Epidemiology of blaCTX-M-Positive Salmonella Typhimurium From Diarrhoeal Outpatients in Guangdong, China, 2010–2017

Salmonella enterica can lead to intestinal diarrhea, and the emergence and spread of cephalosporin-resistant Salmonella have brought great challenges to clinical treatment. Therefore, this study investigated the prevalence and transmission of blaCTX-M genes among S. Typhimurium from diarrhoeal outpatients in Guangdong, China, from 2010 to 2017. A total of 221 blaCTX-M-positive isolates were recovered from 1,263 S. Typhimurium isolates from the facal samples of diarrhoea patients in 45 general hospitals from 11 cities. The most popular CTX-M gene was blaCTX-M-55 (39.6%, 72/182) in the CTX-M-1 group, followed by blaCTX-M-14 (22.5%, 41/182) and blaCTX-M-65 (19.2%, 35/182) in the CTX-M-9 group. The isolates that carried blaCTX-M-9G had significantly higher resistance rates to multiple antibacterials compared with blaCTX-M-1G (p < 0.01). Meanwhile, PFGE analysis not only showed the clonal transmission of blaCTX-M-55/14/65-positve isolates of diarrhoeal outpatients’ origins from different hospitals in Guangdong province, but also the characteristic of blaCTX-M-55/14/65-positve isolates’ bacterial persistence. Multilocus sequence typing (MLST) analysis indicated that these S. Typhimurium isolates possessed ST34 and ST19. Furthermore, genomic Beast phylogenomic analysis provided the evidence of a close relationship of blaCTX-M-positive S. Typhimurium isolates between the outpatients and pork. Most blaCTX-M-55/14/65 genes were transmitted by non-typeable or IncI1/IncFII/IncHI2 plasmids with the size of ranging from ~80 to ~280 kb. Moreover, whole-genome sequencing (WGS) analysis further revealed that blaCTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with the detection rates >50%, and it first reported the emergence of blaTEM-141 in S. Typhimurium. This study underscores the importance of surveillance for blaCTX-M-positive microbes in diarrhea patients.


INTRODUCTION
Salmonella enterica is a zoonotic pathogen of substantial concern to human and animal health (Yin and Zhou, 2018). What's more, it is a leading cause of morbidity and mortality in people worldwide, with approximately 90 million cases of gastroenteritis and 150,000 associated deaths . So far, more than 2,610 Salmonella serovars have been identified, while salmonellosis is caused mainly by S. enterica serovars Typhimurium, Enteritidis and Dublin (Shi, 2015;Mohammed et al., 2017). Nontyphoidal S. Typhimurium is a dominant factor of human gastroenteritis, and improper handling and digestion of inadequately looked food primarily result in the infection. Invasive complications, including meningitis, sepsis and bacteraemia, are very common in infants, the elderly and immunocompromised patients. The disease of S. Typhimurium is usually related to contaminated foods, such as pork and fruits, unpasteurized milk and dairy products, and undercooked eggs (Wegener et al., 2003).
In these potentially life-threatening S. Typhimurium cases, the antibiotics of choice are fluoroquinolones and extended-spectrum cephalosporins (Diard and Hardt, 2017). Third-generation cephalosporins (3GCs) are used across the world to threat infections caused by Salmonella, and subsequently the emergence of resistance attracts particular attention (Whichard et al., 2007). Multidrug-resistant (MDR) Salmonella spp. potentially arising for the selective pressure from sustained antimicrobial exposure are more likely to be the causative agents of invasive disease (Okoro et al., 2015). Moreover, the ESBL-producing strains of Salmonella have been reported in many regions in China, including Beijing, Shanghai, Guangdong, and Shandong (Cao C. et al., 2021). Worse, ESBL-producing S. Typhimurium have increasingly been detected from food animals, even environmental water and human patients (Fu et al., 2020;Ma et al., 2020). Hence, the number of ESBL-Salmon has increased worldwide.
TEM, SHV, and CTX-M were the most prevalent ESBL types. It has commonly been found that ESBL-CTX-M is located on plasmids and considered as the most prevalent type of ESBLs in many European countries (Paterson and Bonomo, 2005). At the same time, there is tremendous diversity of bla CTX-M genotypes isolated from food animals and human populations. Usually, among the reported bacteria with bla CTX-M-55 -positive or bla CTX-M-14 -positive or bla CTX-M-65positive, most are isolated from food and animal sources (Xiang et al., 2015;Zhang et al., 2015;Nadimpalli et al., 2019). A practice was selected for antibiotic resistant S. enterica that can spread to human through contaminated foods. However, this practice is not currently monitored or regulated in Guangdong Province.
Therefore, in this study, ESBL-producing S. Typhimurium isolates, mainly from diarrheal patients, isolated from Guangdong province, and collected at the Guangdong Provincial CDC during the period of 2010-2017, were investigated to gain insight into their public health impacts.

