Antimicrobial Resistance and Genomic Characterization of Salmonella Isolated from Pigeons in China

. Salmonellosis is one of the important bacterial infectious diseases affecting the health of pigeons. Heretofore, the epidemiological characteristics of Salmonella in pigeon populations in China remain largely unclear. The present study investigated the antimicrobial resistance and genomic characteristics of Salmonella isolates in pigeons in different regions of China from 2022 to 2023. Thirty-two Salmonella isolates were collected and subjected to 24 different antimicrobial agents, representing nine categories. The results showed that these isolates were highly resistant to cefazolin (100 % ), gentamicin (100 % ), tobramycin (100 % ), and amikacin (100 % ). Three or more types of antimicrobial resistance were present in 90.62 % of the isolates, indicating multidrug resistance. Furthermore, using whole genome sequencing technology, we analyzed the pro ﬁ les of serotypes, multilocus sequence typing, virulence genes, antimicrobial resistance genes, and plasmid replicons and constructed phylogenetic genomics to determine the epidemiological correlation among these isolates. All strains belonged to Salmonella Typhimurium var. Copenhagen and exhibited ﬁ ve antimicrobial resistance genes and more than 150 Salmonella virulence genes. Moreover, each isolate contained both the IncFIB(S) and IncFII(S) plasmids. In addition, phylogenetic analysis showed that all isolates were very close to each other, and isolates from the same region clustered in the same branch. Overall, our ﬁ ndings provide the ﬁ rst evidence for the epidemiological characteristics of Salmonella in pigeons of China, highlighting the importance of preventing salmonellosis in pigeons.


Introduction
Salmonella is one of the main pathogens leading to the global outbreak of foodborne illness, which poses a serious threat to human health worldwide [1].In China, 70%-80% of bacteria-related foodborne illnesses are caused by Salmonella infection [2].Recent surveys of the disease burden in multiple Chinese provinces have shown that the annual incidence of Salmonella is roughly 245/100,000 [3].Moreover, more than 2,610 Salmonella serotypes have been identified, many of which are pathogenic to both humans and animals [4,5].Notably, Salmonella-infected animals, including mice, wild animals, fowl, and domestic livestock, are important sources of Salmonella contamination in humans.Thus, it is of great importance to monitor the epidemiological profiles of Salmonella infections in animals.
Due to a close contact with humans, pigeons present one important source where humans acquire Salmonella infections.For example, Salmonella in meat pigeons can infect humans via the food chain, while racing pigeons can transmit the pathogen through direct contact and feces [6,7].Salmonellosis can spread not only horizontally but also vertically.For instance, Salmonella can infect breeding pigeons and then spread to the eggs through vertical transmission, resulting in egg necrosis and a decrease in the hatching rate.It can also infect squabs via lactation, leading to infection or even death.Moreover, any age of pigeons can be infected with Salmonella [8], causing a large number of deaths [9].In addition, Salmonella Typhimurium (S.Typhimurium) is one of the most important serotypes of Salmonella Enteritidis, which can infect both humans and a range of animal hosts due to its excellent survival ability under different dietary conditions [10].
In China, the industry for pigeon racing has grown, and meat pigeon consumption has increased in recent years.Accumulating evidence has shown the infection of Salmonella in pigeons, with S. Typhimurium infections as the majority species.For example, S. Typhimurium is reported to be the dominant serotype of pigeon Salmonella isolates in Guangdong and Shanghai, China, and Poland [11][12][13].Infected pigeons often show single or multiple serious clinical symptoms, including diarrhea, weight loss, limps, head and neck skews, and other neurological symptoms [13,14].Furthermore, the misuse and abuse of antibiotics to treat salmonellosis has led to the emergence of multidrug resistance (MDR) bacteria [15,16].Therefore, a comprehensive epidemiological study and MDR profiles are needed to clarify the potential danger of Salmonella infection to pigeons and human health in China.Whole genome sequencing (WGS) can provide accurate information on population dynamics, genome epidemiology, and bacterial genomic characteristics [17].A previous study has used WGS to analyze the serotype, antimicrobial resistance, multilocus sequence typing (MLST), virulence genes, and plasmid replicons of Salmonella strains in the United States [18].However, the WGS profiles of Salmonella isolates in Chinese pigeon populations are still unknown.
In the present study, we investigated the prevalence of Salmonella in racing pigeon clubs and meat pigeon farms across various regions of China.A total of 32 Salmonella strains were isolated from pigeons.Phenotypic resistance results showed that 90.62% of the isolates were MDR.Furthermore, WGS in conjunction with bioinformatics analysis was used to investigate the genomic characteristics of Salmonella isolates.We found that all isolates were Salmonella Typhimurium var.Copenhagen, and the resistance genes, virulence genes, and plasmid types carried by them were also very similar.Overall, these results contribute to a better understanding of the epidemiology and transmission mechanisms of Salmonella in pigeon populations in China.

