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Article

Comparative Analysis between Salmonella enterica Isolated from Imported and Chinese Native Chicken Breeds

by
Wenjian Shi
1,†,
Wenli Tang
2,†,
Yafei Li
1,
Yu Han
1,
Lulu Cui
1,* and
Shuhong Sun
1,*
1
Shandong Key Laboratory of Animal Biotechnology and Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai’an 271018, China
2
Shandong Provincial Key Laboratory of Quality Safety Monitoring and Risk Assessment for Animal Products, Shandong Center for Quality Control of Feed and Veterinary Drug, Jinan 250100, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2023, 11(2), 390; https://doi.org/10.3390/microorganisms11020390
Submission received: 14 December 2022 / Revised: 31 January 2023 / Accepted: 31 January 2023 / Published: 3 February 2023
(This article belongs to the Special Issue Antimicrobial Resistance and Increased Virulence of Salmonella)
Browse Figures
Versions Notes

Abstract

:
Salmonella enterica is considered a significant threat to the global poultry industry and public health. In recent decades, antimicrobial resistance in Salmonella enterica has attracted increasing concern throughout the world. However, limited information is available on Salmonella enterica among different breeds of breeder chickens. Thus, this study aimed to compare the prevalence, serotype distribution, emergence of extended-spectrum beta-lactamases (ESBLs), antimicrobial resistance, and genetic resistance mechanisms in Salmonella enterica among different breeds of breeder chickens. A total of 693 samples (dead embryos, cloacal swabs, water, feed, environmental swabs, and meconium of newly hatched chicks) were selected and cultured for Salmonella from four breeder chicken farms in Shandong province, China, representing one imported and three native breeds, and the isolates were further serotyped. Of the Salmonella isolates, susceptibility to 11 antimicrobials of 5 classes, ESBL screening, and the presence of 21 antimicrobial resistance genes were determined in the present study. Overall, 94 (13.6%) isolates were recovered, which were divided into 3 serotypes (Salmonella Pullorum (n = 36), Salmonella Thompson (n = 32), and Salmonella Enteritidis (n = 26)). The results showed that the prevalence of Salmonella enterica isolates from the imported breeds was higher compared with the three domestic breeds. Eight of the ninety-four isolates were ESBL-positive strains, which were recovered from a domestic breed chicken farm. These eight ESBL-producing isolates were serotyped to Pullorum. Surprisingly, Salmonella Enteritidis (S. enteritidis) and S. pullorum were simultaneously isolated from a single dead embryo observed among one native breed. Meanwhile, among the Salmonella isolates, 53.2% (50/94) were multidrug-resistant strains, and 44.7% (42/94) of the isolates presented resistance to at least five antibiotics. Nearly all of the isolates (97.9%, 92/94) were resistant to at least one antimicrobial; one isolate of S. Thompson was resistant to seven antimicrobial agents belonging to four different classes. The carriage rate of three resistance genes (tetA, tetB, and sul1) among isolates from the imported breeds (87%, 70%, and 65.2%) was higher than that in those from domestic breeds (35.2%, 36.6, and 14.1%). To our knowledge, this is the first report of ESBLs-producing Salmonella isolated from a Chinese native breed of breeder chickens. Our results also highlight that a high prevalence of multidrug-resistant Salmonella enterica contamination is widespread among different breeds of breeder chickens, which is a major risk of food-borne diseases and public health.

