes with other clinically significant ARGs, including qnr genes, was observed in recent studies (Li et al., 2022; Luo et al., 2020). The bla_NDM-1 gene confers resistance to many β-lactam antimicrobials, including carbapenems (Pitout, 2012). The production of NDM by Salmonella spp. has been reported in the United States, China, and Singapore (Rahman et al., 2024; Song et al., 2023; Octavia et al., 2020). Infections caused by bla_NDM-1 producers are difficult to treat, leading to increased morbidity, prolonged hospital stays, higher medical costs, and mortality due to ineffective standard treatments, which significantly affect treatment options and clinical outcomes (Octavia et al., 2020; Rahman et al., 2024; Song et al., 2023). The detection of such multidrug-resistant configurations in human pathogens emphasizes the need for One Health approaches to combat antimicrobial resistance.

The major determinants of AMR in the XDR isolates studied included genes associated with resistance to aminoglycosides, tetracyclines, and sulfonamides. Most of these genes have been documented in previous research. For example, the chromosomally encoded gene aac(6′)-Iaa was identified in S. 1,4,[5],12: i: - (Wei et al., 2024) and S. Typhimurium (Akinyemi et al., 2023) from human origin across multiple countries. Phenotypic resistance to tetracycline correlated with known genetic determinants, particularly the efflux pump gene tetA, which dominated observed resistance patterns (Zhao et al., 2022), a finding consistent with our data. While sulfonamide resistance was primarily associated with the genes sul1 and sul2 (Chen Z. et al., 2020), our study identified dfrA14 as the most prevalent sulfonamide resistance gene, contrasting with the expected predominance of sul1, sul2, and sul3. Notably, the macrolide resistance gene mphA was detected in 43.1% of the XDR Salmonella isolates. Its widespread presence raised clinical concerns, as mphA threatened the effectiveness of azithromycin for treating Salmonella infections (Wang et al., 2023). However, the mechanisms driving mphA dissemination remain unclear, highlighting the need for more in-depth comprehensive molecular epidemiological studies to elucidate transmission pathways. Both fosA3 and fosA7 fosfomycin resistance genes were identified, posing theoretical challenges for managing XDR Salmonella infections. Due to the absence of a fosfomycin-resistant phenotype in our isolates, we can not deterninate the clinical relevance of this fosfomycin resistance genes. These findings highlighted the importance of antimicrobial susceptibility testing when whole-genome sequencing might not provide a definitive assessment (Wang J. et al., 2024). Of particular concern was the detection of the plasmid-mediated mcr-1.1 colistin resistance gene in one S. 1,4,[5],12:i:- isolate. As colistin remains a last-resort antibiotic, this finding signals an alarming escalation in resistance to critically important antimicrobials (Liu J. H. et al., 2024).

The analysis of the four prevalent serovars harboring ARGs revealed that S. 1,4,[5],12:i:-isolates exhibited a broader range of resistance determinants compared to other serovars. In contrast, S. Enteritidis isolates contained fewer ARGs. Interestingly, when we analyzed the genetic factors contributing to FQs resistance, isolates from S. 1,4,[5],12:i:- and S. Typhimurium were primarily characterized by the presence of qnr genes. On the other hand, 87.5% of S. Kentucky isolates displayed double mutations in the gyrA gene (D87N and S83F) and a single mutation in the parC (S80I) gene. These mutations could be significant genetic factors contributing to FQs resistance, which was consistent to the dominant clone of S. Kentucky ST198 in Europe (European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC), 2019). Our findings revealed that the diversity of serovars was associated with different resistance genes and mechanisms. Notably, both tetB and tetR genes coexisted in S. 1,4,[5],12:i:-. Previous studies identified tetB in clinically significant clones of S. Typhimurium and its monophasic variant (Petrin et al., 2023). This suggested that there was genetic heterogeneity among the different serovars, likely a result of their adaptation and evolution in response to varying environmental and antimicrobial pressures. This heterogeneity was reflected in the distinct distribution and transmission patterns of drug resistance genes, which might be facilitated by horizontal gene transfer between isolates.