Bacterial Isolates, Detection of ESBL/pAmpC Genes, and Antimicrobial Susceptibility Testing
A total of 1263 S. Typhimuriums were recovered from facal samples of diarrhoea patients in 45 general hospitals from 11 cities of Guangdong province between 2010 and 2017. These isolates were collected by the Guangdong Provincial Center for Disease Control and Prevention (CDC) in a clinicbased Salmonella infection surveillance of outpatients with diarrhea, as described previously (Zhang et al., 2013). All 1263 S. Typhimurium isolates were incubated on MacConkey agar plates, containing 4 mg/L cefotaxime. The cefotaximeresistant S. Typhimurium isolates were subjected to screening for CTX-M, CTX-M-1G, CTX-M-9G, CMY-2G, SHV, and DHA genes (Supplementary Table S1; Liu et al., 2007), and bla CTX-M-1G/9G -positive isolates were further subjected to determine the subtypes of ESBL-encoding genes, as previously reported (Zhao and Hu, 2013). The DNA sequences and deduced amino acid sequences were compared with the reported sequences from GenBank. Antimicrobial susceptibility testing was performed on all the CTX-M-producing isolates by the agar dilution method, except for colistin with the broth dilution method. The following antimicrobials were tested: cefotaxime, ceftriaxone, ceftazidime, ceftiofur, meropenem, ciprofloxacin, nalidixic acid, sulfamethoxazole/ trimethoprim, gentamicin, amikacin, florfenicol, fosfomycin, azithromycin, doxycycline, olaquindox, tigecycline, and colistin. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI, 2018: M100-S25), and veterinary CLSI (VET01-A4/VET01-S2) guidelines (Humphries et al., 2019), and the resistance breakpoints for colistin were interpreted based on EUCAST (>2 mg/L) criteria, respectively. Escherichia coli ATCC25922 was used as the quality control strain.

Molecular Typing
The genetic relatedness of bla CTX-M -positive S. Typhimurium isolates was analyzed by PFGE with the XbaI digestion of genomic DNA (Palhares et al., 2014). PFGE patterns were analyzed using BioNumerics software (Applied Maths, Sint-Martens-Latem, Belgium) with the Dice similarity coefficient, and a cut-off value of 85% of the similarity values was chosen to indicate identical or similar PFGE types.

WGS and Phylogenetic Analysis
Based on the results of PFGE types and resistance profiles analysis, representative bla CTX-M -positive S. Typhimurium isolates (n = 57) were selected and their genomic DNA were subjected to 250-bp paired-end whole-genome sequencing (WGS), which at a depth of 100X, using the Illumina MiSeq system (Illumina, San Diego, CA, United States), using default parameters, followed by assembling the 150 bp paired-end Illumina reads using SPAdes v3.6.2 (Humphries et al., 2019). Multi locus sequence typing (MLST), antibiotic resistance genes (ARGs), and plasmid replicon types were analyzed using the CGE server. 1 Phylogenetic tree for CTX-M-producing isolates was structured on the basis of the core genome using Harvest version 1.1.2 (Treangen et al., 2014), and the corresponding characteristics of each isolate were visualized using online tool iTOL version 4 (Letunic and Bork, 2019). The population structure of each phylogenetic tree was defined using hierBAPS v6.0 (Cheng et al., 2013).

Conjugation Assay, Gene Location, and Plasmids Analysis
To test the transferability of bla CTX-M genes, conjugation experiment was carried out by the liquid mating-out assay, with the streptomycin-resistant E. coli C600 as the recipient. Transconjugants were selected on MacConkey agar plates that were supplemented with cefotaxime (2 mg/L) and streptomycin (1,500 mg/L). Antimicrobial susceptibility testing was conducted on transconjugants and the bla CTX-M gene was confirmed by PCR, as described above. PCR-based replicon typing was performed for transconjugants, as previously described (Bankevich et al., 2012). To determine the location of bla CTX-M , plasmids from the selected transconjugants were linearized using S1 nuclease and subjected to PFGE, followed by Southern blot hybridization using a digoxigenin-labeled probe specific for bla CTX-M-1G/9G , as previously described (Liu et al., 2007).