1. Introduction

Salmonella spp. are recognized as zoonotic pathogens and one of the leading agents capable of infecting humans and animals [1]. Contaminated poultry and poultry products are essential sources of food-borne illnesses in humans [2,3]. There are several serotypes of Salmonella spp., and more than 3000 serotypes have been classified according to the response of antisera to O and H antigens [4,5] Among Salmonella spp., more than 2500 serovars belong to Salmonella enterica, which has become a severe threat to public health and the poultry industry [6]. The World Health Organization (WHO) estimates that, in low- and middle-income countries, about 600 million people fall ill from unclean food each year, causing 420,000 deaths and costing up to USD 110 billion annually [7]. Salmonella spp. is also one of the leading causes of significant economic losses in China and around the world [8,9]. The predominant Salmonella serotypes prevalent in Chinese native chicken breeds are Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Gallinarum biovar Pullorum [10]. Among them, S. pullorum is widespread and difficult to eliminate because of its vertical transmission [11]. In recent years, there has been some success in implementing rigorous eradication programs for Pullorum, such as attenuated vaccines against host-specific serotypes [12,13]. Many developed countries, such as the United States of America, have been declared free of S. pullorum, but it is still widespread in China [14].
The long-term effects of antibiotics may increase resistance to antimicrobials [15,16,17,18]. Quinolones, third-generation cephalosporins, ampicillin, cotrimoxazole, and doxycycline are used for treating Salmonella infections. These antimicrobial determinants are horizontally transmitted to other pathogens through a variety of mechanisms [16,19], including chromosomally encoded mechanisms or plasmid-mediated resistance mechanisms [20]. Multidrug-resistant (MDR) bacteria pose a serious threat to public health, with humans or animals at risk of infection when exposed to contaminated poultry or products [21,22,23]. The widespread use of tetracycline in animals since the 1950s has resulted in the development of tetracycline resistance in Salmonella spp. [24,25]. Recent studies have shown that Salmonella strains isolated from several countries (China, Thailand, the Republic of Bulgari, and Denmark) carry extended-spectrum β-lactamases (ESBLs), which have attracted much attention around the world [26,27,28,29]. ESBL strains complicate antibiotic therapy [30] as ESBL-encoding plasmids can carry additional β-lactamase genes and other antibiotic resistance genes, which may limit treatment options for ESBL-producing pathogens [31]. Both the US Centers for Disease Control and Prevention (CDC) [32] and the WHO [33] list Gram-negative bacteria that produce beta-lactamases as one of the world’s most pressing threats.
Enterobacteria repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) uses consensus primers to amplify DNA sequences located between repeated sequences for subtyping Gram-negative enteric bacteria. Therefore, acknowledging the genetic relationship between different breeds of breeder chickens MDR Salmonella isolates using the ERIC-PCR strain is crucial for both humans and animals.
Shandong Province is one of the largest Chinese poultry producers, which exports to several countries. High isolation rates of Salmonella recovered from dead embryos and the meconium of newly hatched chicks were confirmed by our previous findings [10,34]. Currently, there are very few studies regarding Salmonella in breeder farms’ chickens in Shandong Province, China. Therefore, in order to fill in the epidemiological gaps concerning the distribution of Salmonella in different breeder chicken farms, four different breeder farms were chosen for the identification and serotyping of Salmonella isolates. Above all, to the best of our knowledge, this is the first report of ESBL-producing S. pullorum being isolated from Chinese native chicken breeds.

2. Materials and Methods

2.1. Sampling Strategy and Isolation and Identification of Salmonella

All animal work was reviewed and approved by the Laboratory Animal Care Committee of Shandong Agricultural University (permit number SDAUA-2021-034) on 10 March 2022. Informed verbal consent was obtained from farmers to allow samples to be collected from their farms.
In July 2020, a total of 693 samples (458 dead embryos, 100 cloacal swabs, 20 water samples, 15 feed samples, 30 environmental swabs, and 70 meconium samples of newly hatched chicks) were selected from four different chicken breeds in Tai’an (White feather broiler), Jinan (Bairi), Rizhao (Langya), and Jining (Luhua) in Shandong Province, China, for Salmonella isolation and identification. The isolation and identification of Salmonella species were performed according to ISO standard 6579:2017 and the Chinese national standard (GB 4789.4-2016) with minor modifications [35,36]. Each sample was added to 4.5 mL of buffered peptone water (BPW, Qingdao Hope Bio-technology Co., LTD., China) and the BPW mixture was incubated at 37 °C for 12 h for pre-enrichment. Approximately 0.5 mL of pre-enriched culture was inoculated into 4.5 mL of Tetrathionate Broth Base (TTB, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) and Selenite Cystine Broth (SC, Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China). Cultures of each TTB and SC broth were inoculated on a Xylose Lysine Desoxycholate Agar base (XLD, Qingdao Hope Bio-technology Co., Ltd., Qingdao, China) and incubated at 37 °C for 48 h [37]. Smooth and round without a black center or large with a black center colonies were confirmed by polymerase chain reaction (PCR) assays with primers designed for the FimW gene (Table S1) [10,38]. Bacterial DNA was extracted using a TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions.
Amplification reactions were carried out at a final volume of 25 µL containing 1 µL of each primer (10 µM), 12.5 µL of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China), 1 μL of genomic DNA, and 9.5 μL of distilled deionized water. PCR of the FimW gene was performed with the following program: 94 °C for 4 min, followed by 24 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C, and a final extension at 72 °C for 10 min. The PCR products were separated through 1% agarose gel electrophoresis and visualized under UV light after staining with ethidium bromide. To confirm the presence of Salmonella, the standard strain of S. enterica (CVCC 3377) purchased from the China Center for the preservation and management of veterinary microorganisms was used as a positive control, and Escherichia coli (ATCC 25922) was used as a negative control.

