Global Spread and Molecular Characterization of CTX-M-Producing Salmonella Typhimurium Isolates

This study aimed to determine the global prevalence and molecular characterization of CTX-M-producing Salmonella Typhimurium isolates. A total of 330 (15.2%, 330/21779) blaCTX-M-positive S. Typhimurium were obtained from the public databases in July 2021. Thirteen variants were found in the 330 members of the blaCTX-M group, and blaCTX-M-9 (26.4%, 88/330) was the most prevalent. The majority of blaCTX-M-positive S. Typhimurium were obtained from humans (59.7%, 197/330) and animals (31.5%, 104/330). The number of blaCTX-M-positive S. Typhimurium increased annually (p < 0.0001). These isolates were primarily found from China, the United Kingdom, Australia, the USA, and Germany. In addition, these isolates possessed 14 distinct sequence types (ST), and three predominated: ST34 (42.7%, 141/330), ST19 (37.0%, 122/330), and ST313 (10.3%, 34/330). The majority of ST34 S. Typhimurium isolates were distributed in China and mainly from swine. However, the majority of ST19 were distributed in the United Kingdom and Australia. Analysis of contigs showed that the major type of blaCTX-M-carrying plasmid was identified as IncI plasmid (52.9%, 27/51) and IncHI2 plasmid (17.6%, 9/51) in 51 blaCTX-M-positive S. Typhimurium isolates. In addition, WGS analysis further revealed that blaCTX-M co-existed with nine antibiotic-resistant genes with a detection rate over 50%, conferring resistance to five classes of antimicrobials. The 154 virulence genes were detected among these isolates, of which 107 virulence genes were highly common. This study revealed that China has been severely contaminated by blaCTX-M-positive S. Typhimurium isolates, these isolates possessed numerous ARGs and virulence genes, and highlighted that continued vigilance for blaCTX-M-positive S. Typhimurium in animals and humans is urgently needed.


Introduction
Salmonella is an important foodborne pathogen that has a nearly ubiquitous distribution among humans, animals, and the open environment. According to the WHO, there over 90 million people are infected by Salmonella annually, and 150,000 people die from Salmonella infection [1]. Over 2600 Salmonella serotypes have been identified and can be referred to as typhoidal or non-typhoidal (NTS); the latter are present in different animal reservoirs and are responsible for self-limiting gastrointestinal syndromes [2]. S. Typhimurium can cause potential threat to human health through the consumption of contaminated food or water [3]. Through estimates of the global invasive non-typhoidal Salmonella disease, a study recently found that S. Typhimurium is emerging in several African countries [4]. After its oral acquisition, S. Typhimurium travels down the intestinal tract and reaches the large intestine, where most of its replication is thought to occur [5].
Treatment of invasive salmonellosis has been compromised due to the emergence of Salmonella isolates with single or multidrug resistance to a number of first-line agents. Consequently, the third generation cephalosporins, such as ceftriaxone, have become treatment modalities of choice for therapy against severe Salmonella infections [6]. However, the increasing prevalence of cephalosporin-resistant Salmonella is also an emerging problem in recent years [7]. Resistance to cephalosporins is mainly due to the acquisition of extended-spectrum β-lactamase genes (ESBLs), among which CTX-M-type enzymes are currently most common-usually located on transmissible plasmids-and can disseminate among the Enterobacteriaceae family [8]. The number of reported cases in various ESBL-producing Salmonella serotypes has been increasing worldwide in recent years, with the predominant CTX-M group that was recently detected in poultry and poultry products in different countries [9,10].
In general, Salmonella virulence factors have a crucial role in systemic infections [11]. Virulence genes are located on various locations of the genome, including Salmonella pathogenicity islands (SPIs) and several mobile genetic elements such as prophages and plasmids [12]. SPI-1 and SPI-2 encode for Type III secretion system (TTSS) and play an important role in delivering different effector proteins into the host that mediates pathogenesis [13]. SPI-1 encodes for genes such as invA, sipABD, and sopBD involved in the invasion of epithelial cells, whereas SPI-2 encodes the genes sifA and ssaR related to the survival and replication of Salmonella within phagocytic cells [14]. In addition, the virulence plasmids carrying virulence genes, such as the spv genes, play a role in Salmonella multiplication within its host cell, increasing the severity of enteritis [15].
In this study, we first employed genomic analysis from a public database to conduct a global survey of epidemic characterization of CTX-M-producing S. Typhimurium. We then characterized the molecular characteristics, diversity, antibiotic-resistant genes, and virulence genotypes of these pathogens.   (Table  S1). It is worth mentioning that the majority of bla CTX-M -positive ST34 S. Typhimurium isolates were distributed in China (n = 94). However, the majority of ST19 were distributed in the United Kingdom (n = 63) and Australia (n = 34) (Figure 2A). In addition, swine were one of the most dominant hosts in the bla CTX-M -positive ST34 S. Typhimurium isolates ( Figure 2B).