Data Availability
All genome assemblies of the 57 bla CTX-M -positive strains were deposited in GenBank and are registered under BioProject accession number PRJNA797940 and PRJNA629650.

Prevalence of CTX-M Genes
A total of 221 (17.5%) isolates displayed resistance to cefotaxime among the 1,263 S. Typhimurium isolates collected in 45 hospitals across 11 cities from Guangdong, China. Of which, 82.4% (182/221) carried one or two bla CTX-M variants. In addition, 20.8% (46) isolates contained bla CMY-2G gene and 16 (7.2%) isolates harbored bla DHA gene, and no bla SHV gene was detected among these isolates. The percentages of cefotaxime-resistant isolates and bla CTX-M -positive isolates had been shifted significantly from 11.5% and 6.1% in 2010 to 24.7% and 17.2% in 2015, but decreased to 15.1% and 13.4% in 2017, respectively ( Figure 1A). The bla CTX-M -positive isolates were identified in 35 hospitals among 11 cities. Of which, Shaoguan had the highest detection power of 25.0%. The mean positive prevalence of bla CTX-M carriages was 13.5% among the 12 cities ( Figure 1C). Among the patients who were found to be positive for bla CTX-M -positive S. Typhimurium isolates, the median age of patients with bla CTX-M -producing isolates was 1 year (range 0-90 years), and 90% of patients were <5 years of age. In addition, 70.3% patients were male (Table 1).
The same scenario was also observed in bla CTX-M-55 , bla CTX-M-14 , and bla CTX-M-65 positive isolates. However, the isolates that carried bla CTX-M-1G , including bla CTX-M-55 , had remarkably higher rates of resistance to ceftazidime compared with bla CTX-M-9G including bla CTX-M-14 and bla CTX-M-65 (p < 0.001). Furthermore, to determine the association between the dominant bla CTX-M genes and the 3GCs susceptibility, MICs of cefotaxime, ceftriaxone and ceftazidime were grouped into three levels, namely low resistance level (≤8 mg/ml), medium resistance level (16-64 mg/ml), and high resistance level (≥128 mg/ml; Figure 2C). The majority of bla CTX-M-55 , bla CTX-M-14 , and bla CTX-M-65 -positive S. Typhimurium isolates showed moderate and high levels resistance to cefotaxime and ceftriaxone. However, the proportion of high levels of resistance to cefotaxime and ceftriaxone in bla CTX-M-55 -positive S. Typhimurium isolates was higher than that of bla CTX-M-14/65 -positive S. Typhimurium. It was obvious that most bla CTX-M-55 -positive isolates are resistant to ceftazidime at high levels. In contrast, most isolates bla CTX-M-14 -positive or bla CTX-M-65 -positive were presented low-level resistant to ceftazidime.

Molecular Typing
The genetic relatedness of bla CTX-M-55 -positive, bla CTX-M-14 -positive and bla CTX-M-65 -positive S. Typhimurium isolates were analyzed by PFGE, respectively. PFGE was successfully performed in 71 bla CTX-M-55 -positive isolates and distributed into 26 pulsotypes. The 22 isolates in Type III were obtained in nine hospitals across four cities during 2014-2016. Similarly, the 17 isolates in Type VII were originated in six hospitals from four cities during 2010-2015 (Supplementary Figures S1A, S2). The clonal transmission of bla CTX-M-55 -positive strains was observed at different hospitals in the same city between 2014 and 2016. A total of 21 different pulsotypes were detected among 41 bla CTX-M-14 -positve isolates, and Type I was predominant (n = 9, 21.95%; Supplementary Figures S1B, S2). The clonal transmission of bla CTX-M-14 -positive strains was observed at the same hospitals in the same city in 2012. Most importantly, all 35 bla CTX-M-65positve isolates were distributed into 15 pulsotypes, and the most predominant Type VIII contained 19 isolates (54.3%) and was originated from nine hospitals in seven cities during 2013-2017 (Supplementary Figures S1C, S4). The spread of bla CTX-M-65 -positve isolates' clones from Guangzhou and Jieyang was observed.

