Antimicrobial resistance profiles of Salmonella isolates from human diarrhea cases in China: an eight-year surveilance study

Salmonella is widely known as one of the most common foodborne pathogens, and antibiotics remain effective in clinical therapies against its infections. To guide better clinical antibiotic treatment, we analyzed the antimicrobial resistance (AMR) profiles of a nation-wide collection of 36,822 Salmonella isolates derived from sporadic diarrhea cases in China from 2014 to 2021. A panel of 15 antibiotics, including 10 critically important and 5 highly important antimicrobial agents for human medicine based on the WHO CIA List, was selected for AMR surveillance. Salmonella enterica serovar Typhimurium, Enteritidis, I 4,[5],12:i:-, London, and Stanley were turns to be the top five serotypes from human diarrhea cases in China. Antimicrobial susceptibility testing revealed that the majority of the isolates (87.2%) were resistant to at least one antimicrobial agent, and 66.5% were multidrug resistant (MDR). Salmonella isolates were found to be highly resistant to ampicillin (73.4%) while sensitive to imipenem (98.73%). Over the eight years, the isolates were demonstrated generally an increase in resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and azithromycin, but displayed varied resistance profiles in terms of serotypes. Stanley (0.66–27.18%) and Agona (0.78–45.30%) had lower resistance rates compared to Typhimurium (1.11–85.6%), Enteritidis (1.55–91.29%), and I 4,[5],12:i:(1.02–94.28%). In conclusion, our results provide systematic data on the resistance characteristics of Salmonella isolates from human diarrhea cases in China. Furthermore, this data identifies priorities for the clinical treatments of antibiotics.


Introduction
Foodborne diseases continue to be a significant cause of human morbidity and mortality worldwide. Among the 22 common foodborne diseases, nontyphoidal Salmonella enterica causes the highest burden [1]. In China, data from national foodborne disease surveillance revealed that Salmonella was among the top three pathogenic causes of bacterial foodborne outbreaks [2]. The isolation rate of Salmonella from sporadic diarrheal cases was highest among the four major bacterial pathogens (Vibrio parahaemolyticus, Salmonella, diarrheagenic Escherichia coli, and Shigella) in the foodborne disease active surveillance [3].
Outbreaks associated with resistant Salmonella are frequently reported worldwide [4]. In China, to monitor trends of antimicrobial susceptibility among foodborne pathogens, AMR surveillance has been included in the National Foodborne Disease Surveillance Plan since 2014. All 32 Chinese provincial CDC laboratories are required to conduct antimicrobial susceptibility tests (ASTs) and submit the minimum inhibitory concentrations (MICs) to the National Molecular Tracing Network for Foodborne Disease Surveillance (TraNet) [5]. The China National Center for Food Safety Risk Assessment (CFSA) manages and analyzes all AMR data and reports it to the National Health Commission annually.
This study, provides the first summary of AMR data for 36,822 Salmonella isolates from sporadic diarrhea cases between 2014 and 2021 in China. Additionally, we analyzed the trends and changes of AMR and serotype in these isolates during an eight-year period. The data presented herein would provide better evidence to support clinicians and policy-makers during their clinical therapies and efforts to combat Salmonella infections.

Geographic distribution
From 2014 to 2021, a total of 31 Chinese provincial CDCs reported the MICs of 36,822 Salmonella isolates derived from human sporadic diarrhea cases to the CFSA. The number of isolates reported by each province varied from 91 in Chongqing to 9,174 in Guangdong (Fig. 1) (Fig. 2). Among all MDR isolates, 53.9% (13,194/24,478) exhibited resistance to over five classes of antimicrobial agents.
The trend analysis based on the linear regression model showed that the isolates exhibited upward resistance trends to ampicillin, chloramphenicol, trimethoprimsulfamethoxazole, and azithromycin, and the resistance rates increased by 2.2%, 1.8%, 1.7%, and 0.4% for each additional year, respectively. While decreasing resistance rates were observed for gentamicin, nalidixic acid, and ciprofloxacin, with the resistance rates decreasing by 1.4%, 1.1% and 0.6% for each additional year, respectively. Resistance to colistin E decreased from 27.5% in 2019 to 15.9% in 2021 (Fig. 3); however, three-year data could not fit the linear regression model to conduct statistical tests.
The AMR of Salmonella isolates varied among serotypes, with significant differences in resistance rates and MDR rates were observed. For instance, S. Stanley and S. Agona had lower resistance rates than S. Typhimurium, I 4, [5],12:i:-, Enteritidis, and London (Fig. 4).