Ethics Statement.
This study was carried out in accordance with standard procedures without any operations on living animals.Dead pigeons with clinical symptoms of Salmonella infection were used as a sample source for this study.

Sample Collection.
Between June 2022 and April 2023, 215 samples were gathered for this investigation, comprising 92 samples of racing pigeons and 123 samples of meat pigeons.This study was not a random sampling, but a sample of sick pigeons with clear clinical symptoms was selected through clinical diagnosis.These collected samples showed weight loss, head and neck deviation, arthritis, diarrhea, and other single or comprehensive symptoms.Details of the sample collection are shown in Table 1.These samples were collected from four cities in Hebei Province, two cities in Shaanxi Province, Henan Province, Hunan Province, Jiangsu Province, Fujian Province, Ningxia Hui Autonomous Region, and Shanghai (Figure 1).

Isolation and Identification of Salmonella
Isolates.The procedure for Salmonella isolation is depicted as follows: Following aseptic necropsy, tissue samples from the liver, heart, kidney, lung, and intestine were collected for Salmonella isolation (Figure S1).To increase the number of bacteria in tissue samples, the proper amounts of the tissue samples were inoculated in a selenite cystine solution (Haibo biology, HB4085, China) and cultured for 18 hr at 37°C.Then, the red precipitate's culture solution was smeared on agar medium containing xylose-lysine-deoxycholic (XLD) (Haibo biology, HB4105, China) acid and cultured at 37°C for 24 hr to identify potential Salmonella colonies.Finally, characteristic black center colonies or colorless translucent colonies were chosen to identify Salmonella isolates using 16S rDNA PCR (Table S1 and Figure S2).(R).In order to facilitate the analysis of the results, the sensitive intermediate-resistant strains were identified as having antimicrobial resistance, and the strains with three or more antimicrobial resistances were identified as having MDR.The quality control strain utilized was Escherichia coli ATCC 25922.

Genomic DNA Extraction and Whole Genome Sequencing.
The isolated strains were shaken overnight at 37°C in Luria-Bertani (LB) (Haibo biology, HB0128, China) liquid medium, and then centrifuged to obtain a bacterial precipitate.Bacterial genomic DNA was extracted using a commercial TIANmp Bacterial DNA kit (Tiangen Biotech, China) according to the manufacturer's instructions.After passing the DNA sample quality test, a sequencing library was generated using the Rapid Plus DNA Lib Prep Kit for Illumina (Cat.No. RK20208).WGS of all isolates was performed at Novogene Bioinformatics Technology Co., Ltd.(Tianjin, China) using Illumina platforms and the PE150 strategy.
Additionally, an evolutionary tree based on the SNPs of the core genomes was built using the maximum likelihood method (Roary and FastTree).In this study, the phylogenetic tree, corresponding serotype, ST (sequence type), province, antimicrobial resistance genes, and insertion sequences were visualized using the Interactive Tree of Life (https://itol.embl.de)web server.