Phylogenetic Analysis of bla CTX-M-55/14/65 -Positive Salmonella Typhimurium
The population structure was further analyzed by constructing phylogenetic trees based on the core genomes of the 57 bla CTX-M -positive isolates. Bayesian analysis displayed that all isolates were classified into four different lineages. The major Lineage I belong to ST34 and Lineage II-IV belong to ST19.
To  on the basis of 32,165 core genome single nucleotide polymorphisms (cgSNPs) among 138 isolates (Figure 4). These 138 isolates were mainly distributed in Guangdong province (n = 134) and other provinces (n = 4), such as Shanghai, Hebei, Jiangxi, and Zhejiang. Notably, these 138 isolates were primarily ST34 and ST19 members and originated from diverse sample types, including humans (patients, synviol fluid and blood culture), food (beef, chicken, feces, pork) and the environments (

DISCUSSION
In this study, the detection rate of bla CTX-M -positive S. Typhimurium from diarrhoeal outpatients increased from 2010 to 2015 in Guangdong Province, China. It was speculated that bla CTX-M -positive S. Typhimurium outbreaks are linked to FIGURE 3 | Phylogenetic analysis of bla CTX-M -positive S. Typhimurium isolates in this study (n = 57). Bayesian evolutionary tree was constructed using core-genome SNPs. Each isolate is labeled with the city of isolation year, hospital, ST and cluster. The red-filled squares indicate the possession of the indicated antimicrobial resistance genes (ARGs), the blue-filled squares indicate plasmid (Inc type), and the green-filled squares indicate virulence genes. the consumption of food animal or raw meat, particularly pork. Firstly, previous studies showed that the swine is one of the major reservoirs for Salmonella (Wang et al., 2007;Jackson et al., 2013). Secondly, the data from China's National Nutrition Survey also displayed that the total pork intake of Chinese residents increased by 73% from 1992 to 2012 (He et al., 2015). It's worth noting that the overall percentage of cephalosporin use had an upward trend from 2012 to 2017 in hospitals (Branch, 2020), which can give us some hints that the transmission of bla CTX-M may be relevant to the selective pressure of cephalosporin antibiotics. Then, it was obvious that the detection rate of cefotaxime-resistant S. Typhimurium in Guangdong province steadily decreased from 2016 to 2017. Meanwhile, according to CHINET bacterial resistance monitoring, the detection rate of cefotaxime-resistant Enterobacter decreased gradually from 2015 to 2017. Therefore, the long-term monitoring of cephalosporin usage and the prevalence of the bla CTX-Mpositive Salmonella are necessary for public health.
In this study, nine bla CTX-M variants were detected in 182 bla CTX-M -producing isolates, and the most predominant was bla CTX-M-55 , followed by bla CTX-M-14 and bla CTX-M-65 , which is consistent with previous studies (Zhang et al., 2018). Currently, bla CTX-M-55 -positive Salmonella has been reported as the dominant genotype in other countries, including Germany, Cambodia, Korea and Vietnam, and was frequently detected in food animals, especially in poultry and pork (Nguyen et al., 2016;Kim et al., 2017;Lay et al., 2021;Pietsch et al., 2021); bla CTX-M-14 -positive Enterobacteriaceae has been reported as the dominant genotype in some countries, including China, South Korea, Japan, and Spain, and was frequently detected in food, especially in retail chicken meat and pork (Bai et al., 2016;Shigemura et al., 2020;Wang et al., 2021); bla CTX-M-65 -positive Salmonella has been found in China, the United States, and Germany, and was commonly found in food animal sources, especially in chicken (Brown et al., 2018;Martínez-Puchol et al., 2021;Pietsch et al., 2021).
The bla CTX-M-65 -positive S. Typhimurium isolates with the same profile were found in the three hospitals (GZ5, JY1, and DG3), which indicated that the clonal dissemination of bla CTX-M-65 -positive S. Typhimurium occurred in hospital. Furthermore, a few bla CTX-M-55/14/65 -positive S. Typhimurium isolates from 2010 to 2017 showed 100% homology by PFGE analysis, which suggested that the possible long-term outbreaks were caused by clonal transfer of bla CTX-M-55/14/65 -positive S. Typhimurium strains within the hospital. WGS demonstrated that these S. Typhimurium isolates belonged to ST34 and ST19. In fact, it has been shown that ST34 and ST19 are common S. Typhimurium STs responsible for infections worldwide, especially in China (Woh et al., 2021). It has been proved previously that the ST34 S. Typhimurium isolates with the highest percentage of MDR are mainly recovered from diarrhea patients (Biswas et al., 2019;Jiu et al., 2020;Luo et al., 2020;Sun et al., 2020). Furthermore, ST19 has been found mostly in human clinical Salmonella isolates, but also in animals and the environment, and successful in South African and China. ST19 was only occasionally found in United States and Mexico and coexists with quinolone resistance genes qnrS (Kariuki and Onsare, 2015;Gómez-Baltazar et al., 2019;Monte et al., 2020;Yao et al., 2020;Xiaoting et al., 2021).
