For a few years, nontyphoidal Salmonella (NTS) infection has been among the most common etiological causes of diarrheal and foodborne diseases (Balasubramanian et al., 2019). It is estimated that over 2.8 billion cases can be attributed to Salmonella infection. Children under 5 years old bear the burden of the major disease, especially in low-income regions (Havelaar et al., 2015; GBD 2017 Non-Typhoidal Salmonella invasive disease collaborators, 2019; Gilchrist and MacLennan, 2019). NTS generally causes self-limiting diarrheal illness, but may typically lead to severe invasive NTS for children's lack of immunity (Abuaita et al., 2021; Rana et al., 2021). Infection with different NTS serovars can present various epidemiological and clinical manifestations. Clinically, acute gastroenteritis with the onset of fever, vomiting, abdominal pains, and various diarrhea are the classic symptoms of NTS (Chen et al., 2013; Aoki et al., 2017). Antimicrobial agents may be required in the cases of invasive NTS (Crump et al., 2015). However, with the widespread use and abuse of antibiotics, especially quinolones and third-generation cephalosporins, drug-resistant bacteria can spread through antimicrobial-resistant genes (ARGs), and multiple drug-resistant (MDR) strains have constantly appeared (McDermott et al., 2018; Kariuki et al., 2019). Furthermore, the increasing rate of antimicrobial resistance poses a serious threat to healthcare systems and public health.

So far, over 2,500 Salmonella enteric serovars have been recognized, and S. Enteritidis and S. Typhimurium are the most prevalent serotypes (Michael and Schwarz, 2016; Jazeela et al., 2020). In recent years, emerging Salmonella serovars, such as S. I 4,[5],12:i:- have increased rapidly and spread globally. The traditional serotypes determined by a combination of O antigen and H antigens have some limitations. Currently, molecular typing technologies, such as multilocus sequence typing (MLST) and whole genome sequencing (WGS) have been applied due to their strong specificity, ease of operation, short detection time, and low cost. MLST has been widely used in epidemiology investigation because of its simplicity and repeatability. In addition, it can achieve network data sharing and comparison (Foley et al., 2006; Tang et al., 2019).

At the same time, with the decreasing cost of genome sequencing and the continuous advancement of bioinformation analysis technology, WGS is gradually applied to the outbreak investigation and epidemiological analysis at the forefront of disease prevention and control (Guinea et al., 2021; Papić et al., 2021). WGS-based methods, such as core genome multilocus sequence typing (cgMLST), include all core genes loci present in all given isolates. The complete analysis offers a high-resolution readily maintained and easily-shared database only using the same or similar online templates (Maiden et al., 2013).

Considering the important hazard of Salmonella in children, the widespread antibiotic resistance of Salmonella, and the absence of data from north China, the object of this study is to explore the serotypes, antimicrobial resistance, and presence of ARGs in the strains isolated from diarrheic children under 5 years old in Jilin Province. These analyses can be used for risk assessments of diarrheic children and for establishing prevention and control measures against foodborne diseases.

Our study combined clinical epidemiology and genomic data to depict the characteristics of NTS infections in northern China. In line with recent findings (Li et al., 2014; Zhan et al., 2019), S. Enteritidis and S. I 4,[5],12:i:- are the leading Salmonella serovars. S.I 4,[5],12:i:- currently represents the common serotype among human cases in many countries (Seixas et al., 2018; He et al., 2020). This serovar first appeared in the 1990s but increased considerably in the last 10 years, especially in Europe and Asia (Kawakami et al., 2019). The appearance of S. I 4,[5],12:i:- was always associated with foodborne disease outbreaks and accompanied by the MDR strains (Mølbak et al., 2014; Elnekave et al., 2018). Therefore, monitoring trends in various serovars over time and developing public health methods may help in focusing on preventative strategies. Our study has made a detailed epidemiological description based on symptoms of diarrhea cases. However, due to no certain evidence of suspected food and Salmonella diarrheas onset, the exact exposed food was missing. It was also impossible to identify the possible sources of infection for every sporadic case.