Prevalence and Serotypes of Salmonella in Pigeons.
Among all meat pigeon samples (n = 123), 32 Salmonella isolates were identified, mainly distributed in Henan, Hebei, Hunan, and Shanghai in China.Of the 96 racing pigeon samples collected from five provinces, no Salmonella isolate was found.In the entire pigeon population, the prevalence of Salmonella was 14.9%, while the prevalence in meat pigeons was 26%.Interestingly, every isolate shared the same serotype and MLST pattern and had similar copies of the seven housekeeping genes: aroC, hemD, purE, thrA, dnaN, sucA, and hisD.All isolates were Salmonella Typhimurium var.
3.5.Salmonella Virulence Genes.In order to investigate the virulence gene profile of Salmonella isolates, we analyzed the genome with the complete VFDB dataset.A total of 156 genes associated with the virulence and pathogenicity mechanisms of Salmonella were found in the 32 Salmonella strains.The number of virulence genes in each isolate ranged from 151 to 156, and there was little difference in virulence genes among isolates (Figure 5).Moreover, the distribution of S. Typhimurium virulence genes isolated from various geographical locations was nearly the same, with only one to six virulence gene differences.There were 1-6 virulence gene differences among different isolates, including safD, stdC, ssaI, ssaM, sseG, and sspH2.All Salmonella isolates had plasmids harboring the spvB gene, the stress adaptation gene sodCI, the enterotoxin gene stn, and the serum resistance gene rck, all of which are critical components of the virulence system of Salmonella.In addition, the typical virulence genes from Salmonella pathogenic islands 1 and 2 (SPI-1 and SPI-2) were present in all Salmonella strains analyzed.

