Pathogenicity of Multidrug-Resistant Salmonella typhimurium Isolated from Ducks.

Buffered peptone water (BPW), tetrathionate broth (TTB-enriched broth), selenocysteine (SC)-enriched broth, and xylose-lysine deoxycholate (XLD) were purchased from HaiBo BioTech Ltd. (Qingdao, China). Antibiotic susceptibility test plates were purchased from Hangzhou Microbial Reagent Co., Ltd. (Hangzhou, China). Diagnositic Sera Kit for Salmonella was purchased from Ningbo Tianrun Bio-pharmaceutical Co., Ltd. (Ningbo, China). Using the method described previously [ 20 ], 13,621 samples (361 duck embryos and 13,260 cloacal swabs) for Salmonella isolation and identification were collected from 239 duck farms in Shandong Province between September 2022 and November 2023, and the samples were stored in a low-temperature environment and transferred to the laboratory. In short, each sample was added to 4.5 mL of BPW and then 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 TTB and SC, respectively. Cultures of each TTB and SC broth were inoculated on an XLD Agar base and incubated at 37 °C for 48 h [ 22 ]. The confirmation of smooth and round colonies without a black center or large with a black center was achieved by polymerase chain reaction (PCR) assays with primers designed for the FimW gene [ 20 ]. All Salmonella strains were tested for resistance to 15 antimicrobial drugs by the broth-diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) [ 23 ]. The antibiotics used were ceftriaxone (CTR, 30 μg), ceftazidime (CAZ, 30 μg), ampicillin (AMP, 10 μg), amoxicillin (AMX, 20 μg), meropenem (MEM, 10 μg), tetracycline (TE, 30 μg), doxycycline (DOX, 30 μg), amikacin (AMK, 30 μg), enrofloxacin (ENR, 15 μg), ofloxacin (OFX, 30 μg), azithromycin (AZI, 30 μg), sulfamethoxazole (SXT, 25 μg), and sulfamonomethoxine (SMM, 30 μg). Polymyxin B (PB) susceptibilities were determined using the microbroth dilution assay [ 24 ]. MDR was defined as resistance to three or more classes of antimicrobial agents. Salmonella serotype was detected using the Diagnostic Sera Kit for Salmonella (Ningbo Tianrun Bio-pharmaceutical Co., Ltd., Ningbo, China) according to the instructions. Genomic DNA was extracted with the Genomic DNA Purification Kit (Tiangen Biotech, Beijing, China), and DNA templates were stored at −20 °C before use [ 25 ]. Eight virulence genes ( invA , hilA , sipC , sopE , spvC , ssaR , ssrA , and stnP1 ) were detected in all isolates, as previously described [ 26 ]. Ten PCR products were randomly selected and sequenced for each gene, and the sequences were then analyzed by sequence comparison using the NCBI database to verify the accuracy of the sequences. AS previously described [ 27 ], the overnight cultures of S. typhimurium strain were diluted to an OD 600 of 1000-fold in 3 mL of fresh LB broth in polystyrene tubes at 37 °C with shaking for 12 h. After incubation, the growth medium was decanted, and the tubes were washed three times with sterile PBS buffer and air-dried. Biofilms were quantified using a crystal violet assay by adding 250 μL of 0.05% crystal violet solution to each well. Plates were incubated for 15 min at room temperature and rinsed with distilled water. The crystal violet was dissolved in 200 μL of 95% ethanol, and biofilm formation was analyzed at OD 570 . Each strain of S. typhimurium was tested in three independent replicates. The freshly cultured bacterial solution was adjusted to an OD 600 of 0.1, and 1 μL was pipetted into the semi-solid LB solid medium at a depth of approximately 0.5 mm using a 10 μL of pipette and then incubated in a constant temperature incubator at 37 °C for 4–24 h. The growth of the strain was observed, and the diameter of the strain was measured [ 28 ]. Each strain of S. typhimurium was tested in three independent replicates. Firstly, the infection model of broiler ducklings was constructed to obtain the LD 50 of the leg muscle injection dose as 3.1 × 10 8 CFU. To further explore the pathogenicity of S. typhimurium to broiler ducklings, 24 one-day-old Cherry Valley ducklings were purchased from Ningyang Juxiang Breeding Co., Ltd., Tai’an City, Shandong Province, China. The ducklings were randomly divided into 2 groups and placed in a clean and tidy environment at 32 °C with unrestricted access to food and drinking water. S. typhimurium strain 22SD07 was cultured to logarithmic growth stage, washed with PBS, and diluted to 10 8 CFU/mL. After 3 days, the challenge doses were 10 8 CFU/mL each (≈0.32 LD 50 ) of the 100 μL bacterial solution for the intraperitoneal injection group, and controls were injected with the same dose of saline. The ducklings were weighed and recorded at 1, 3, and 7 dpi, and the heart, liver, spleen, cecum, and bursa were collected at 3 and 7 dpi from 6 ducklings to calculate the visceral tissue indexes, part of which were stored in the −80 °C and paraformaldehyde for subsequent pathological sectioning. Heart, liver, spleen, thymus, and bursa were collected for the determination of Salmonella counts. The immune organ index was calculated as follows: immune organ index (g/kg) = immune organ weight (g)/live weight of chicks (kg). Tissue samples were weighed and homogenized in PBS, and serial dilutions of the homogenates were plated onto XLD plates (37 °C, 16 h) for bacterial enumeration. The expression of cytokines, including interleukin-4 (IL-4), IL-6, IL-10, IFN-γ, and tumor necrosis factor (TNF-α), was analyzed by qPCR. RNA from liver, spleen, and cecum was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA synthesis was performed using 1 μg of RNA template via the Transcriber First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland). Appropriate primer sets were used for qPCR ( Table 1 ) on the ABI 7500 Detection System (Applied Biosystems, Carlsbad, CA, USA) using 18S as an internal reference. The PCR procedure consisted of 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and then 60 °C for 30 s. The results were analyzed using GraphPad Prism (Version 8.0.1) and expressed as mean ± SD. Student’s t -test was used to assess the differences between two groups. Results were considered significant when * p < 0.05, ** p < 0.01, and *** p < 0.001.

