Antimicrobial Resistance and Genomic Characterization of <i>Salmonella</i> Isolated from Pigeons in China

Zhang, Yuhua; Lu, Zheng; Zhao, Haoyu; Li, Shuangyu; Zhuang, Hong; Wang, Juan; Li, Ruichao; Zheng, Weibo; Zhu, Hongwei; Xie, Peng; Hu, Yibin; Zhou, Caiyuan; Mao, Qian; Sun, Leilei; Li, Shanshan; Wang, Wenhui; Wang, Fang; Pan, Wei; Wang, Chengbao

doi:https://doi.org/10.1155/2024/3315678

Transboundary and Emerging Diseases

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2024 | Article ID 3315678 | https://doi.org/10.1155/2024/3315678

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

Yuhua Zhang,¹Zheng Lu,¹Haoyu Zhao,¹Shuangyu Li,¹Hong Zhuang,¹Juan Wang,¹Ruichao Li,²Weibo Zheng,³Hongwei Zhu,³Peng Xie,¹Yibin Hu,¹and Caiyuan Zhou¹ et al.

Academic Editor: Zongfu Wu

Received17 Feb 2024

Revised09 Apr 2024

Accepted16 Apr 2024

Published06 May 2024

Abstract

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 profiles 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 five 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 findings provide the first evidence for the epidemiological characteristics of Salmonella in pigeons of China, highlighting the importance of preventing salmonellosis in pigeons.

1. 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 [11–13]. 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.

2. Materials and Methods

2.1. 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.

2.2. 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).

2.3. 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).

2.4. Antimicrobial Susceptibility Testing

According to the Clinical Laboratory Standard Institute (CLSI) guidelines, antimicrobial susceptibility profiles were assessed by broth microdilution using the Sensititre™ automated antimicrobial susceptibility system (Thermo Fisher Scientific, United Kingdom) and the Gram-negative GN4F plate. The 24 antimicrobial agents tested were as follows: penicillins (ampicillin: AMP, 8–16 μg/mL; piperacillin: PIP, 16–64 μg/mL; ticarcillin/clavulanic acid: TCC, 8/2−64/2 μg/mL; ampicillin/sulbactam: SAM, 4/2−16/8 μg/mL; piperacillin/tazobactam: TZP, 8/4−128/4 μg/mL), carbapenems (meropenem: MEM, 0.5–8 μg/mL; ertapenem: ETP, 0.25–8 μg/mL; imipenem: IPM, 0.5–8 μg/mL; doripenem: DOR, 0.5–4 μg/mL), cephalosporins (cefazolin: CZO, 1–16 μg/mL; cefepime: FEP, 4–32 μg/mL; ceftazidime: CAZ, 1–16 μg/mL; ceftriaxone: CRO, 0.5–32 μg/mL), monobactams (aztreonam: ATM, 1–16 μg/mL), aminoglycosides (gentamicin: GEN, 2–8 μg/mL; amikacin: AMK, 8–32 μg/mL; tobramycin: TOB 2–8 μg/mL), tetracyclines (tetracycline: TET, 4−8 μg/mL; minocycline: MIN, 1–8 μg/mL; tigecycline: TGC, 1–8 μg/mL), quinolones (ciprofloxacin: CIP, 0.5–2 μg/mL; levofloxacin: 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 (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.

2.5. 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.

2.6. Bioinformatic Analyses

After performing quality checks on the raw reads with Fastp (v0.23.1) [19], the reads were assembled with SPAdes 4.0.1 [20] using the “careful” command option. After genome assembly, SeqSero1.2 (https://cge.food.dtu.dk/services/SeqSero/) [21] and MLST2.0 (https://cge.food.dtu.dk/services/MLST/) were used to predict the serotype and sequence type of the isolated strains on the data platform of the Genome Epidemiology Center [22]. PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/) was additionally employed to find the plasmid type with a minimum coverage of 60% and a nucleotide identity of 95% [23]. In addition, the virulence factor database (VFDB) (http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi?func=VFanalyzer) manages the virulence factors of bacterial pathogens, and we conducted virulence gene prediction for 32 Salmonella isolates here [24]. In order to conduct a comprehensive analysis of resistance genes as much as possible, the resistance genes were detected using ResFinder 4.4.1 (https://cge.cbs.dtu.dk/services/ResFinder/) with settings of threshold of 95% and minimum length of 60%. Moreover, using homology and single nucleotide polymorphism (SNP) model prediction of resistance groups on the comprehensive antibiotic resistance database platform, the Resistance Gene Identifier (https://card.mcmaster.ca/analyze/rgi) software was used to identify the AMR gene [25]. 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.