Discussion
As an important zoonotic pathogen, Salmonella is seriously harmful to humans and animals, causing huge economic losses.The pigeon breeding industry is the fourth major poultry breeding industry in China [26].However, so far, the pigeon industry has not yet established a strict biosafety management system, and there are also defects in disease prevention and immunization procedures.Therefore, once a Salmonella infection occurs, it will cause serious damage to pigeons.The present study investigated the epidemiological, antimicrobial resistance, and genomic characteristics of Salmonella isolated from pigeons in various parts of China using WGS data in conjunction with accurate bioinformatics techniques, which provides a scientific basis for the spread of Salmonella in pigeons in China.
In this study, 32 strains of Salmonella from pigeons were all Salmonella Typhimurium var.Copenhagen.MLST analysis showed that all strains belong to ST128.Previous studies have reported that S. Typhimurium is the most common serotype of Salmonella in animals [27].Moreover, the Copenhagen variant is the most prevalent variant of S. Typhimurium in pigeons [28].It is reported that the sequence type of S. Typhimurium isolated from pigeons from China and Poland was also ST128.These results, including ours, indicate that the serotype and sequence type of Salmonella isolates may be a stable lineage in the pigeon population.Furthermore, we showed that the prevalence of S. Typhimurium in the overall pigeon population was 14.9%, and in meat pigeons, it was 26%.In line with our finding, two previous studies reported 26.1% and 21% prevalence of S. Typhimurium in the meat pigeon population in the markets of Shanghai and Beijing, respectively [12,29].
The prevalence of salmonellosis in pigeons in Italy, Egypt, and Poland was reported to be 0.9%, 13.3%, and 5.5%, respectively [13,30,31].The prevalence of salmonellosis in pigeons varies in different countries, which may be related to different sampling methods.But it is worth noting that the situation of Salmonella infection in meat pigeon populations in China may be more severe, which needs extensive investigation in pigeon populations.
Antibiotics are the most commonly therapy for the treatment of bacterial diseases, including pigeon salmonellosis.In the field of veterinary medicine, the widespread and sustained use of antimicrobial agents has led to the development of AMR in animals and livestock [32].The present study evaluated the phenotypic and genotypic AMR profiles of 32 Salmonella strains in pigeons.In order to identify accurate epidemiological information about the antimicrobial susceptibility of Salmonella, all isolates were tested for 24 different antimicrobial agents.In the present study, the phenotypic resistance results of amikacin, gentamicin, and tobramycin were consistent with the aminoglycoside resistance gene aac (6 ′ )-Iaa, and there was no significant correlation between the resistance of other types of antibiotics and the resistance genes carried.Antimicrobial resistance genes do not necessarily mean phenotypic resistance and vice versa.The occurrence of antimicrobial resistance is not only determined by the presence of antimicrobial resistance genes but also affected by various factors, such as enzyme activation, target modification or protection, and biofilm formation [33].We found that all strains exhibited a common resistance to gentamicin, amikacin, tobramycin (aminoglycosides), and cefazolin (first-generation cephalosporins), although they come from different regions.The result was also observed in the antimicrobial susceptibility test of Salmonella isolated from humans [34].We also found that some strains were resistant to tigecycline, but no plasmid-mediated tigecycline resistance genes tet (X3) and tet (X4) were found.It is speculated that the causes of antimicrobial resistance are related to other tigecycline resistance mechanisms, such as overexpression of efflux pumps, mutation of cell membrane porin, and ribosome protection mechanisms [35].Notably, we showed that 90.62% of Salmonella isolates in pigeons were MDR and could resist at least three types of antimicrobial agents.Considering the limited antibiotics available to pigeons at present, infection with MDR strains may lead to ineffective treatment.This reminds us to continuously monitor antimicrobial resistance in pigeon populations and use antibiotics reasonably.
The antimicrobial resistance genes predicted by the two data platforms are slightly different.Therefore, we combined the antimicrobial resistance genes and chromosome mutation results with a threshold consistency higher than 95% in the two data platforms for statistics.Antimicrobial resistance can be more accurately predicted when the two methods are combined.We detected five antimicrobial resistance genes in Salmonella isolates from pigeons.Among them, the most common resistance gene is aac (6 ′ )-Iaa, which encodes aminoglycoside drugs.Moreover, phenotypic resistance studies confirmed that all strains were resistant to aminoglycosides Transboundary and Emerging Diseases (gentamicin, tobramycin, and amikacin).Consistent with a previous study [36], all isolates were positive for the gene aac (6 ′ )-Iaa encoding aminoglycoside resistance.In addition, we found that the efflux pump of the mdsABC complex encodes the antimicrobial resistance genes mdsA and mdsB.MdsABC is a unique multidrug transport factor for Salmonella.MdsA and mdsB can promote resistance to various antibacterial drugs, including phenicol antibiotic, cephalomycin, monobactam, carbapenem, and cephalosporin.This indicates that its presence can lead to resistance to multiple antibiotics [37][38][39][40].Furthermore, partial regulatory systems contribute to bacterial resistance to heavy metals.In Salmonella, the response to gold ions is mediated by a specific metal regulatory factor golS, which controls the expression of the RND (resistance-nodulation-division) efflux pump gesABC.The CpxR/CpxA system (cell-envelope stress-responding system) promotes the gold resistance of Salmonella by controlling gols-dependent gesABC transcription, enabling microorganisms to resist contaminated environments [41][42][43].Notably, the sdiA gene exists in all isolated strains.To our best knowledge, there is no study to clarify the role of the sdiA gene in antimicrobial resistance.The sdiA gene of Salmonella regulates the rck gene, which mediates the adhesion and invasion of Salmonella to epithelial cells and the body's resistance to complement [44,45].In addition, we observed mutations caused by amino acid substitutions in gyrA, from serine to phenylalanine at codon 83 (S83F) and from aspartic acid to asparagine at codon 87 (D87N).Single point mutations in the quinolone resistance-determining region of gyrA reduce the sensitivity of fluoroquinolones (e.g., ciprofloxacin).However, antimicrobial resistance may require two or more mutations in the quinolone-determining region of gyrA, gyrB, parC, and parE [46][47][48].Plasmids, transferred between different bacterial species and clones, can carry antimicrobial resistance or virulence genes.Obtaining plasmids carrying antimicrobial resistance or virulence genes is transmitted to each other between bacteria of different and geographical origins, which may alter the prevalence of virulence or multidrug-resistant bacterial cloning [49,50].The present study found IncFIB(S) and IncFII(S) plasmids in all isolates.IncFIB(S) and IncFII(S) plasmids are Salmonella virulence plasmids that may carry virulence-related genes spv, rck (resistance to complement killing), and pef (plasmid-encoded fimbriae) [51].However, antimicrobial genes were not found on the contigs carrying the IncFIB(S) and IncFII(S) sequence replicons.This may have been due to the absence of these genes on virulence plasmids [23].
Virulence genes are closely related to the ability of Salmonella to invade host cells and spread in vivo [52].In this study, we found 156 genes related to Salmonella virulence and pathogenic mechanisms in the genomes of 32 Salmonella isolates in pigeons.All isolates carried the typical virulence factors of Salmonella pathogenicity islands 1 and 2, indicating the strong pathogenicity of these isolates.There were 1-6 virulence gene differences among different Salmonella isolates; however, the serum resistance gene rck, the stress adaptation gene sodCI, the enterotoxin gene stn, and plasmid-carrying spvB gene were detected in each Salmonella isolate.It is reported that rck functions as a "gene toxin" that affects the DNA integrity of epithelial cells, in addition to its roles as a "cyclomodulin" that affects the cell cycle machinery and as an invasion factor [53].The sodCI gene decreases external oxidative damage to host cells by encoding phagocytic superoxide dismutase [54].The enterotoxin stn gene has a significant role in the pathogenicity of S. Typhimurium infection, which can maintain the composition and integrity of the bacterial cell membrane by regulating the localization of the OmpA membrane [55].The spvB virulence gene of Salmonella acts as an intracellular ADP-ribosylation toxin, which can rearrange the host cytoskeleton to increase Salmonella invasion and promote systemic transmission [56,57].
Overall, the Salmonella strains prevalent in Chinese pigeon populations were Salmonella Typhimurium var.Copenhagen, although these isolates were isolated in different regions and at different times.The analysis of the whole genome sequence characteristics showed that these strains had highly similar antimicrobial resistance genes and virulence genes along with the same sequence type, serotype and plasmid replicons.In addition, the genetic relationship between isolates from different regions is very close, indicating that there may be a pandemic clonal transmission of pigeon salmonellosis in China.Studies have reported that the Salmonella Typhimurium var.
Copenhagen can be transmitted to humans and other animals [7,9,58].This suggests that Salmonella originating from pigeons may pose a threat to human health.Therefore, the prevention and control of salmonellosis should be strengthened in the pigeon industry.