Salmonella is one of the most important zoonotic pathogens, and the widespread use of antibiotics in the poultry industry has led to the emergence of MDR Salmonella [ 1 , 2 ]. The indiscriminate and unregulated use of antibiotics has led to an increase in the prevalence of MDR Salmonella in animals and humans, and MDR Salmonella can be transmitted to humans through animal products or the environment, posing significant public health and food safety concerns [ 3 , 4 ]. It can be transmitted in the environment through animal feces [ 5 ] and to humans through various processes used in the production of food of animal origin [ 6 ], posing a serious threat to food safety and public health [ 7 ]. S. typhimurium is MDR Salmonella with a high detection rate in epidemiological surveys of poultry farms in recent years—which not only causes chick mortality but can also colonize adult chickens for a long time, leading to asymptomatic persistent infections—and is also one of the most important serotypes of salmonella transmitted from animals to humans in most parts of the world [ 8 , 9 ]. The prevalence of Salmonella isolated from fresh duck meat in the southern retail market of China has been reported. The results show that a high level of Salmonella contamination is detected in duck meat (151/365, 41.4%). Among them, S. typhimurium accounted for 6/151 [ 10 ]. Kang et al. found that S. typhimurium was distributed in various production stages in the duck production chain [ 11 ]. Zhao conducted an epidemiological investigation on Salmonella isolated from several duck farms in Shandong Province and its surrounding areas, among which Kottbus and S. typhimurium were the dominant serotypes [ 12 ]. The Import and Export Food Safety Inspection and Monitoring Programme has adopted a zero-tolerance policy for Salmonella in food safety inspection and monitoring programs. With increasing awareness of the evolution of antimicrobial resistance (AMR) in Salmonella, longitudinal surveillance programs have been initiated by governments and researchers, providing valuable epidemiological data for risk assessment and medication guidelines [ 13 ]. Biofilms are dynamic communities of heterogeneous microorganisms that transition from a free-swimming planktonic state to a sessile state embedded in the extracellular matrix [ 14 , 15 ]. Several AMR phenotypes and genes, particularly those conferring resistance to fluoroquinolones, have been reported to be associated with biofilm formation [ 16 ]. In addition, biofilm formation is closely associated with pathogenicity, and reports on the relationship between AMR pathogenicity and biofilms are inconsistent, suggesting that the true relationship is unclear [ 17 ]. Virulence factors enable bacteria to enter, adhere to, and replicate in host cells. Virulence factors also enable bacteria to overcome host defenses and cause disease [ 18 ]. Salmonella has several known virulence factors present in Salmonella pathogenicity islands (SPIs), phages, bacterial clusters, and plasmids. Most virulence genes involved in pathogenesis are located in SPI-1 and SPI-2 pathogenicity islands in the Salmonella genome [ 18 ]. Duck meat is a popular meat product, and China is the world’s largest producer and consumer of farmed waterfowl [ 19 ]. Our previous findings confirmed the high isolation rate of Salmonella from dead embryos and meconium of newly hatched chicks [ 20 ] and that infection with S. typhimurium alters the intestinal microbiota by increasing the abundance of Rumatococcaceae and Serratiaceae [ 21 ]. In this study, we analyzed the epidemiology, drug resistance characteristics, and virulence genes of S. typhimurium in 239 large-scale duck farms in selected areas of Shandong Province. Furthermore, the S. typhimurium strain with strong biofilm formation ability was tested in animal experiments to investigate its pathogenicity to ducklings.

