Antimicrobial Resistance in Non-typhoidal Salmonella from Retail Foods Collected in 2020 in China

Objective: Non-typhoidal Salmonella (NTS) is a major cause of human salmonellosis globally. Food animals are major NTS reservoirs. An increase in antimicrobial resistance (AMR) in foodborne NTS has led to clinical treatment failures. Here, to examine the prevalence and perform characterization of foodborne NTS with AMR in China, we tested the antimicrobial susceptibility of 1,256 NTS isolates cultured from retail foods in 2020 in China. Methods: The antimicrobial susceptibility of 26 antimicrobial agents representing 12 classes was evaluated with the broth-microdilution method; the presence of ten mcr genes was screened with multi-PCR. The complete closed genomes of mcr -gene-carrying isolates were generated by hybrid assembly through whole genome sequencing on both the PacBio and Illumina platforms. Genomic features and genetic environments of the mcr-1 gene were analysed. Results: The overall drug resistance rate was 92.28%, and the multi-drug resistance (MDR) rate was 76.53%. A total of 341 AMR profiles were determined, and resistance was highest to nalidixic acid (63.38%). Among 887 NTS isolates with MDR, 232 showed co-resistance to cefotaxime and ciprofloxacin, and 25 were resistant to ten classes of antimicrobial agents. The resistance of NTS isolated from different regions varied. Isolates from raw chicken sources most frequently showed resistance. Four NTS carried the mcr-1 gene and represented four different serotypes. Four mcr-1 gene-bearing plasmids from the four Salmonella isolates were classified into two replicon types (IncI2 and IncHI2A). Two mcr-1 genes in IncI2 type plasmids were found to be located between a PAP2 family protein-encoding gene and a relaxase-encoding gene, whereas the other two mcr-1 gene structures in IncHI2A type plasmids showed variations in the presence of insertion sequences. Conclusion: Our data demonstrated severe AMR among foodborne NTS isolated from food in China, thus highlighting the importance of antimicrobial susceptibility surveillance to decrease the spread of AMR, particularly to critical drugs in human medicine.


INTRODUCTION
Foodborne diseases remain a global public health challenge posing a major burden. In 2010, 31 hazards in unsafe food caused 600 million cases of foodborne illnesses and 420,000 deaths worldwide; 40% of these deaths occurred among children younger than 5 years of age [1]. The most frequent causes of these foodborne illnesses were diarrhoeal agents, which were responsible for 230,000 deaths. Non-typhoidal Salmonella (NTS), a major cause of foodborne infections, gives rise to more than 93 million cases of gastroenteritis annually and 155,000 deaths globally, thus resulting in approximately 4 million disability-adjusted life years [2]. In the United States, 1.35 million illnesses, 26,500 hospitalizations and 420 deaths have been estimated to be attributable to NTS, thus leading to more than $400 million in medical costs each year [3]. In 2019, 27 European Union member states reported 5,175 foodborne outbreaks, among which NTS was the most commonly identified agent and accounted for 17.9% of the total outbreaks [4]. From 2002 to 2017, China reported 2,815 foodborne disease outbreaks associated with meat and meat products, thus resulting in 52,122 illnesses, 25,361 hospitalizations and 96 deaths, among which NTS was the most common cause of outbreaks (420/2815, 14.92%) and hospitalizations (7641/25,361, 30.13%). Hence, NTS is the most frequently reported bacterial species causing human gastrointestinal infections globally. Food animals, mainly poultry, serve as a major reservoir of NTS, and contaminated animal-based products are frequently associated with human salmonellosis.