Conclusions
Using WGS combined with bioinformatics analysis methods, this study provides accurate information on the epidemiology of Salmonella in pigeon flocks from different regions of China.Even if there are several limitations to the present study, such as a possible insufficient sample size, the results nevertheless carry significant reference value.We found that Salmonella typhimurium Copenhagen variants are the common serotype prevalent in pigeons in different regions of China.Notably, 90.62% of the isolates showed multidrug resistant, which deserves more investigations.Meanwhile, there are large number of virulence genes carried by the isolated strains, indicating a strong pathogenicity and virulence and consequently, a serious harm to pigeon industry.Overall, the present study reveals the urgency of strengthening the research and monitoring of Salmonella epidemiology in pigeons.

FIGURE 1 :
FIGURE 1: Distribution of sampling locations in eight provinces and regions of China.

FIGURE 2 :
FIGURE 2: Distribution of Salmonella isolates studied according to sampling year, province, host, sequence type, and serotype.

TABLE 1 :
The source and prevalence statistics of Salmonella collected samples in this study.
LVX, 1−8 μg/mL), folate pathway inhibitors (trimethoprim/sulfamethoxazole: TMS, 2/38−4/76 μg/mL), and nitrofuran (nitrofurantoin: NIT, 32-64 μg/mL).The minimum inhibitory concentration (MIC) for each antibiotic was interpreted using CLSI standards and NARMS breakpoints, and the MIC values were categorized as susceptible (S), intermediate (I), or resistant Heatmap of tree hierarchical clustering of virulence genes and isolated strains.This figure shows the prediction of the virulence gene profile of the studied isolates.The x-axis shows the isolate ID numbers, and the y-axis shows the identified selected virulence genes.Blue cells indicate the presence of a gene; gray cells indicate the absence of a gene.