3. Results

3.1. 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. Copenhagen ST128 (n = 32) (Figure 2).

3.2. Phenotypic Antimicrobial Resistance

All Salmonella isolates were subjected to testing for 24 different antimicrobial drugs in nine different categories, and the results are shown in Table S2 and Figure S3. In order to facilitate the statistical results, we classified intermediate drug-resistant strains as drug-resistant results. We found high resistance of isolates to gentamicin, tobramycin, cefazolin, and amikacin (100%; 32/32), followed by minocycline (84.375%; 27/32), tetracycline (78.125%; 25/32), and tigecycline (50%; 16/32). Moreover, all isolates were susceptible to cefepime, ceftriaxone, doripenem, ertapenem, aztreonam, and levofloxacin (Figure 3(a)). In addition, 90.62% of the strains were resistant to at least three types of antibiotics, indicating MDR (Figure 3(b)).

(a)

(b)

Figure 3

Phenotypic antimicrobial susceptibility (a) and distribution of multiple resistance among the studied Salmonella isolates (b). Abbreviations: AMP, ampicillin; PIP, piperacillin; TCC, ticarcillin/clavulanic acid; SAM, ampicillin/sulbactam; TZP, piperacillin/tazobactam; MEM, meropenem; ETP, ertapenem; IPM, imipenem; DOR, doripenem; CZO, cefazolin; FEP, cefepime; CAZ, ceftazidime; CRO, ceftriaxone; GEN, gentamicin; AMK, amikacin; TOB, tobramycin; TET, tetracycline; MIN, minocycline; TGC, tigecycline; CIP, ciprofloxacin; LVX, levofloxacin; TMS, trimethoprim/sulfamethoxazole; NIT, nitrofurantoin; and ATM, aztreonam.

3.3. Genotypic Antimicrobial Resistance

Analyzing the antimicrobial resistance genes, 32 Salmonella isolates were identified to carry five identical antimicrobial resistance genes. All isolates contained aminoglycoside acetyltransferase aac (6^′)-Iaa resistance genes, efflux mechanism genes mdsA and mdsB, genes encoding gold resistance golS, and biofilm-related genes sdiA (Figure 4). Moreover, genomic mutations conferring quinolone resistance were detected in 26 (81.25%) isolates, and two single mutations of the gyrA gene were observed: gyrA (D87N) (n = 18) and gyrA (S83F) (n = 8) (Figure 4).

3.4. Plasmid Replicons

Using the PlasmidFinder tool, we detected the plasmids carried by Salmonella strains. These isolates carried the same plasmid replicon, and a total of two plasmids were detected in 32 isolates. The plasmids of isolates were IncFII(S) and IncFIB(S) (Figure 4). Interestingly, the IncFll(S) and IncFIB(S) plasmids were carried by different isolates from pigeons from different provinces, indicating that these plasmids have widespread distribution across different geographical regions.

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.

4. 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 (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 [37–40]. 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 [41–43]. 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 [46–48].

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.

5. 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.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Yuhua Zhang, Zheng Lu, Haoyu Zhao, Shuangyu Li, and Hong Zhuang contributed equally to this study.

Acknowledgments

This study was supported by grants from Xi’an IRIS Livestock Technology Co. Ltd. (K4030218170), China Postdoctoral Science Foundation (2017M610659, 2018T111113), Shaanxi Provincial Natural Science Foundation (2022JQ-158), the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (grant numbers: SKLVEB2016KFKT014), and the Fundamental Research Funds for the Central Universities (2452019053).