Discussion
The global burden of AMR has led to triggered the development of coordinated national and global action plans [7,8]. Increasingly, governments around the world have launched and strengthened AMR surveillance programs in both animals and humans [9][10][11][12][13]. In China, the AMR surveillance program for animals was launched in 2008 and for humans in 2005 [14]. In 2014, AMR surveillance for foodborne pathogens from human diarrhea cases was launched. The main goal of the AMR surveillance program in TraNet was to track the trend in antibiotic resistance of selected foodborne pathogens, identify important resistance patterns, and assess the effect of interventions. This is the first time that the Chinese human diarrhea surveillance data has been officially released.
Fluoroquinolones, third-generation cephalosporins, and macrolides are commonly used to treat human Salmonella infections. Over 70% of human Salmonella isolates had decreased susceptibility to ciprofloxacin, and the proportion was far higher than the 5.8% shown by the NARMS in the US [15]. Enteritidis was the most common serotype in both China and the US, a high proportion (> 90%) of human Enteritidis isolates with DSC collected in this study indicated that ciprofloxacin may be unsuitable for the treatment of Enteritidis infection in China. A similar resistance rate to ciprofloxacin were observed in Salmonella isolates from China (16.2%) and EU (14.1%) [16]. Moreover, 39.8% of the tested Salmonella isolates with DSC were susceptible to nalidixic acid, implying a high presence of plasmid-mediated quinolone resistance (PMQR) in human Salmonella infections [15]. The use of fluoroquinolones may further facilitate the transmission of PMQR determinants in patients during treatment. Nearly 20% of human Salmonella isolates from diarrhea cases were resistant to third-generation cephalosporins in China, and the resistant proportion was much higher than that in the US (2.7%) and EU (0.8%). However, no upward trend in resistance to third-generation cephalosporins was observed in this study, indicating that these antimicrobials are still effective for the treatment of most Salmonella infections. Resistance to third-generation cephalosporins is mainly mediated by ESBLs and AmpCtype β-lactamases. bla CTX-M was the most prevalent gene among partial ESBL-producing Salmonella isolates in China, according to the whole-genome sequencing (WGS) analysis (unpublished data). Although it is still unclear to what extent isolates with ESBL from animals are directly responsible for human infections, some countries have placed voluntary restrictions on the use of these critically important antimicrobials for disease prophylaxis in food-producing animal husbandry under the One Health perspective [17,18]. It should be noted that the rate of human azithromycin-resistant Salmonella isolates (7.9%) in China was not only higher than that reported in the US and EU (< 1%), but also showed a gradual upward trend, suggesting that a prudent use of azithromycin is urgently needed in the treatment of Salmonella infections in Chinese clinical settings.
Another important aim of TraNet is to assess the effectiveness of interventions. Previous studies have shown that restricting the use of antibiotics in foodproducing animals is associated with a reduction in antibiotic-resistant bacteria in both animals and humans [19,20]. Our surveillance data also indicated a downward resistant trend in colistin resistance in Salmonella isolates from diarrhea cases. This decrease further demonstrates that the ban on colistin as an animal growth promoter in 2017 had a significant effect on reducing colistin resistance in bacteria of human origin, which is consistent with the decreasing colistin resistance of E. coli from animals and humans in China after the colistin withdrawal policy was implemented [20,21]. Therefore, continuous surveillance is necessary for early warning of emerging AMR in human foodborne pathogens in hospital settings.
AMR varies by serotypes in different countries/regions. The serotype distribution of sporadic human isolates in China was similar to that in the EU (top 3: Enteritidis, Typhimurium, and I 4, [5],12:i:-,), but different from that  [22]. Given the interconnections of AMR amongst the environment, animals, and humans, a national integrated surveillance system across different sectors is needed to better understand the ecology of resistant bacteria, implement evidence-based control measures, and promote effective stewardship.