Discussion
At present, it is concerning that the increasing incidence of infections is caused by ESBL-producing organisms, especially Salmonella spp., because they are resistant to most of the β-lactam antimicrobials and other antimicrobial classes [16,17]. During the last decade, the most encountered ESBL genes were the CTX-M enzyme family, primarily carried by transferable plasmids and transposons [18]. In this study, all 330 bla CTX-Mpositive S. Typhimurium were collected from humans and animals, especially swine. These isolates were distributed across 15 countries. The countries possessing the greatest amount of bla CTX-M -positive S. Typhimurium isolates were China, UK, Australia, USA, and Germany [19][20][21][22][23]. In addition, we noticed an increase in quantity of bla CTX-M -positive S. Typhimurium isolates from 2014 to 2020. This may be related to the increased rate of Salmonella after 2009 in food animals [24].
This group of 330 bla CTX-M -positive S. Typhimurium isolates possessed 14 distinct ST, with three predominant: ST34, ST19, and ST313. Previous studies have shown that ST19, ST34, and ST313 are commonly found STs of S. Typhimurium. Recently, one of the most prevalent S. Typhimurium, ST34, was widely reported in Europe, North America, Asia, and Australia [25]. In addition, ST34 S. Typhimurium isolates have acquired strong biofilm-forming ability and were often reportedly carrying the colistin-resistant genes (mcr-1, mcr-3, mcr-5, and mcr-9) and even the bla NDM gene [25][26][27]. ST19 and ST34 were the most common STs in Asia. However, ST19 isolates were resistant to fewer antibiotic classes than ST34 isolates [27]. Thus far, ST19 has been reported from humans, reptiles, ovine, swine, poultry, food, and bovine from France, Mexico, China, Germany, Scotland, Portugal, Qatar, Korea, Ireland, the United States, the United Kingdom, and Denmark, according to Enterobase [28]. ST313 is a relatively newly emerged sequence type and closely related to the ST19 group of S. Typhimurium, which has caused a devastating epidemic of bloodstream infections across sub-Saharan Africa [29].
The bla CTX-M genes were mainly located on IncI plasmid in the S. Typhimurium isolates, followed by IncHI2 and IncA/C plasmids. Additionally, 17 types of Inc plasmids were identified among the bla CTX-M -positive S. Typhimurium isolates. IncHI2 and IncHI2A were the most prevalent type of plasmid, followed by IncFIB and IncFII plasmids. IncI plasmids were closely associated with the spread of several ESBL genes. The bla CTX-M gene was located on IncI1 plasmids in all of the isolates, which were obtained from slaughterhouses located in seven districts in France [30]. A previous study has shown that IncA/C and IncHI2 were the most common types of plasmids among ESBL-producing Salmonella, and they often carried multiple antibiotic-resistant genes, including strB, qnrS, tet(A), sul, and dfrA14 [31]. Additionally, the numerous types of plasmids may serve as important vehicles for the spread of ARGs [32].
In addition to bla CTX-M , a total of 26 ARGs were identified among the S. Typhimurium isolates and conferred resistance to ten classes of antibiotics, including quinolone, aminoglycoside, β-lactam, sulphonamide, trimethoprim, tetracycline, phenicol, macrolide, fosfomycin, and colistin. Consistent with a previous study, most of the bla CTX-M -positive isolates carried acquired-resistance determinants associated with three or more drug classes [33]. Notably, a few bla CTX-M genes co-existed with mcr-1, mcr-3, mcr-9, and bla NDM-5 , conferring resistance to colistin and carbapenem, which were considered as the last-resort antibiotics used for the treatment of infections caused by multidrug-resistant Gram-negative bacteria [34].
An important trait of Salmonella is that they can invade, survive, and multiply in a host cell in the presence of genetic determinants for virulence. Virulence genes have been extensively studied in Salmonella [35]. In this study, a total of 154 virulence genes were identified in the 330 bla CTX-M -positive S. Typhimurium isolates, and most of the isolates carried 107 virulence genes. Of these, the detection rate of some virulence genes ranged from 26.4% to 31.8%, including grvA, spvB, spvC, spvR, pefA, pefB, pefC, pefD, and rck. The gene grvA (for Gifsy-2-related virulence) is located on the prophage Gifsy-2; grvA null mutant showed an increase in the virulence of serovar Typhimurium in mice [36]. SpvB facilitated Salmonella survive and replicate within macrophages via perturbing cellular iron metabolism [37]. The protein encoded by the spvC gene has a phosphorylated threonine lyase activity that inhibits MAP phosphokinase. The pathogenicity of Salmonella strains greatly increases when both spvB and spvC genes co-exist in the bacteria [38]. The spvR encodes a positive activator for the following four genes, spvABCD [39]. Plasmid-encoded fimbriae (Pef) of Salmonella are among the fimbriae whose expression have been observed in animals. Pef fimbriae biogenesis depends on the pef operon, located on the virulence plasmid of S. Typhimurium. This operon encodes the major Pef fimbriae subunit pefA, the usher protein pefC, required for the assembly of the fimbriae, and the pefD periplasmic chaperone for pefA [40]. Recent research has shown that rck protein is able to induce the Salmonella entry mechanism. rck mimics natural host cell ligands and triggers engulfment of the bacterium by interacting with the epidermal growth factor receptor [41].
There are several limitations of this study. First, the increase in genomes in the pubic repository may be related to the development of sequencing technology rather than an actual increase. Second, not all genomes of isolates will upload to the database. Finally, the phenotypes of antibiotic resistance and virulence genes and the transfer of resistance genes were not evaluated in this study.