Our genomic Beast tree analysis provided evidence for the closer relationship among bla CTX-M -positive strains from the outpatients in this study and pork. Pig has been singled out as the most likely reservoir for the amplification and spread of Enterobacteriaceae that are resistant to ESBL and other antibiotics (Nordmann and Poirel, 2016), The same major bla CTX-M , as presented in this study, was also detected in isolates from a pig farm in China (Zhang et al., 2019). Therefore, our study provides strong genome epidemiology-based evidence that the consumption of pork is the likely contamination source of bla CTX-M -positive S. Typhimurium.
In the current study, most bla CTX-M-55/14/65 genes identified were carried by IncI1, IncHI2, and IncFII plasmids, which indicated that plasmids belonged to bla CTX-M-55/14/65 -positive isolates and were diverse. Among them, IncI1 has become one of the most common plasmid families in contemporary Enterobacteriaceae from both human and animal sources. In clinical epidemiology, IncI1 ranks first as the confirmed vehicle of the transmission of extended spectrum beta-lactamase and AmpC genes in isolates from food-producing animals (Carattoli et al., 2021). The second, HI2, followed by FII plasmid, was found to be associated with the transfer of the mcr-1 and ESBL encoding genes all over the world, especially in European and African countries. The coexistence of mcr-1 and ESBL encoding genes in HI2 plasmids was less reported in China in recent years (Biswas et al., 2019;Wang et al., 2020). Worryingly, as the vector of drug resistance gene, FII plasmid not only carries mcr-1, but also is one of the common carriers of NDM gene (Wu et al., 2019).
WGS analysis further revealed that bla CTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with detection rates >50%. Of note, mcr-1, conferring resistance to the last-resort antibiotic colistin, was detected in seven bla CTX-M -positive S. Typhimurium isolates. To begin with, the coexistence of mcr-1 and bla CTX-M-55 was first reported in the literature from colistin-resistant clinical source E. coli isolates in Ecuador in 2016 (Ortega-Paredes et al., 2016). Next, the coexistence of mcr-1 and ESBL encoding genes (including bla CTX-M-55/14 ) has been found in Tunisian from chicken, in China from food animal (including pigs, cattle and chickens) and in France from human E. coli (Birgy et al., 2018;Hassen et al., 2020;Shafiq et al., 2021). The coexistence of mcr-1 and bla CTX-M in Salmonella isolates was mostly reported from food animal sources and found in Asian countries, including China, Cambodia and Laos (Ma et al., 2017;Lay et al., 2021). Last but not least, the coexistence of mcr-1, bla  , and bla  in Klebsiella pneumoniae ST485 Clinical Isolates appeared in China , which further alerted us to the dangers of multidrug-resistant strains.

CONCLUSION
In summary, our study investigated the epidemiology of S. Typhimurium in Guangdong province, China. which could supplement important local epidemiological data. Among them, ST34 S. Typhimurium dominated the cefotaxime-resistant strains and the major resistance mechanism of cefotaxime-resistant Salmonella produced the CTX-M-type ESBLs, in which bla CTX-M-55 was most prevalent. Obviously, the prevalence of bla CTX-M -positive S. Typhimurium carried multiple resistance genes, which indicated the potential risk of Salmonella infections. In the current study, bla CTX-M-55/65 -positive S. Typhimurium isolates were found from different outpatients with community acquired diarrhoea at same hospital, which suggested the nosocomial cloning transmission. This study underscored the importance of surveillance for bla CTX-M -positive microbes in patients and indicated a high likelihood for the spread of cephalosporin resistance from pig chain to humans.

DATA AVAILABILITY STATEMENT
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 in the article/Supplementary Material.

ETHICS STATEMENT
Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
QJ wrote the first draft of the manuscript. QJ and L-xF contributed to conception and design of the study. B-xK, D-sW, DW, M-gW, R-yS, J-eL, and ZS performed the statistical analysis. All authors contributed to manuscript revision, read, and approved the submitted version.