2.2. Serological Identification and Biochemical Identification of Salmonella Isolates

All isolates were serotyped by slide agglutination of O and H antigens according to the instructions provided by the manufacturer of the antiserum that we used (Ningbo Tianrun Bio-technology Co., LTD., China). The serotypes could then be determined according to the Kauffmann–White classification scheme [39]. For the identification of S. gallinarum and S. pullorum, dulcitol fermentation and ornithine decarboxylation tests were conducted according to a previous study [40]. Salmonella biochemical identification tubes were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, China). PCR was performed on isolates identified as S. pullorum using a specific IpaJ gene for S. pullorum (Table S1); the standard strain of S. pullorum (CVCC 526) purchased from the China Center for the Preservation and Management of Veterinary Microorganisms was used as a positive control and S. enteritidis (CVCC 3377) was used as a negative control.

2.3. Antimicrobial Susceptibility Testing

According to the Clinical and Laboratory Standards Institute (CLSI) protocol [41], the Kirby–Bauer drug-sensitive disk method was used to test the sensitivity of Salmonella isolates to 11 antimicrobials of 5 classes, including β-lactames: ampicillin (AMP), 10 μg; amoxicillin (AMX), 20 μg; ceftazidime (CAZ), 10 μg; and cefoxitin (FOX), 30 μg; tetracyclines: tetracycline (TET), 30 μg and doxycycline (DOX), 30 μg; quinolones: ofloxacin (OF), 5 μg; aminoglycosides: gentamicin (GM), 10 μg and streptomycin (STR), 10 μg; and sulfonamides: sulfamethoxazole–trimethoprim (SXT), 25 μg. Briefly, bacterial suspensions were obtained from overnight cultures, adjusted to the 0.5 McFarland turbidity standard, and the organisms were then evenly spread on the surface of a Muller Hinton agar plate using a cotton swab. After about 15 min, the disks were applied to the plates and incubated at 37 °C for 18 h. Finally, the diameter of the inhibition zone was measured using a ruler. As per standardized international terminology created by the European Centre for Disease Control (ECDC) and CDC, multidrug-resistant (MDR) bacteria were defined as having acquired nonsusceptibility to at least one agent in three or more antimicrobial categories [42].

2.4. ESBL Screening

Cefotaxime and ceftazidime were used as the initial screening standards, and strains resistant to both cefotaxime and ceftazidime were selected for ESBL screening. The double-disk synergy method was designed for drug susceptibility testing. ESBLs have the property of being inhibited by clavulanate. Bacteriostatic rings were found in resistant strains around the mixed discs of third-generation cephalosporins and clavulanate. If clavulanate increased the bacteriostatic ring by more than 5 mm, the isolates were presumed to produce ESBLs. Escherichia coli (ATCC 25922) and Klebsiella quasipneumoniae (ATCC 700603) were used as negative and positive controls, respectively.

2.5. Molecular Detection of Antimicrobial Resistance-Associated Genes

All isolates were screened for the presence of 21 antimicrobial resistance genes. The following genes encode resistance to β-lactamases: blaVIM, blaSHV, blaNDM, blaCTX-M, blaTEM, blaIMP, blaMIR, and blaDHA; aminoglycosides: AAC2 and AAC4; quinolones: qnrC and oqxA; tetracyclines: tetA, tetB, and tetC; sulfonamides: sul1 and sul2; and colistin: Mcr-1, Mcr-2, Mcr-3, and Mcr-4. For each gene, PCR was performed in a final 25 μL reaction mixture system containing 12.5 μL of 2 × Taq Master Mix (Vazyme Biotech co., Ltd., Nanjing, China), 10 μM of forward and reverse primers, 1 μL of genomic DNA, and 9.5 μL of distilled deionized water. The primers used for 21 antimicrobial resistance genes are detailed in Table S2. The PCR conditions and size of the PCR products were obtained as previously described (Table S3) [28,43,44,45,46,47,48,49,50].

2.6. ERIC-PCR

The primers (F: 5′-ATG TAA GCT CCT GGG GAT TCA C-3′, R: 5′-AAG TAA GTG ACT GGG GTG AGC G-3′) were used for ERIC-PCR [51]. The PCR was performed in a 25 μL solution containing 1 μL of each primer (10 μM), 2 μL of genomic DNA, 8.5 μL of distilled deionized water, and 12.5 μL of 2 × Taq Master Mix (Vazyme Biotech, Nanjing, China). The PCR conditions were as follows: 1 cycle at 94 °C for 7 min, followed by 35 cycles of 45 s at 94 °C, 1 min at 52 °C, and 8 min at 65 °C, and a final extension at 65 °C for 10 min. The same reaction mixture without a DNA template was used as the negative control. ERIC-PCR products were electrophoresed on 1.5% agarose gel, and the gel images were analyzed using a Kodak Gel Logic 212 Imaging System (Rochester, NY, USA). The product sizes were estimated using a DNA ladder 5000 (Vazyme Biotech, Nanjing, China).
The ERIC patterns of each isolate were coded as 1 (presence) or 0 (absence) for DNA bands. Dendrograms of each isolate were constructed using the unweighted pair–group method with an arithmetic average (UPGMA). Additionally, isolates were considered to have the same ERIC patterns with more than 80% genetic similarity, and isolates with more than 90% similarity were treated as the same strains.