Supplementary Materials

Figure S1: part of the samples in this study were dissected for clinical symptoms. Figure S2: the results of PCR amplification of 16S rDNA gene of some Salmonella isolates. Figure S3: phenotypic antimicrobial resistance heat map of Salmonella isolates studied. Table S1: PCR primers used in this study. Table S2: summary of detailed data involved in this study. (Supplementary Materials)

References

S. Yan, Z. Jiang, W. Zhang et al., “Genomes-based MLST, cgMLST, wgMLST and SNP analysis of Salmonella Typhimurium from animals and humans,” Comparative Immunology Microbiology and Infectious Diseases, vol. 96, Article ID 101973, 2023.
View at: Publisher Site | Google Scholar
B. Tang, M. Elbediwi, R. B. Nambiar et al., “Genomic characterization of antimicrobial-resistant Salmonella enterica in duck, chicken, and pig farms and retail markets in Eastern China,” Microbiology Spectrum, vol. 10, no. 5, Article ID e0125722, 2022.
View at: Publisher Site | Google Scholar
Y.-J. Li, Y.-F. Yang, Y.-J. Zhou et al., “Estimating the burden of foodborne gastroenteritis due to nontyphoidal Salmonella enterica, shigella and vibrio parahaemolyticus in China,” Plos One, vol. 17, no. 11, Article ID e0277203, 2022.
View at: Publisher Site | Google Scholar
J. Gong, P. Kelly, and C. Wang, “Prevalence and antimicrobial resistance of Salmonella enterica Serovar Indiana in China (1984–2016,” Zoonoses and Public Health, vol. 64, no. 4, pp. 239–251, 2017.
View at: Publisher Site | Google Scholar
D. Monte, M. A. Nethery, R. Barrangou, M. Landgraf, and P. J. Fedorka-Cray, “Whole-genome sequencing analysis and CRISPR genotyping of rare antibiotic-resistant Salmonella enterica serovars isolated from food and related sources,” Food Microbiology, vol. 93, Article ID 103601, 2021.
View at: Publisher Site | Google Scholar
M. C. V. de Oliveira, B. Q. Camargo, M. P. V. Cunha et al., “Free-ranging synanthropic birds (Ardea alba and Columba livia domestica) as carriers of Salmonella spp. and diarrheagenic Escherichia coli in the vicinity of an Urban Zoo,” Vector-Borne and Zoonotic Diseases, vol. 18, no. 1, pp. 65–69, 2018.
View at: Publisher Site | Google Scholar
L. Teske, M. Ryll, D. Rubbenstroth et al., “Epidemiological investigations on the possible risk of distribution of zoonotic bacteria through apparently healthy homing pigeons,” Avian Pathology, vol. 42, no. 5, pp. 397–407, 2013.
View at: Publisher Site | Google Scholar
S. Yousef and R. Mamdouh, “Class I integron and beta-lactamase encoding genes of multidrug resistance Salmonella isolated from pigeons and their environments,” Cellular and Molecular Biology, vol. 62, no. 14, pp. 48–54, 2016.
View at: Google Scholar
W. Rabsch, H. L. Andrews, R. A. Kingsley et al., “Salmonella enterica serotype Typhimurium and its host-adapted variants,” Infection and Immunity, vol. 70, no. 5, pp. 2249–2255, 2002.
View at: Publisher Site | Google Scholar
C. V. Jawale and J. H. Lee, “An immunogenic Salmonella ghost confers protection against internal organ colonization and egg contamination,” Veterinary Immunology and Immunopathology, vol. 162, no. 1-2, pp. 41–50, 2014.
View at: Publisher Site | Google Scholar
M. L. Yang, S. Q. Zheng, K. Peng, W. Wang, Y. H. Huang, and J. Peng, “Isolation and Identification of pigeon-derived Salmonella typhimurium and pathogenic analysis,” Acta Veterinaria et Zootechnica Sinica, vol. 54, no. 11, pp. 4880–4888, 2023.
View at: Google Scholar
D. M. Liang, X. B. Xu, W. F. Wang et al., “Isolation, identification and analysis of serotype and drug resistance of Salmonella from pigeon in Shanghai,” China Animal Husbandry & Veterinary Medicine, vol. 2016, no. 43, pp. 3322–3328, 2016.
View at: Google Scholar
E. Kaczorek-Lukowska, P. Sowinska, A. Franaszek, D. Dziewulska, J. Malaczewska, and T. Stenzel, “Can domestic pigeon be a potential carrier of zoonotic Salmonella?” Transboundary and Emerging Diseases, vol. 68, no. 4, pp. 2321–2333, 2021.