The emergence and spread of NTS with antimicrobial resistance (AMR) have become major public health concerns over the past two decades. The presence of extended spectrum beta-lactamase genes in NTS plasmids and reports of carbapenemase-containing NTS isolates are particularly concerning [5,6], because both confer resistance to highly important antimicrobial agents. The acquisition of genes conferring AMR to both antimicrobial agents on foodborne NTS along the food chain is increasing. Treatment options for salmonellosis in animals and humans have been hindered by AMR in NTS. Data from China have indicated that the prevalence of NTS with multi-drug resistance (MDR) increased from 20-30% in the 1990s to 70% in the early 2000s; moreover, the overall incidence of foodborne NTS with AMR exceeded 70% between 2015 and 2016, and was notably observed in strains carrying plasmids with the mcr-1 gene, which mediates resistance to colistin [7]. Food workers who are infected with NTS with AMR after consuming or handling contaminated food may serve as reservoirs, thus posing a high risk of further food contamination. To decrease the prevalence of NTS in foods and consequently the burden of human salmonellosis, China implemented a nationwide foodborne pathogen monitoring and control program. In this study, the antimicrobial susceptibility of 1256 NTS isolates cultured from retail foods in 2020 in China was tested. All isolates were subsequently screened for the presence of mcr genes through polymerase chain reaction (PCR), which was followed by whole genome sequencing of mcr gene-positive strains to provide further confirmation. Our aim was to gain genomic insight into antimicrobial mechanisms.

Bacterial strains
A total of 1256 foodborne NTS isolates were cultured from various retail foods, primarily meat and meat-based products, collected from 30 provinces (municipalities or autonomous regions) in China in 2020. The presumptive colonies were confirmed to be Salmonella according to both their morphology and invA gene amplification by PCR, as described previously; those with negative amplification were further validated with GN card and Vitek2 compact (BioMérieux, France) analysis [8]. All isolates confirmed to be Salmonella were preserved in brain heart infusion broth with 40% (v/v) glycerol (HopeBio, Qingdao, China) at -80°C before analysis. Escherichia coli ATCC ® 25922 was used as the control in antimicrobial susceptibility testing (AST).
Whole genome sequencing DNA extraction and whole genome sequencing were conducted for mcr-gene-carrying isolates to obtain complete genomes. Briefly, single colonies of NTS isolates were cultured in brain heart infusion broth and incubated at 37°C overnight. A TIANamp bacterial DNA extraction kit (DP302, TIANGEN BIOTECH, Beijing, China) was used to extract the bacterial genomic DNA according to the manufacturer's instructions, and library preparation was then performed with an NEBNext ® Ultra DNA Library Prep Kit for Illumina (NEB#E7370) and sonication fragmentation (350 bp insert). Sequencing was performed commercially on the Illumina HiSeq platform with a PE 150 sequencing strategy (Novogene, Beijing, China) and a HiSeq X Ten Reagent Kit v2.5 (Illumina, San Diego, CA). The mcr gene carrying isolates were also sequenced on the SMRT ® Pacific Biosciences (PacBio) Sequel platform (Tianjin Biochip Corporation, Tianjin, China), with a 10-kbp template library preparation step with a PacBio ® Template Prep Kit. SMRT Analysis v2.3.0 was used for de novo assembly according to the RS Hierarchical Genome Assembly Process (HGAP) workflow v3.0. Subsequently, Consed version 28.0 was used to manually inspect and trim duplicate ends to generate single, complete and closed sequences for each chromosome and plasmid. For data error correction, Pilon v1.23 was used with Illumina MiSeq sequencing read data. The closed genomes were then annotated with prokka (version 1.14.6).

Bioinformatic analysis
The predicted serotypes and multi-locus sequence typing (MLST) types were identified with the Salmonella In Silico Typing Resource (SISTR). Plasmid replicon types (Incompatibility groups or Inc groups) were identified through the Center for Genomic Epidemiology (CGE) website with PlasmidFinder (v2.0). All gene, plasmid and chromosome sequences used in this study were managed, aligned and analysed in Geneious prime (v2023.1.2) software. The genetic environments of the mcr-1 gene were analysed and displayed with Easyfig (v2.2.2).