2.7. Statistical Analysis

The statistical software package SPSS 23.0 (SPSS, Inc., Chicago, IL, USA) was used for data analysis. To compare the differences between the results obtained from imported and native breeds, a chi-square test was used.

3. Results

3.1. Prevalence of Salmonella Isolates

As is shown in Table 1, a total of 94 Salmonella isolates were recovered from 693 samples, in which Salmonella was detected from dead embryos, cloacal swabs, feed, and the meconium of newly hatched chick samples. The separation proportion of Salmonella isolated from different sample types during the study was from 0 to 38.3%. Among the isolates, Salmonella strains were observed in 38.3% (23/60) of those from Farm A, 6.3% (15/238) of those from Farm B, 13.9% (32/230) of those from Farm C (twenty- nine from the dead embryos, one from the cloacal swabs and two from the feed) and 14.5% (24/165) of those from Farm D. The prevalence of Salmonella was 38.3% (23/60) from a foreign breed (white feather broiler). Moreover, a high prevalence (34.3, 24/70) of Salmonella was also observed in the meconium of newly hatched chicks. The detection results of some samples amplifying the FimW gene are shown in Figure 1.

3.2. Distribution of Salmonella Serotypes

With regard to the serotyping of Salmonella isolates, three serotypes of Salmonella enterica were identified among the 94 Salmonella strains (Figure 2). The isolates from three native breeds of breeder chickens contained three serotypes, and the isolates from foreign breed chicken (white feather broiler) contained only one serotype. The predominant serotypes were Salmonella enterica serovar Gallinarum biovar Pullorum (S. pullorum) and Gallinarum (S. gallinarum) isolated from native breed chickens (36/94, 38.3%), followed by Salmonella Thompson (32/94, 34.0%) and S. enteritidis, isolated from both domestic and foreign breed farms (26/94, 27.7%). According to the White–Kauffmann–Le Minor scheme, the 36 Salmonella strains were identified as S. pullorum and S. gallinarum. Combined with the results of dulcitol fermentation, the ornithine decarboxylation test, and the PCR assay, these 36 isolates were further confirmed to be S. pullorum. In particular, S. enteritidis and S. pullorum were simultaneously isolated from a single dead chicken embryo in one native breed of breeder chicken. The detection results of some samples amplifying the Ipaj gene are shown in Figure 3.

3.3. Antimicrobial Susceptibility and ESBL Production

In this study, resistance to at least one antibiotic was observed in 92 isolates (97.9%). The studied isolates had high resistance to streptomycin (83.0%, 78/94), ampicillin (75.5%, 71/94), and amoxicillin (73.4%, 69/94), while all isolates were susceptible to ofloxacin (see Table S4). Meanwhile, the results showed that the resistances (ampicillin, tetracycline, and doxycycline) of Salmonella enterica isolated from imported breeds (100%, 82.6%, and 65.2%) were higher than the resistances of Salmonella enterica isolated from three domestic breeds (67.6%, 12.7%, and 8.4%) (p = 0.012, p < 0.01 and p < 0.01), respectively (Table 2), while lower resistance was observed against ceftazidime (0 for the imported breed and 21.1% for the native breeds, p < 0.01). Among the 94 Salmonella enterica isolates, eight (8.5%) were ESBL-producing strains. Notably, all eight ESBL-positive isolates belonged to S. pullorum, which was recovered from a native breed of breeder chickens (Table 3). Meanwhile, high resistance rates were detected for ceftazidime (100%) and streptomycin (87.5%). All eight strains were resistant to at least one antimicrobial, while only one (12.5%, 1/8) isolate was MDR. Moreover, the distribution of antimicrobial resistance varied between the confirmed serotypes among these Salmonella strains (Table 4). Furthermore, compared with the Pullorum serotype, Thompson and Enteritidis isolates showed quite high resistance (Table 5).
The MDR statistics showed that 53.2% (50/94) of strains had multidrug resistance to three or more drug classes. The MDR rates of Salmonella enterica isolated from imported breeds (73.9%, 17/23) were much higher than those collected from three native breeds (46.5%, 33/71) (Figure 4). Nearly 85.9% (61/71) of isolates among the three native breeds were resistant to at least two antibiotics, while the resistance rate of Salmonella strains in the imported breed was 100% (23/23). Additionally, by comparing the rates of isolates from three domestic chicken breeds (38.0%, n = 27) that were resistant to five antibiotics, our results showed that the rates for the strains of Salmonella from foreign breeds (52.2%, n = 12)) were much higher. On the contrary, two isolates collected from native breeds were resistant to seven antimicrobial agents.