The emergence of Salmonella infections with MDR has become a major threat to public health worldwide. In our study, 203 (78.1%) isolated NTS were classified as MDR among 260 strains, and resistance occurred to antibiotics commonly used in treatment. A similar incidence rate was presented in studies by Duong et al. (2020) . In addition, four strains from children under the age of 1 were resistant to at least seven antimicrobials, and these results highlighted the need for effective surveillance of antimicrobials management in China. At present, the third generation cephalosporins antimicrobials are the new preferred antimicrobial therapy instead of quinolones in clinical trials for Salmonella infection (Kariuki et al., 2015). Different from the high resistance of quinolones, 14.2 and 2.7% resistant rates were observed in cefotaxime and ceftazidime, respectively. Similarly, a relatively low resistant rate was also observed in Brazil (Viana et al., 2020) and Greece (Mellou et al., 2021).

The consistency between genomic and phenotypic investigations has been successfully verified in our study. Resistance genes, such as bla_TEM−1B, bla_OXA−1, bla_CTX−M−55, and bla_CTX−M−14 that belonged to β-lactams were detected. The highest frequency of bla_TEM−1B of strains was consistent with studies in eastern China (Zheng et al., 2021). Extended-spectrum β-lactamases (ESBLs) bla_CTX−M−55 were also detected. The carbapenems are the critical “last resort” antimicrobials to Salmonella infections for their stability toward ESBLs (Fernández et al., 2018), and in our study, the strains remain susceptible in phenotype and genotype.

According to the data (Hopkins et al., 2005; Zhan et al., 2019), mutations in chromosome and plasmid-mediated resistance genes all contributed to quinolones resistance, which is commonly used quinolones for Salmonellosis. Similar to a previous report in Taiwan (Fang et al., 2021), mutations in parC and gyrA were more likely to cause the resistance of quinolones.

Moreover, resistance to quinolones can be acquired by plasmid-mediated quinolone resistance (PMQR) genes, such as oqxAB. As shown in Supplementary Table 5, 28 of 170 quinolones resistance strains harbored PMQR genes. As for plasmid-mediated genes, the qnrS1 gene was also detected in retail food (Yang et al., 2020), which may prove the horizontal transfer of plasmids. Previous studies reported that quinolones were strongly linked to the overuse of antibiotics in food animals (de Jong et al., 2012; Redgrave et al., 2014). Meanwhile, quinolones have just been out of use in food-producing animals in China since 2018 (Cuypers et al., 2018). Moreover, previous findings also described quinolones as therapy producing resistance to food and water-borne infections (Threlfall, 2002; Molina et al., 2016). Hence, further efforts are warranted to control the spread of resistance trends from multiple sources. In our study, we found that bla_TEM−1B and oxqAB coexisted in four MDR strains. This suggested that the common existence of these genes leads to resistance to both quinolone and β-lactams. Further efforts to explore the transmission and influence of multiply classes of antibiotics coexistence will be of interest.

Multilocus sequence typing was used for the epidemiology investigation of NTS in our study. ST11 and ST34 were the predominant types, which was in line with the distribution of animal farms and retailed foods in China (Yang et al., 2019; Li et al., 2020). These findings support the widespread transmission of Salmonella strains among different regions. MLST and cgMLST showed a high level of molecular traceability. The tree diagrams based on MLST and cgMLST indicated that human Salmonella cases with unrelated epidemiological backgrounds were apt to be sporadic (Tönnies et al., 2021). In this study, consistency between MLST sequence type and phenotypic serotype was found to provide evidence for the genetic structure of Salmonella. However, 13 strains were not distinguished. It indicates that MLST cannot distinguish all isolates. Compared with MLST, cgMLST based on WGS presented a relatively high resolution, but more equipment and time were necessary. The combination of the two types can provide the origin and evolution basis of Salmonella strains and reveal the evolution and variation.

In this article, we analyzed the epidemiological and molecular characteristics of NTS isolated from diarrheic children. Besides, S. Typhimurium and S. I 4,[5],12:i:- were the dominant serotypes. A considerable increasing AMR prevalence rate was observed, providing evidence between resistant phenotype and genotype. MLST and cgMLST were performed for serotype clustering, while cgMLST had high resolution and good repeatability. In conclusion, our study showed the genetic diversity of NTS isolates from diarrheic children and suggested the monitoring of antibiotic resistance and dominant STs of Salmonella in children.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI - PRJNA817369.

WZ, CZ, and JW: conceptualization. KL, BS, and JS: experiment investigation. XL, XS, and WZ: writing, original draft preparation, and revising. JW provided critical comments on this work. All authors have read and agreed to the manuscript.

The authors thank the funding support from the National Natural Science Foundation of China (grant number 82073603) and the Jilin Province Science and Technology Development Plan Item (grant number 20210204139YY).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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