View at: Publisher Site | Google Scholar
M. Vereecken, P. De Herdt, R. Ducatelle, and F. Haesebrouck, “The effect of vaccination on the course of an experimental Salmonella typhimurium infection in racing pigeons,” Avian Pathology, vol. 29, no. 5, pp. 465–471, 2010.
View at: Publisher Site | Google Scholar
B. Walther, K. Tedin, and A. Lübke-Becker, “Multidrug-resistant opportunistic pathogens challenging veterinary infection control,” Veterinary Microbiology, vol. 200, pp. 71–78, 2017.
View at: Google Scholar
Y. Xu, X. Zhou, Z. Jiang, Y. Qi, A. Ed-Dra, and M. Yue, “Antimicrobial resistance profiles and genetic typing of Salmonella serovars from chicken embryos in China,” Antibiotics-Basel, vol. 10, no. 10, Article ID 1156, 2021.
View at: Publisher Site | Google Scholar
Y. Wang, Y. Liu, N. Lyu et al., “The temporal dynamics of antimicrobial-resistant Salmonella enterica and predominant serovars in China,” National Science Review, vol. 10, no. 3, 2023.
View at: Publisher Site | Google Scholar
S. Pornsukarom, A. H. M. van Vliet, and S. Thakur, “Whole genome sequencing analysis of multiple Salmonella serovars provides insights into phylogenetic relatedness, antimicrobial resistance, and virulence markers across humans, food animals and agriculture environmental sources,” Bmc Genomics, vol. 19, no. 1, Article ID 801, 2018.
View at: Publisher Site | Google Scholar
S. Chen, Y. Zhou, Y. Chen, and J. Gu, “fastp: an ultra-fast all-in-one FASTQ preprocessor,” Bioinformatics, vol. 34, no. 17, pp. i884–i890, 2018.
View at: Publisher Site | Google Scholar
A. Bankevich, S. Nurk, D. Antipov et al., “SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing,” Journal of Computational Biology, vol. 19, no. 5, pp. 455–477, 2012.
View at: Publisher Site | Google Scholar
S. Zhang, Y. Yin, M. B. Jones et al., “Salmonella serotype determination utilizing high-throughput genome sequencing data,” Journal of Clinical Microbiology, vol. 53, no. 5, pp. 1685–1692, 2015.
View at: Publisher Site | Google Scholar
M. V. Larsen, S. Cosentino, S. Rasmussen et al., “Multilocus sequence typing of total-genome-sequenced bacteria,” Journal of Clinical Microbiology, vol. 50, no. 4, pp. 1355–1361, 2012.
View at: Publisher Site | Google Scholar
A. Carattoli, E. Zankari, A. Garcia-Fernandez et al., “In silico detection and typing of plasmids using plasmid finder and plasmid multilocus sequence typing,” Antimicrobial Agents and Chemotherapy, vol. 58, no. 7, pp. 3895–3903, 2014.
View at: Publisher Site | Google Scholar
B. Liu, D. Zheng, Q. Jin, L. Chen, and J. Yang, “VFDB 2019: a comparative pathogenomic platform with an interactive web interface,” Nucleic Acids Research, vol. 47, no. D1, pp. D687–D692, 2019.
View at: Publisher Site | Google Scholar
B. P. Alcock, W. Huynh, R. Chalil et al., “CARD 2023: expanded curation, support for machine learning, and resistome prediction at the comprehensive antibiotic resistance database,” Nucleic Acids Research, vol. 51, pp. D690–D699, 2023.
View at: Publisher Site | Google Scholar
X. Li, X. M. Wang, Y. X. Shao, Z. Wang, F. Ji, and J. G. Huang, “Research progress on pigeon salmonellosis and prevention and control measures of its antibiotic substitutes,” China Animal Husbandry & Veterinary Medicine, vol. 48, no. 7, pp. 2584–2593, 2021.
View at: Google Scholar
Y. Li, Q. Yang, C. Cao et al., “Prevalence and characteristics of Salmonella isolates recovered from retail raw chickens in Shaanxi Province, China,” Poultry Science, vol. 99, no. 11, pp. 6031–6044, 2020.
View at: Publisher Site | Google Scholar
C. Dorn, A. Schroeter, and R. Helmuth, “Report on Salmonella isolates submitted to the German National Veterinary Salmonella reference laboratory in the year 1999,” Berliner Und Munchener Tierarztliche Wochenschrift, vol. 115, no. 7-8, pp. 252–258, 2002.
View at: Google Scholar