Materials
A total of 21,779 assembled genomes of Salmonella Typhimurium were downloaded from the NCBI database in 8 July 2021 https://www.ncbi.nlm.nih.gov/assembly. The detailed information (collected year, hosts, and location) was obtained from the pathogens database (https://www.ncbi.nlm.nih.gov/pathogens, accessed on 8 July 2021).

Methods
All the genomes of S. Typhimurium were applied to a filter for the presence of bla CTX-M and other antibiotic-resistant genes using ResFinder (https://cge.cbs.dtu.dk/services/ ResFinder/ accessed on 29 July 2021). The contigs carrying bla CTX-M genes were extracted for the identification of plasmid types by PlasmidFinder software. Multilocus sequence types (MLST) and replicating type of plasmid were identified using MLST and Plas-midFinder (https://cge.cbs.dtu.dk/services accessed on 29 July 2021). Virulence factors were identified using the Virulence Factor Database (http://www.mgc.ac.cn/VFs/main. htm accessed on 13 September 2021). The heatmaps were visualized with the "pheatmap" package. The histogram, stack bar diagram, pie diagram, and line chart were plotted with "ggplot2" and colour was used with the "RColourBrewer" package. The χ 2 test and Fisher's exact test were used to perform the statistical analysis. For all models, we considered p < 0.01 statistically significant and then performed 2-sided probability on those results by using SPSS version 23.0.

Conclusions
In conclusion, we identified 330 bla CTX-M -positive S. Typhimurium isolates from the public database. Thirteen variants were found in the 330 members of the bla CTX-M group, and bla CTX-M-9 was the most prevalent. The majority of bla CTX-M -positive S. Typhimurium were obtained from humans and animals. The number of bla CTX-M -positive S. Typhimurium increased annually. These isolates were primarily detected in China, the United Kingdom, Australia, the USA and Germany. In addition, these isolates possessed 14 distinct ST, and three were predominant: ST34, ST19, and ST313. The majority of ST34 S. Typhimurium isolates were distributed in China and were mainly from swine. However, the majority of ST19 were distributed in the United Kingdom and Australia. The major types of bla CTX-Mcarrying plasmid were identified as IncI plasmid and IncHI2 plasmid. In addition, bla CTX-M co-existed with nine antibiotic-resistant genes with a detection rate of over 50%, conferring resistance to five classes of antimicrobials. The 154 virulence genes were detected among these isolates, of which 107 virulence genes were highly common. This study provides new insights into clinical antibiotic therapy for Salmonella infection and provides a foundation for further scientific research.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antibiotics10111417/s1, Figure S1: The analysis of hosts carrying the most common CTX-M allelic variants; Table S1: The detection rate of 154 virulence genes in 330 bla CTX-M -positive S. Typhimurium isolates.
Author Contributions: Methodology, software, data curation, and writing, L.G.; review and editing, Y.Z.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available in supplementary material.

Conflicts of Interest:
The authors declare no conflict of interest.

Abbreviation
The lists of abbreviation used in this study.

MLST Multilocus sequence types WGS
Whole genome sequencing Inc Incompatible ARGs Antibiotic resistance genes WHO World Health Organization ESBL Extended-spectrum β-lactamase SPI Salmonella pathogenicity island TTSS Type III secretion system NCBI National Center for Biotechnology Information