3.4. Characterization of Antimicrobial Resistance Genes

The carriage status of Salmonella strains for 21 antimicrobial resistance genes was screened by PCR (Table 6). Among these, only one (blaTEM) of eight genes encoding resistance to β-lactams was detected in 97.9% (92/94) of the isolates. The frequency of the blaTEM gene among Salmonella enterica recovered in foreign breed farms (100%) was higher than those in native breeds (97.2%) (p = 0.841). Among the 85 β-lactam-resistant isolates, all strains harbored the blaTEM gene. However, the AAC2 gene encoding resistance to aminoglycosides was only detected in one isolate collected from an imported breed (p = 0.038). With regard to tetracyclines, two (tetA and tetB) genes were detected in the 28 tetracycline-resistant strains. The rates of three genes (tetA, tetB, and sul1) harbored among isolates from imported breeds of breeder chickens (87%, 70%, and 65.2%) were higher than those of isolates from three domestic breeds (35.2%, 36.6, and 14.1%) (p < 0.01, p = 0.001, and p < 0.01). Furthermore, all instances of two (sul1 and sul2) genes encoding resistance to sulfonamides were detected in the 29 sulfonamide-resistant isolates. Additionally, different serotype isolates carried different resistance genes. Compared with the Enteritidis serotype, Thompson and Pullorum isolates showed higher resistance to tetA and tetB (Table 7). In addition, no gene encoding resistance to colistin was detected in any of the Salmonella isolates.

3.5. ERIC-PCR Analysis

In the present study, the ERIC-PCR results showed that 94 isolates were genotyped into 17 different banding patterns (Figure 5), and isolates belonging to the same serotype were grouped together. The 20 isolates with the serotype Thompson showed a higher correlation, whereas Pullorum and Enteritidis isolates showed genetic diversity. Among the isolates, strains from the same farm were relatively closely clustered. A maximum of 20 Thompson strains belonging to one pattern could be observed. Moreover, 23 isolates recovered from imported breeding chickens were divided into 5 clusters and 2 single isolates, while 71 Salmonella strains were classified into 10 clusters, which were recovered from three local breed chicken farms.