G. D. Xie, H. P. Luo, X. Ren, Y. W. Chen, W. Yu, and S. H. Cui, “Isolation, identification and drug resistance analysis of Escherichia coli and Salmonella in Beijing pigeon meat,” Journal of Food Safety and Quality, vol. 10, no. 1, pp. 48–53, 2019.
View at: Google Scholar
A. Gargiulo, T. P. Russo, R. Schettini et al., “Occurrence of enteropathogenic bacteria in urban pigeons (Columba livia) in Italy,” Vector Borne and Zoonotic Diseases (Larchmont, N.Y.), vol. 14, no. 4, pp. 251–255, 2014.
View at: Publisher Site | Google Scholar
K. M. Osman, M. Mehrez, A. M. Erfan, and A. Nayerah, “Salmonella enterica isolated from pigeon (Columba livia) in Egypt,” Foodborne Pathogens and Disease, vol. 10, no. 5, pp. 481–483, 2013.
View at: Publisher Site | Google Scholar
B. Tang, A. Siddique, C. Jia et al., “Genome-based risk assessment for foodborne Salmonella enterica from food animals in China: a one health perspective,” International Journal of Food Microbiology, vol. 390, Article ID 110120, 2023.
View at: Publisher Site | Google Scholar
Z. Xu, M. Wang, C. Zhou et al., “Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009–2016: the diversity of contamination and the resistance evolution of multidrug-resistant isolates,” International Journal of Food Microbiology, vol. 333, Article ID 108790, 2020.
View at: Publisher Site | Google Scholar
L. J. Wu, Y. Luo, G. L. Shi, and Z. Y. Li, “Prevalence, clinical characteristics and changes of antibiotic resistance in children with nontyphoidal Salmonella Infections from 2009–2018 in Chongqing, China,” Infection and Drug Resistance, vol. 14, pp. 1403–1413, 2021.
View at: Publisher Site | Google Scholar
J. Zhu, J. Lv, Z. Zhu et al., “Identification of TMexCD-TOprJ-producing carbapenem-resistant gram-negative bacteria from hospital sewage,” Drug Resistance Updates: Reviews and Commentaries in Antimicrobial and Anticancer Chemotherapy, vol. 70, Article ID 100989, 2023.
View at: Publisher Site | Google Scholar
J. Chen, A. Ed-Dra, H. Zhou, B. Wu, Y. Zhang, and M. Yue, “Antimicrobial resistance and genomic investigation of non-typhoidal Salmonella isolated from outpatients in Shaoxing city, China,” Frontiers in Public Health, vol. 10, Article ID 988317, 2022.
View at: Publisher Site | Google Scholar
F. Campioni, F. P. Vilela, G. Cao et al., “Whole genome sequencing analyses revealed that Salmonella enterica serovar Dublin strains from Brazil belonged to two predominant clades,” Scientific Reports, vol. 12, no. 1, Article ID 10555, 2022.
View at: Publisher Site | Google Scholar
I. M. Martins, A. A. Seribelli, R. T. Machado et al., “Invasive non-typhoidal Salmonella (iNTS) aminoglycoside-resistant ST313 isolates feature unique pathogenic mechanisms to reach the bloodstream,” Infection Genetics and Evolution, vol. 116, Article ID 105519, 2023.
View at: Publisher Site | Google Scholar
S. Song, S. Hwang, S. Lee, N. C. Ha, and K. Lee, “Interaction mediated by the putative tip regions of MdsA and MdsC in the formation of a Salmonella-specific tripartite efflux pump,” Plos One, vol. 9, no. 6, Article ID e100881, 2014.
View at: Publisher Site | Google Scholar
F. P. Vilela, P. R. D. Dos, M. W. Allard, and J. P. Falcao, “Genomic analyses of drug-resistant Salmonella enterica serovar Heidelberg strains isolated from meat and related sources between 2013 and 2017 in the south region of Brazil,” Current Genetics, vol. 69, no. 2-3, pp. 141–152, 2023.
View at: Publisher Site | Google Scholar
K. O. Akinyemi, C. O. Fakorede, J. Linde et al., “Whole genome sequencing of Salmonella enterica serovars isolated from humans, animals, and the environment in Lagos, Nigeria,” Bmc Microbiology, vol. 23, no. 1, Article ID 164, 2023.
View at: Publisher Site | Google Scholar
S. K. Checa, M. Espariz, M. E. Audero, P. E. Botta, S. V. Spinelli, and F. C. Soncini, “Bacterial sensing of and resistance to gold salts,” Molecular Microbiology, vol. 63, no. 5, pp. 1307–1318, 2007.
View at: Publisher Site | Google Scholar