Antimicrobial resistance of NTS from food samples NTS isolates were recovered from six categories of foods in this study. Among the food categories, resistance to any of the 26 tested compounds was most frequently observed in raw chicken sources (approximately 93.85% resistant to one or more agent class, 565/602), followed by other raw poultry sources (92.04%, 104/113) and raw duck (88.19%, 254/288). NTS cultured from prepared meat exhibited the same trend of resistance to one class of drug as that observed in raw poultry meat (93.55%, 58/62 for red meat sources; 93.85%, 122/130 for poultry meat sources). MDR was most frequently found in isolates from prepared poultry meat (76.15%, 99/130) and raw chicken (74.42%, 448/602), followed by prepared red meat (72.58%, 45/62), other raw poultry  and resistance phenotypes against a panel of 26 antimicrobial compounds are shown in Table 3. Of note, three strains, 2020s302, 2020s327 and 2020s329, cultured from two prepared chicken samples and one prepared beef sample, respectively, were from the same region (Quzhou, Zhejiang province), whereas strain 2020s542 was isolated from prepared chicken meat in Suizhou, Hubei province. All four mcr-1 positive isolates showed resistance to ampicillin, ceftriaxone, cefotaxime and colistin, and susceptibility to cefoxitin, ertapenem, imipenem, meropenem, amikacin, tigecycine and nitrofurantoin. The four mcr-1 positive strains showed an MDR phenotype for at least three classes of antimicrobial compounds. The strains 2020s302, 2020s327, 2020s329 and 2020s542 were resistant to three, nine, ten and eight classes of antimicrobial agents, respectively, with AMR profiles   Table 4.
To better understand the genetic environment of the mcr-1 loci of the plasmids bearing the mcr-1 gene, we compared and analysed sequences extracted from various plasmids from this study and previous studies, belonging to two replicon types (Fig 2). The analysis revealed that the mcr-1 genes in five IncI2 type plasmids (pCFSA244-2, pCFSA664-3, pHNSHP45, p2020s542-3 and p2020s302-1) were located between a PAP2 family protein-encoding gene (yellow arrow) and a relaxase-encoding gene (dark green arrow). In plasmid pHNSHP45 (KP347127), an IS30 family element ISApl1 was followed by a relaxase-encoding gene downstream. In plasmids p2020s542-3 and p2020s302-1 in this study, together with pCFSA244-2 and pCFSA664-3, the mcr-1 genes were found to have a PAP2 family proteinencoding gene distal to the right site of the mcr-1 gene, without any insertion sequences (ISs).
In comparison with these IncI2 type plasmids, seven IncHI2A plasmids (two plasmids in this study and five other plasmids from previous studies) did not have a relaxase-encoding gene upstream of the mcr-1 gene, but encoded some hypothetical proteins and contained some open reading frames (ORFs). Beyond the ORF differences, the main difference in the gene structures near mcr-1 among the plasmids was the varying presence of insertion sequences. pCFSA1096 had no IS; pCFSA122-1, pCFSA629, pHNSHP45-2 (KU341381) and p2020S329-2 contained only one ISApl1; and a tellurium resistance gene cluster was located downstream of the PAP2 encoding gene in pCFSA629, pHNSHP45-2 and p2020S329-2. Moreover, pWW012 (CP022169), the mcr-1-carrying plasmid from our previous study, and p2020s329-2, from the present study, contained an IS-mcr-1-PAP2-IS module, which is an ISApl1-f lanked composite transposon (Tn6330). Notably, the PAP2 encoding gene of p2020s329-2 had exactly the same sequence as the same gene in pWW012 but in an opposite orientation.