4. Discussion

Salmonella is recognized as one of the most important zoonotic agents in the world, which can be transmitted horizontally and through fertilized eggs [52,53]. Based on this, in order to better evaluate the actual contamination by Salmonella enterica, we obtained various samples (dead embryos, cloacal swabs, environment swabs, water, animal feed, and meconium of newly hatched chicks) from four different breeds of breeder chickens.
In this study, the prevalence of Salmonella enterica from one imported breed flock and three native breed poultry farms in Shandong Province was 13.6% (94/693); the isolation rate was similar to that in a study on chickens (14.3%) [54], and higher than that from breeder chicken hatcheries in Shandong Province (6.7%) [53]. Additionally, the prevalence of Salmonella in the foreign breed (38.6%) was much higher than that collected in Henan Province (11.48%) [52]. In fact, the differences in sample types, geographical locations, sampling years, or isolation methods resulted in a complex and varied isolation comparison.
Three serotypes of Salmonella enterica were identified among the 94 Salmonella strains according to the Kauffmann–White scheme, and the distribution of serotypes among each breeder chicken was significantly different (Figure 1). Salmonella Gallinarum biovar Pullorum collected from two of the three native breed flocks was the most common serotype (38.3%), followed by Thompson (34%) and Enteritidis (27.6%). This finding is consistent with our previous reports [14,55]. These results indicate that, although Pullorum is well controlled in many developed countries, it remains commonly present in Chinese native breeder chickens. S. pullorum is ubiquitous in our country and is one of the most serious threats to our poultry industry [56]. Importantly, the prevalence of Pullorum has exceeded 30% in Chinese native breeds of breeder chickens [57] for a long time, resulting in prevention and control work facing great difficulties. S. enteritidis is considered one of the most commonly reported pathogens responsible for Salmonella infections in humans [58]. Thus, it is important to continuously monitor the prevalence and serovar distribution of Salmonella in our domestic breeder chicken farms.
In recent decades, molecular subtyping techniques have been used to widely investigate genetic diversity. Genetic relatedness among Salmonella isolates from four different breeds of breeder chicken was analyzed using ERIC-PCR, and the 94 isolates were classified into 17 clusters in this study. In our study, a high level of genetic diversity was observed among the 94 isolates, indicating that there was no significant genetic correlation between the four different breeder chicken farms.
In the current study, most isolates showed high resistance to streptomycin (83.0%, 78/94), ampicillin (75.5%, 71/94), and amoxicillin (73.4%, 69/94), which was similar to some previous studies [52,59,60], suggesting that high selective pressure by exposure to regular use of these antibiotics may be one of the main reasons for the emergence of such antimicrobial-resistant Salmonella strains [61]. Interestingly, isolates collected from different breeds of chickens showed different levels of antimicrobial resistance. The overall average antimicrobial resistance of Salmonella enterica isolated from three domestic breeds was higher than that of imported breeder flocks, while our study revealed that Salmonella, whether from imported breed or native breed chickens, presented low resistance to quinolones regarding the principal antimicrobial agents used in salmonellosis. In addition, 53.2% (50/94) of the isolates were MDR; this result indicated a higher resistance frequency than that described in another investigation [62], which may limit the therapeutic options for Salmonella infection. Meanwhile, Enteritidis (100%, 26/26) and Thompson (87.5%, 28/32) showed a high MDR rate in our study; this result was similar to those of other studies [23,60,63]. It is likely to spread along the food production chain with other Enterobacteriaceae through horizontal gene transfer, posing a threat to public health. Of note, the presence of multidrug-resistant isolates may pose a potential transmission risk to public health through the food chain. Antibiotics have been commonly used to prevent and treat diseases in humans and livestock in the past. However, the indiscriminate use of antibiotics in farming, relying on selective pressure, will lead to increased resistance in Salmonella and other bacteria, and MDR bacteria might present a significant challenge to the whole poultry industry.
Recent studies showed that the prevalence of ESBL-producing bacteria is increasing [64,65]. In the present study, ESBL positivity was detected in 8.5% (8/94) of the Salmonella strains from one native breed in a breeder farm. However, none of them were multidrug-resistant. To our knowledge, this is the first time that ESBL-producing S. pullorum has been isolated from a native breed in a breeder chicken farm (Luhua). According to our investigation, the contaminated chickens, poor breeding conditions, and irregular breeding techniques may explain the production of ESBL.
In addition, we found that most of the isolates in this study (97.9%, 92/94) carried the blaTEM gene, which is consistent with the high rate of β-lactam resistance. Among the 85 β-lactam-resistant isolates, all strains harbored the blaTEM gene, which is regarded as one of the reasons responsible for β-lactam resistance. Our results showed that tetracycline and sulfonamide resistance genes were commonly distributed in most Salmonella isolates from different breeds, and this was also correlated with their resistance phenotypes. Tetracycline was discovered in the 1950s and is widely used as a feed additive because of its antibacterial and growth-promoting properties [66]. Additionally, among the tetracycline-resistant strains, the tetA and tetB genes were detected in all 28 resistant strains; this may be closely related to the abuse of tetracycline. Our study also detected sul1 and sul2 genes encoding resistance to sulfonamides in sulfamethoxazole–trimethoprim-resistant isolates. Specifically, tetracycline and sulfonamide AMR genes were detected, but no phenotypic resistance was observed among several strains. Mismatches in genotype and phenotype AMR were also observed in other studies [67,68,69,70]. Indeed, antimicrobial phenotype resistance was not related only to the presence or absence of antimicrobial resistance genes. Several other mechanisms, such as enzyme activation, target modification/protection, and resistance genes, may be considered “silent”; the regulation of resistant gene expression or even a change in the cell wall may affect the phenotype resistance [71]. In contrast, nearly 85.1% of isolates presented a high resistance to aminoglycosides, while only one of them carried the AAC2 gene encoding resistance to aminoglycosides. Colistin is considered to be the last-resort antibiotic for the treatment of MDR Gram-negative bacteria [72]; none of the four genes encoding colistin resistance were detected in any of the strains.

5. Conclusions

In conclusion, multidrug-resistant (MDR) Salmonella enterica strains were highly prevalent among different breeds of breeder chickens in Shandong Province, China. This is the first report in which ESBL-producing S. pullorum was isolated from a native breed in a breeder chicken farm. In addition, our data found that multidrug-resistant S. pullorum and S. Thompson are widespread in domestic chicken breeding farms, posing a serious threat to public health. These results highlight the importance of the continuous monitoring of antimicrobial resistance in Salmonella enterica among breeder chicken farms in China.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11020390/s1, Table S1: Primers used for confirming Salmonella and S. pullorum. Table S2: Primer sequences of antimicrobial resistance genes. Table S3: PCR conditions and size of PCR products among 21 antimicrobial resistance genes. Table S4: characteristics of 94 Salmonella isolates.