S. Cerminati, G. F. Giri, J. I. Mendoza, F. C. Soncini, and S. K. Checa, “The CpxR/CpxA system contributes to Salmonella gold-resistance by controlling the GolS-dependent gesABC transcription,” Environmental Microbiology, vol. 19, no. 10, pp. 4035–4044, 2017.
View at: Publisher Site | Google Scholar
J. L. Smith, P. M. Fratamico, and X. Yan, “Eavesdropping by bacteria: the role of SdiA in Escherichia coli and Salmonella enterica serovar Typhimurium quorum sensing,” Foodborne Pathogens and Disease, vol. 8, no. 2, pp. 169–178, 2011.
View at: Publisher Site | Google Scholar
J. Volf, M. Sevcik, H. Havlickova, F. Sisak, J. Damborsky, and I. Rychlik, “Role of SdiA in Salmonella enterica serovar Typhimurium physiology and virulence,” Archives of Microbiology, vol. 178, no. 2, pp. 94–101, 2002.
View at: Publisher Site | Google Scholar
D. J. Eaves, L. Randall, D. T. Gray et al., “Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 10, pp. 4012–4015, 2004.
View at: Publisher Site | Google Scholar
G. P. Macias-Farrera, J. R. de Oca, J. Varela-Guerrero et al., “Antibiotics susceptibility of quinolones against Salmonella spp. strains isolated and molecularly sequenced for gyrA gene,” Microbial Pathogenesis, vol. 114, pp. 286–290, 2018.
View at: Publisher Site | Google Scholar
L. M. Weigel, C. D. Steward, and F. C. Tenover, “gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae,” Antimicrobial Agents and Chemotherapy, vol. 42, no. 10, pp. 2661–2667, 1998.
View at: Publisher Site | Google Scholar
K. T. Aung, W. C. Khor, K. H. Ong et al., “Characterisation of Salmonella Enteritidis ST11 and ST1925 Associated with Human Intestinal and Extra-Intestinal Infections in Singapore,” International Journal of Environmental Research and Public Health, vol. 19, no. 9, Article ID 5671, 2022.
View at: Publisher Site | Google Scholar
B. K. Khajanchi, N. A. Hasan, S. Y. Choi et al., “Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources,” Bmc Genomics, vol. 18, no. 1, Article ID 570, 2017.
View at: Publisher Site | Google Scholar
I. Rychlik, D. Gregorova, and H. Hradecka, “Distribution and function of plasmids in Salmonella enterica,” Veterinary Microbiology, vol. 112, no. 1, pp. 1–10, 2006.
View at: Publisher Site | Google Scholar
S. A. Dos, R. G. Ferrari, and C. A. Conte-Junior, “Virulence factors in Salmonella Typhimurium: the sagacity of a Bacterium,” Current Microbiology, vol. 76, no. 6, pp. 762–773, 2019.
View at: Publisher Site | Google Scholar
J. Mambu, E. Barilleau, L. Fragnet-Trapp et al., “Rck of Salmonella typhimurium delays the host cell cycle to facilitate bacterial invasion,” Frontiers in Cellular and Infection Microbiology, vol. 10, Article ID 586934, 2020.
View at: Publisher Site | Google Scholar
F. C. Fang, M. A. DeGroote, J. W. Foster et al., “Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 13, pp. 7502–7507, 1999.
View at: Publisher Site | Google Scholar
M. Nakano, E. Yamasaki, A. Ichinose et al., “Salmonella enterotoxin (Stn) regulates membrane composition and integrity,” Disease Models & Mechanisms, vol. 5, no. 4, pp. 515–521, 2012.
View at: Publisher Site | Google Scholar
M. L. Lesnick, N. E. Reiner, J. Fierer, and D. G. Guiney, “The Salmonella spvB virulence gene encodes an enzyme that ADP-ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells,” Molecular Microbiology, vol. 39, no. 6, pp. 1464–1470, 2001.
View at: Publisher Site | Google Scholar
L. Sun, S. Yang, Q. Deng et al., “Salmonella Effector SpvB disrupts intestinal epithelial barrier integrity for bacterial translocation,” Frontiers in Cellular and Infection Microbiology, vol. 10, Article ID 606541, 2020.
View at: Publisher Site | Google Scholar
D. Haag-Wackernagel and H. Moch, “Health hazards posed by feral pigeons,” The Journal of Infection, vol. 48, no. 4, pp. 307–313, 2004.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2024 Yuhua Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

150

Downloads

58

Citations