DISCUSSION
AMR poses an important, complex, and high-priority global public health challenge. China has one of the largest food animal production economies worldwide. To decrease the potential consequences of foodborne AMR risk to humans, animal and plant health, China has implemented a national AMR monitoring system. The status of resistance in Salmonella is assessed annually in many samples, primarily retail meat products. In this study, we characterized NTS isolates cultured in 2020, which were tested for resistance to a panel of 26 antimicrobial agents. The drug resistance rate of food-borne NTS in 2020 was 92.28%, and the MDR rate was 76.53%, in agreement with those reported in 2015 [7]. However, a higher resistance frequency was found for certain antimicrobial agents, such as cephems, quinolones, f luoroquinolones, lipopeptides, penicillins and aminoglycosides, which have a long history of use in food production chains in China.
Quinolones are the preferred first-line drugs for clinical treatment or prevention/prophylaxis of Salmonella disease. The frequency of drug resistance to nalididic acid and ciprof loxacin was 63.38% and 26.51%, respectively-values slightly higher than those obtained in 2016 (52.5% and 21.3%) [14]. Therefore, much greater attention should be paid to the continuing increase in quinolone resistance, which could lead to a risk of clinical treatment failure. AMR varied among regions and food categories. Foodborne NTS showed regional differences in drug resistance, ranging between 100% and 78.57% in this study. A total of 341 AMR profiles were found in the tested NTS isolates, thus indicating high polymorphism. Additionally, more than 90% of the Salmonella isolates were resistant to at least one antimicrobial agent, and resistance to commonly used compounds including ampicillin, ampicillin-sulbactam, nalidixic acid and tetracycline was observed among substantial numbers of study isolates. For example, the frequency of resistance to ciprof loxacin (26.51%) and extended-spectrum cephalosporins, including ceftazidime (17.75%) and cefotaxime (25.64%), was much higher than values reported for NTS cultured from raw chicken carcasses between 2011 and 2012 (16.47% for ciprof loxacin, 4.71% for ceftazidime and 11.18% for cefotaxime) [8]. Given NTS from poultry sources in this study tended to be more resistant than that from other sources FIGURE 2 | Genetic environments associated with the mcr-1 gene in different bacterial plasmids. The figure was generated in Easyfig (v2.2.5). Plasmids marked with "pCFSA" were carried by the mcr-1 positive Salmonella isolates, according to our previous research [1,2], and the plasmid pWW012 belonged to a Salmonella isolate, according to previous research from our laboratory (accession number: CP022169) [3], whereas plasmids pHNSHP45 and pHNSHP45-2 (accession number: KP347127 and KU341381) belonged to Escherichia coli strain SHP45, the first reported isolate bearing the mcr-1 gene [4]. Replicon types are shown in two groups for all plasmids. Confirmed and putative open reading frames (ORFs) are indicated by block arrows, their orientations are indicated by different colours, and arrow size is proportional to the predicted ORF length. The mcr-1 gene is indicated by a red arrow, whereas genes encoding mobile elements (insertion sequence, IS) are indicated by blue arrows. Regions of homology among plasmids, ranging from 67% to 100% sequence identity are indicated by the graded shaded regions between sequences. and also than that from ten years ago, Salmonella isolated from raw chicken samples collected after 2020 might have higher level of drug resistance. Hence, administration and management of the use of antimicrobial agents in the food production chain is essential. Carbapenems are not used in Chinese agriculture, nor are they approved for use in food-producing animals in any country. No carbapenemase-producing Salmonella was found in the present study, thus suggesting that carbapenems may still be effective when tested in vitro. However, resistance to carbapenem compounds must be monitored, because these compounds might have suboptimal efficacy in the clinical treatment of Salmonella infection in vivo in some cases.