A total of 44 Salmonella strains were isolated from 361 dead embryos and 13,260 cloacal swabs with a positivity rate of 0.32% (44/13,621). Eight of these strains were S. typhimurium , designated 22SD01-22SD08. The results of the antimicrobial susceptibility analysis of 8 S. typhimurium varied in resistance to 14 antibiotics, as shown in Table 2 . MDR strains accounted for 37.5% (3/8). Among them, the highest rate of resistance to DOX and AK reached 75% (6/8) and 62.5% (5/8), respectively ( Table 3 ). Notably, SD2207 had a resistance profile of CTX-DX-CTR-TE-AMX-AMP-CAZ. The identification of eight virulence genes in S. typhimurium strains is shown in Table 1 . Apart from 22SD04, which carried six virulence genes ( invA , sipC , sipA , ssaR , ssrA, and stnp1 ), the other seven strains carried invA , sipC , sipA , ssaR , ssrA , stnp1, and hilA . The biofilm formation ability of different strains of S. typhimurium was first assessed. Different clinical strains showed different abilities to form biofilms, with strain 22SD07 showing the most robust biofilm-forming capacity ( Figure 1 A). In addition, the results of the motility assay are shown in Figure 1 B, where the motility ring of strains on the semi-solid flat dish showed no significant difference. Therefore, the S. typhimurium strain 22SD07 was selected for further investigation. As shown in Figure 2 , 22SD07 did not affect the body weight of the ducklings at 1 and 3 dpi, but the weight of the ducklings was reduced at 7 dpi ( p > 0.05) ( Figure 2 A). Additionally, it significantly increased the heart index at 7 dpi and liver index at 3 dpi ( Figure 2 B,C), while it decreased the bursa index significantly at 3 and 7 dpi ( Figure 2 E). As shown in Figure 3 , the colonization of S. typhimurium in the liver and spleen was observed by measuring the bacterial load of S. typhimurium at 3 and 7 dpi. The results showed the bacterial load of Salmonella in the liver reached 10 3.574±0.153 cfu/g, while that in the spleen reached 10 3.942±0.2122 cfu/g. However, the load of S. typhimurium in the liver and spleen was lower at 7 dpi than at 3 dpi. The bacterial load of Salmonella in the liver decreased to 10 0.8132±0.5267 cfu/g, while that in the spleen decreased to 10 0.6799±0.5069 cfu/g. After the S. typhimurium infection, the splenic tissue is poorly demarcated between white and red marrow, with punctate cellular necrosis (black arrows), fragmented nuclei, and eosinophilic red staining of the cytoplasm ( Figure 4 A). The connective tissue of the lamina propria of the ileum is densely packed, with erythrocytes visible in multiple capillaries ( Figure 4 B). The jejunal enterochromaffin epithelium is incomplete, with occasional detachment of the enterochromaffin epithelium and pitting necrosis, cytoplasmic nuclear consolidation, and eosinophilic red staining of the cytoplasm ( Figure 4 C). In the cecum, there are large aggregates of lymphocytes (black arrows), a shortening of the glandular ducts, a decrease in the number of goblet cells, and loose cytoplasm in the intestinal glandular epithelium ( Figure 4 D). The relative expressions of cytokines in the liver, spleen, and cecum at 7 dpi were examined. The relative expression of IL-10 ( p 0.05) and IL-6 ( p > 0.05) mRNA in the liver of the S. typhimurium group was lower than in the control ( Figure 5 B). In the spleen, the relative expression of TNF-α ( p < 0.05) was significantly higher than in the control at 7 dpi. In the cecum, the relative expression of TNF-α ( p < 0.01), IL-6 ( p < 0.05), and IFN-γ ( p < 0.01) mRNA in the cecum of the infected group was significantly higher than in the control at 3 dpi.