The dissemination of AMR was, in part, attribuable to the horizontal transfer of ARGs, frequently facilitated by plasmids (Castañeda-Barba et al., 2024). We conducted a further analysis of the chromosomal and plasmid-borne distribution of ARGs in 51 XDR Salmonella isolates from Guizhou province using WGS data. Our findings revealed that over half of the isolates harbored at least one plasmid, with IncHI2/IncHI2A plasmids being particularly prevalent, which indicated their widespread distribution among XDR human Salmonella isolates in this region. Notably, the carriage rate of IncHI2/IncHI2A plasmids in China significantly exceeded those reported in other countries (Sun et al., 2024). These plasmids were known to facilitate interbacterial ARG transfer through conjugation, accelerating the dissemination of resistance and posing a serious public health threat (Jiang et al., 2020). Several crucial ARGs identified on IncHI2/IncHI2A plasmids in Guizhou included tetA, PMQR genes, bla_CTX-M, aph(3′)-Ia, mphA, sul1-3, dfrA14, bla_TEM-1, mcr-1.1, and bla_NDM-1. The accumulation of such ARGs on these plasmids enabled bacterial resistance to first-line antimicrobial, complicating clinial treatment (Wang and Dagan, 2024). Of particular concern was the detection of IncFIA(HI1)-HI1A-HI1B(R27) hybrid plasmid, which have been linked to the spread of high-risk ARGs like tet (X4) in Enterobacterales from both human and nonhuman sources (Yue et al., 2024; Zhai et al., 2024). These plasmids were classified as high-risk due to their association with clinically significant resistance mechanisms. These findings highlight the urgent need for sustained surveillence of plasmid-mediated ARGs to identify emerging resistance trends and inform targeted interventions.

Molecular typing for Salmonella isolates within the same serovars is critical for outbreak investigation and bacterial epidemiology. With the widespread application of WGS, WGS-based high-resolution subtyping methods have become popularized in tracing bacterial transmission and identifying outbreaks. Among these methods, cgMLST is the most widely used approach for the strain-level differentiation of Salmonella. This method offers high discriminatory power, making it invaluable in epidemiological and outbreak investigations (Zhao et al., 2022). CgMLST is analyzed variations in core genome loci (defined as genes present in 95–98% of the reference isolates used to establish the allele scheme) to construct phylogenetic relationships (Dangel et al., 2019). In this study, cgMLST analysis of 51 XDR Salmonella isolates revealed significant genetic diversity, with 39 distinct cgSTs identified. No evidence of localized outbreaks was observed in Guizhou. Four isolates of cgST190343 exhibited identical resistance profiles and carried IncHI2/IncHI2A plasmids with the same ARGs patterns. These isolates were originated from two distinct cities in 2023, indicating either the same source of infection or potential cross-regional transmission. The cgMLST results demonstrated robust discriminatory resolution, underscoring its utility in public health surveillance. By contributing genomic data from these XDR Salmonella isolates to global databases, we can enhance resources for pathogen tracking and strength international disease surveillance systems. Such efforts enable faster, more targeted public health interventions during outbreaks and improve preparedness for emerging antimicrobial resistance pathogens.

Our study presents the first data on the WGS-based characterization of XDR Salmonella isolates from humans between 2019 and 2023 in Guizhou province, China. The detection rate of XDR Salmonella isolates increased from 3.8% in 2019 to 7.4% in 2023. S. 1,4,[5],12:i:- isolates exhibited a broader range of resistance determinants than other serovars. Notably, one bla_NDM-1 positive isolate was detected for the first time in Guizhou. Meanwhile, XDR Salmonella isolates carried multiple ARGs on plasmids, which could promote the acquisition and dissemination of ARGs. This study showed that implementing continuous dynamic surveillance will enhance our understanding of the epidemiology of XDR Salmonella in this region and help public health officials identify emerging trends and potential threats.