Author Contributions

Conceptualization, S.S. and L.C.; Funding acquisition, S.S.; Methodology, L.C., W.S., Y.L. and Y.H.; Project administration, S.S.; Writing—original draft, L.C. and W.S.; Writing—review and editing, L.C., W.S. and W.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Program of Shandong Province (2022CXGC010606) and Agricultural Breed Project of Shandong Province (2020LZGC013).

Institutional Review Board Statement

The animal study was reviewed and approved by Animal Care and Use at Shandong Agricultural University (permit number SDAUA-2021-034). Written informed consent was obtained from the owners for the participation of their animals in this study.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Microorganisms 11 00390 g001 550
Figure 1. PCR assays for the detection of Salmonella spp. isolates. M: molecular weight standards of DL 2000; Lane 1–8: Eight Salmonella strains; +: positive control (S. enteritidis CVCC 3377); −: Negative control (Escherichia coli ATCC 25922).
Figure 1. PCR assays for the detection of Salmonella spp. isolates. M: molecular weight standards of DL 2000; Lane 1–8: Eight Salmonella strains; +: positive control (S. enteritidis CVCC 3377); −: Negative control (Escherichia coli ATCC 25922).
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Microorganisms 11 00390 g002 550
Figure 2. Serotypes of Salmonella enterica isolates (n = 94) recovered from different chicken breeds.
Figure 2. Serotypes of Salmonella enterica isolates (n = 94) recovered from different chicken breeds.
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Microorganisms 11 00390 g003 550
Figure 3. PCR assays for the detection of Salmonella spp. isolates. M: molecular weight standards of DL 2000; Lanes 1–8: Eight Salmonella strains; +: positive control (S. pullorum CVCC 526); −: Negative control (S. enteritidis CVCC 3377).
Figure 3. PCR assays for the detection of Salmonella spp. isolates. M: molecular weight standards of DL 2000; Lanes 1–8: Eight Salmonella strains; +: positive control (S. pullorum CVCC 526); −: Negative control (S. enteritidis CVCC 3377).
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Microorganisms 11 00390 g004 550
Figure 4. Prevalence of multidrug resistance among 94 Salmonella enterica isolates. 0–7: The number of antimicrobials to which the isolates have resistance; (): The number of isolates with resistance to antimicrobials.
Figure 4. Prevalence of multidrug resistance among 94 Salmonella enterica isolates. 0–7: The number of antimicrobials to which the isolates have resistance; (): The number of isolates with resistance to antimicrobials.
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Microorganisms 11 00390 g005 550
Figure 5. Genetic relatedness of 94 Salmonella isolates based on ERIC-PCR patterns. ERIC-PCR dendrogram exhibiting the genetic relatedness of Salmonella enterica isolated from four different breeds of breeder chickens. The clustering was performed using UPGMA.
Figure 5. Genetic relatedness of 94 Salmonella isolates based on ERIC-PCR patterns. ERIC-PCR dendrogram exhibiting the genetic relatedness of Salmonella enterica isolated from four different breeds of breeder chickens. The clustering was performed using UPGMA.
Microorganisms 11 00390 g005
Table
Table 1. Number of samples and prevalence of Salmonella in Poultry farms (n = 693).
Table 1. Number of samples and prevalence of Salmonella in Poultry farms (n = 693).
LocationFarmBreedType of SampleNo. of SampleNo. of Positive Sample (%)
Tai’anFarm AWhite feather broilerDead embryos6023 (38.3)
JiningFarm BBairiDead embryos23815 (6.3)
RizhaoFarm CLangyaDead embryos10029 (29)
Cloacal swabs1001 (1)
Water15-
Feed152 (13.3)
JiningFarm DLuhuaDead embryos 60-
Meconium of newly hatched chicks7024 (34.3)
Environmental swabs30-
Water5-
Grand total69394 (13.6)
Table
Table 2. Numbers and percentages of Salmonella enterica isolates resistant to tested antibiotics.
Table 2. Numbers and percentages of Salmonella enterica isolates resistant to tested antibiotics.
Antibiotic Classes Antibiotic Number (%) of Resistant Isolates