The widespread and inappropriate use of antimicrobials has accelerated the emergence and spread of AMR [ 29 ]. Although infections caused by antimicrobial-resistant Salmonella have been reported worldwide, cases in developing countries are increasing significantly and at an alarming rate [ 30 ]. The clinical use of antibiotics is one of the main means of treating Salmonella disease, and with increasing farm density, the irrational use of antibiotics has led to the emergence of MDR strains. In this study, we examined 13,621 samples of duck cloacal swabs and dead embryos collected from duck farms located in Shandong Province between September 2022 and November 2023, and 44 strains of Salmonella were isolated and identified. The S. typhimurium isolates were then subjected to antimicrobial susceptibility testing to determine their resistance profile. The results showed that all S. typhimurium recovered from ducks had a high rate of resistance to DOX and AMK, which could be attributed to the overuse and misuse of tetracycline and aminoglycoside antibiotics in the treatment of duck diseases [ 31 ]. The rates of resistance to other antibiotics were not high, reflecting the fact that the amount of antibiotics used in duck meat production may be lower than in chicken farming. However, it cannot be ignored that indiscriminate use of antibiotics may increase the number of MDR Salmonella strains and the rate of antibiotic resistance. More importantly, the gradual increase in MDR Salmonella strains may cause public health problems [ 32 ]. All eight S. typhimurium strains carried invA , sipC , sipA , ssaR , ssrA , and stnp1 . The major virulence factor of Salmonella is invA , which encodes a protein that makes Salmonella highly pathogenic. Many studies have shown that there is a correlation between the virulence genes contained in Salmonella and their own virulence, mainly due to the interaction between a large number of virulence genes [ 33 , 34 ]. During Salmonella invasion, the bacterial effectors SipC , SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake [ 35 ]. Both the T3SS translocator SipC and effector SipA are critical for Salmonella infection by subversion of the host cell cytoskeleton, but the precise molecular interplay between them remains unknown. It has been shown that SipA binds along the F-actin grooves with a unique binding pattern using cryoelectronic microscopy. SipA stabilizes F-actin through charged interface residues and appears to prevent inorganic phosphate release through the closure of the “back door” of the adenosine 5′-triphosphate pocket. Moreover, SipC promotes the binding of SipA to F-actin [ 36 ]. In addition, a variety of virulence genes are clustered on the so-called Salmonella Pathogenicity Islands (SPIs); SPI-2 presents the ssaR and sifA genes, which are necessary for the survival and subsequent multiplication of the bacterium and also play an important role in systemic infection [ 37 , 38 ]. The SsrAB system is encoded by the ssrAB operon located in SPI-2. SsrA is the sensor kinase, and SsrB is the response regulator that directly controls the expression of target genes [ 39 ]. The SsrAB two-component system is the central positive regulator for the SPI-2 genes and other functionally related genes located outside SPI-2 [ 40 , 41 ]. Biofilm formation is a key strategy for the survival of Salmonella under unfavorable environmental conditions [ 42 ]. Some bacterial cell surface components, such as cellulose, flagella, and hyphae, have been shown to contribute to the attachment of Salmonella to different surfaces [ 43 ]. In this study, the S. typhimurium strain known as 22SD07 was highly biofilm-forming, and drug susceptibility tests showed it to be MDR. So, we further explore its pathogenicity to ducklings. An injection dose of 10 8 CFU S. typhimurium (0.32 LD 50 ) was selected by leg muscle injection, and diseased ducks with obvious clinical characteristics were obtained from this infection experiment in Cherry Valley broiler ducklings. The results showed that the bacterial content of the organs had decreased significantly but was still present at 7 dpi ( Figure 3 ). It has been shown that the number of heterophilic cells in the spleen is significantly increased in susceptible chicken breeds infected with Salmonella [ 44 , 45 ]. Salmonella uses virulence factors to gain entry into the intestinal epithelium and survive in mucosal macrophages, leading to an acute inflammatory process [ 46 ]. Inflammatory bowel disease is an idiopathic inflammatory disease of the intestines affecting the ileum, rectum, and colon. The main manifestations are diarrhea, abdominal pain, and bloody stools. The pathological changes induced by Salmonella include enteritis and septicemias. The intestinal mucosa displays punctate hemorrhage, thickening of the cecum wall, intestinal mucosa congestion, enlargement of the mesenteric lymph node, necrosis, and other characteristics. It has been demonstrated that infection with avian S. typhimurium increases mortality and intestinal diseases and leads to gastroenteritis related to human food-borne diseases [ 45 ]. The pathological section results of this experiment showed notable inflammatory alterations and tissue damage. The most significant changes were observed in the spleen, which exhibited an indistinct boundary between white and red medulla, infiltration of lymphocytes, and cytoplasmic eosinophilic red staining ( Figure 4 A). Hemorrhages were observed in the jejunum, ileum, and spleen, accompanied by connective tissue hyperplasia and cell infiltration ( Figure 4 B–D). Additionally, cellular necrosis was evident in the jejunum and spleen. The mucosal barrier is destroyed by inducing desquamation of the epithelium, the loss of mucin-producing goblet cells, and the overall distorted villi shape (becoming shorter and wider), as findings have been reported previously [ 47 , 48 ]. This mucosal destruction and inflammation lead to the interference of intestinal function, promote the progress of Salmonella typhimurium colonization, and cause a loss of weight gain [ 49 , 50 ].

The isolation rate of S. typhimurium in some duck farms in Shandong Province was 0.06% (8/13,621), and MDR S. typhimurium accounted for 37.5% (3/8), all carrying six kinds of virulence genes, including invA , rck , sipC , pefA , sipA, and ssaR . S. typhimurium 22SD07 caused decreased body weight, increased organ index, and intestinal villous shedding.