Polymyxins are important lipopeptide antibiotics that serve as the last line of defence against multidrug-resistant Gram-negative bacterial infections. The clinical utility of polymyxins is currently facing a highly concerning threat with the global spread of mobile colistin resistance (MCR) and the relevant mcr genes, which are the main determinant of polymyxin resistance in Escherichia coli. High prevalence of these genes in agriculture persists globally, and particularly in China, owing to high polymyxin usage. The transferability of mcr is of considerable concern, because of the potential of multidrug-resistant Gramnegative bacteria to acquire mcr-bearing plasmids and thus evade antimicrobial treatment with the last-line polymyxins. The mcr-1 gene was first reported in November 2015 in China [15]. Although the use of colistin as a feed additive for animals has been banned for agriculture purposes in China since 30 April 2017 [16], NTS carrying the mcr-1 gene was isolated from lettuce, beef and pork products in various foods at a frequency of 1.07% (3/280) in 2017, and from goose eggs and field snails at a frequency of 0.69% (4/579) in 2018 (data not published). No mcr-1 positive foodborne NTS was detected in 2019. In the present study, mcr-1 in foodborne NTS collected in 2020 was detected at a low level of 0.32% (4/1256), similarly to the 0.23% (6/2555) in isolates reported by Hu et al. [17]. Compared with our previous data on food sources of mcr-1-bearing Salmonella isolates (pork, chicken, egg, and dumpling sources) [17], the four strains of Salmonella carrying the mcr-1 gene in this study were cultured from either poultry or beef, thereby indicating that Salmonella, as a reservoir of the mcr-1 gene, may have complex diversity in food sources, and the mcr-1 positive clone may be largely limited to meat. Additionally, the widespread presence of mcr-1 positive Salmonella in chicken, beef, pork, egg and vegetables also suggested potential transmission via the food chain, particularly by chickens.
The most common mcr-1 gene locus structure is a 2609 bp DNA sequence consisting of an mcr-1 gene and a putative PAP2 super family protein gene, along with two copies of ISApl1, which is a member of the IS30 family; this structure forms the composite transposon Tn6330, which is ISApl1-flanked and is also believed to mediate the initial mcr-1 gene mobilization event [18,19]. Although Tn6330 was commonly found among many mcr-1-bearing isolates, the mcr-1 gene can also be disseminated through just a single end of ISApl1 or other means not involving this IS element. Dissemination can occur with different plasmid replicon types, including IncI2-, P-, X4-and HI-type plasmids, thus contributing to four general mcr-1 structures identified to date [18,20]. In this study, the mcr-1 region located on p2020s327-1 had a highly similar single-ended Tn6330 variant structure and tellurium resistance gene coding region to those in two other mcr-1 locus structures on pCFSA629 and pHNSHP45-2, from a S. Typhimurium and an E. coli isolate, respectively. Moreover, both the mcr-1 regions belonging to p2020s329-2 and pWW012 (S. Typhimurium) contained more than one IS. However, the IS from pWW012 had the standard Tn6330 structure, except for one difference in p2020s329-2, in which the PAP2 encoding gene had an opposite orientation, possibly because of genetic rearrangement caused by the loss and gain of ISApl1 from the transposon during multiplication. Plasmid-to-chromosomal transfer of mcr-1 has also been suggested to have occurred recently, and Tn6330 on the chromosome might provide a relatively stable mcr-1 state, and the loss of ISApl1 from the transposon may occur during the mobilization event, thus posing an additional challenge in preventing the spread of colistin resistant bacteria [21].
This report showed that all four mcr-1 positive Salmonella isolates had MDR phenotypes, three of which were resistant to more than seven (or as many as ten) of the 12 examined antibacterial classes. Because polymyxins are the last therapeutic option for life-threatening infections caused by Gram-negative 'superbugs', all possible effort must be made to minimize the emergence of resistance, particularly that due to mcr. Hence China should consider integrated monitoring and surveillance of foodborne antimicrobial use as well as AMR in humans, animals and plants/crops, on the basis of a "One Health" approach-a strong multi-sectoral collaborative and institutional system [22]. Furthermore, more studies should focus on mechanisms and transmission of resistance of food-borne Salmonella to important antimicrobial drugs, to provide a theoretical basis for the rational use of antimicrobial drugs and governmental supervision to ensure food safety.