(n = 94)		p Value
Imported BreedNative Breed
(n = 23)(n = 71)
β-lactamesAmpicillin23 (100)48 (67.6)0.012 *
Amoxicillin20 (87)49 (69)0.150
Cefoxitin0 (4.3)3 (4.2)0.040 *
Ceftazidime 015 (21.1)0.000 **
TetracyclinesTetracycline19 (82.6)9 (12.7)0.000 **
Doxycycline15 (65.2)6 (8.4)0.000 **
QuinolonesOfloxacin00-
AminoglycosidesGentamicin1 (4.3)29 (40.8)0.000 **
Streptomycin19 (82.6)59 (83.1)0.964
SulphonamidesSulfamethoxazole-trimethoprim029 (40.8)0.000 **
p value: * p < 0.05; ** p < 0.01.
Table
Table 3. Characterization of ESBL-producing isolates.
Table 3. Characterization of ESBL-producing isolates.
NumberSourceBreedSerotypeResistant PhenotypeResistant Genes
1Meconium of newly hatched chicksLuhuaS. pullorumSTR—CAZblaTEM—tetB
2Meconium of newly hatched chicksLuhuaS. pullorumSTR—CAZblaTEM
3Meconium of newly hatched chicksLuhuaS. pullorumSTR—CAZblaTEM—tetA—tetB
4Meconium of newly hatched chicksLuhuaS. pullorumTET—STR—AMX—AMP—CAZblaTEM—tetB
5Meconium of newly hatched chicksLuhuaS. pullorumSTR—AMX—AMP—CAZblaTEM—tetB
6Meconium of newly hatched chicksLuhuaS. pullorumSTR—CAZblaTEM—tetA—tetB—sul1—sul2
7Meconium of newly hatched chicksLuhuaS. pullorumSTR—CAZblaTEM—tetA—tetB
8Meconium of newly hatched chicksLuhuaS. pullorumCAZblaTEM
Table
Table 4. Resistance rates of different serovar Salmonella isolates toward 10 antimicrobial agents.
Table 4. Resistance rates of different serovar Salmonella isolates toward 10 antimicrobial agents.
SerotypesAntibiotics
SXTGMSTROFAMPAMXDOXTETCAZFOX
Pullorum0075%042%39%3%11%53%6%
Thompson91%91%91%094%100%6%6%00
Enteritidis04%85%0100%88%69%85%0%4%
SXT: sulfamethoxazole–trimethoprim; GM: gentamicin; STR: streptomycin; OF: ofloxacin; AMP: ampicillin; AMX: amoxicillin; DOX: doxycycline; TET: tetracycline; CAZ: ceftazidime; FOX: cefoxitin.
Table
Table 5. Rates of multidrug resistance among three different serotypes (%).
Table 5. Rates of multidrug resistance among three different serotypes (%).
SerotypesAntibiotic Agents
01234567
Pullorum100100944027500
Thompson0002046560100
Enteritidis0064027391000
0–7: The number of antimicrobials to which the serotype has resistance.
Table
Table 6. Prevalence of antimicrobial resistance genes among Salmonella isolates.
Table 6. Prevalence of antimicrobial resistance genes among Salmonella isolates.
Antimicrobial Resistance GenesNumber (%) of Resistant Isolates
(n = 94)		p Value
Imported BreedNative Breed
(n = 23)(n = 71)
blaTEM23 (100)69 (97.2)0.841
AAC21 (4.3)00.038 *
tetA20 (87)25 (35.2)0.000 **
tetB16 (70)26 (36.6)0.001 **
sul115 (65.2)10 (14.1)0.000 **
sul202 (2.8)0.094
p value: * p < 0.05; ** p < 0.01.
Table
Table 7. Detection rates of antimicrobial resistance genes among the three different serotypes (%).
Table 7. Detection rates of antimicrobial resistance genes among the three different serotypes (%).
SerotypesAntimicrobial Resistance Genes
blaTEMAAC2tetAtetBsul1sul2
Pullorum100061.161.122.22.8
Thompson1003.884.669.265.40
Enteritidis93.8012.56.203.1
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MDPI and ACS Style

Shi, W.; Tang, W.; Li, Y.; Han, Y.; Cui, L.; Sun, S. Comparative Analysis between Salmonella enterica Isolated from Imported and Chinese Native Chicken Breeds. Microorganisms 2023, 11, 390. https://doi.org/10.3390/microorganisms11020390

AMA Style

Shi W, Tang W, Li Y, Han Y, Cui L, Sun S. Comparative Analysis between Salmonella enterica Isolated from Imported and Chinese Native Chicken Breeds. Microorganisms. 2023; 11(2):390. https://doi.org/10.3390/microorganisms11020390

Chicago/Turabian Style

Shi, Wenjian, Wenli Tang, Yafei Li, Yu Han, Lulu Cui, and Shuhong Sun. 2023. "Comparative Analysis between Salmonella enterica Isolated from Imported and Chinese Native Chicken Breeds" Microorganisms 11, no. 2: 390. https://doi.org/10.3390/microorganisms11020390

APA Style

Shi, W., Tang, W., Li, Y., Han, Y., Cui, L., & Sun, S. (2023). Comparative Analysis between Salmonella enterica Isolated from Imported and Chinese Native Chicken Breeds. Microorganisms, 11(2), 390. https://doi.org/10.3390/microorganisms11020390

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