Limitations
We acknowledge four limitations of the study. First, all Salmonella isolates were cultured from selected sentinel hospitals in China, and only a fraction of diarrheal cases were sampled in these hospitals, so the conclusions might not fully represent the Salmonella resistance profiles in China. Second, not all isolates were clearly identified due to insufficient data. I 4, [5],12:i:-was underreported in previous years because the second-phase flagellar antigen was not detected. Resistance profiles by different serotypes might have been influenced by this, especially the serovar Typhimurium and I 4, [5],12:i:-. Third, although standard procedures were applied in participating laboratories, the selected antimicrobials and concentration ranges in the panels changed, and not all the isolates were tested against the same antimicrobials. For instance, it was inappropriate to assess the trend of colistin E because of the lack of AST data before 2019. Fourth, WGS analysis, including confirmation of Salmonella serotypes, core genome multilocus sequence typing, and determination of virulent and AMR genes (ARGs), was not used in TraNet to facilitate surveillance until 2019. Isolates collected before 2019 were not subjected to the prediction of ARGs; therefore, this study only focused on phenotypes.
There is a need to combine AMR testing and WGS techniques to link the resistance of food and human isolates.

Conclusion
We described an eight-year dataset of the AMR profile of Salmonella isolates from active foodborne disease in China, and found that Salmonella AMR varied by serotype. Although the isolates displayed downward resistance trends to nalidixic acid, gentamicin, ciprofloxacin, and colistin, the resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and azithromycin presented upward trends. Compared with the surveillance data in the US and EU, the resistance profiles in human Salmonella isolates in China suggest that prudent use of antibiotics for treating Salmonellacaused foodborne diseases is needed.

Human Salmonella Isolates
Based on the national foodborne disease surveillance plan in China, sentinel hospitals were chosen by each provincial health administration to conduct laboratory-based active foodborne disease surveillance. At least 10 stool specimens per hospital every month were collected from sporadic diarrheal cases to identify the five most common foodborne pathogens, including Salmonella species, Shigella species, Vibrio. parahaemolyticus, DEC, and norovirus. According to the guidelines and techniques of the national foodborne disease surveillance manual, all isolates were ultimately sent to provincial CDCs for final identification, molecular subtyping, and AMR testing. Then, provincial CDC laboratories were instructed to submit the epidemiological information and experimental data of isolates to the CFSA via TraNet. In this study, all Salmonella isolates were derived from sporadic diarrhea cases under the framework of active foodborne disease surveillance, and isolates from foodborne outbreaks were not included.

Antimicrobial susceptibility testing (AST)
The broth microdilution method was applied to test the antimicrobial susceptibility in participating TraNet laboratories. The Clinical and Laboratory Standards Institute (CLSI) recommended antibiotics and commonly used antibiotics in clinical therapy. A panel of 15 antibiotic agentsfrom 11 classes was selected for surveillance, including ampicillin, ampicillin/sulbactam, cefazolin cefotaxime, ceftazidime, cefoxitin, tetracycline, chloramphenicol, nalidixic acid, ciprofloxacin, azithromycin, gentamicin, trimethoprim-sulfamethoxazole, imipenem, and colistin. All antibiotics were grouped into critically important and highly important antimicrobials based on the World Health Organization (WHO) List of Critically Important Antimicrobials for Human Medicine (WHO CIA List) [23] ( Table 2). The MIC for each antimicrobial was used to categorize isolates as susceptible, intermediate (if applicable) or resistant based on CLSI M100-S32 clinical breakpoints. Escherichia coli ATCC 25,922 was included to ensure the reliability and reproducibility of AST.

Data analysis
All reported data were audited and checked and then exported and managed using Microsoft Excel 2016. All variable values were reported as counts or percentages (%). Statistical analyses, such as resistance rate, MDR profile, and resistance trend analysis based on a linear regression model, were conducted with R version 4.1.2. Isolates that were resistant to at least one of the tested antimicrobials were considered resistant isolates, and MDR was defined as "resistance to three or more classes of antimicrobials".