Research Note: Changes in pathogenic characteristics and drug resistance of Salmonella in poultry meat in Jiading District, Shanghai from 2019 to 2021

Abstract

To investigate the contamination status, serotype distribution, and drug resistance of Salmonella in poultry sold in Jiading District, Shanghai. Four types of raw poultry meats (chickens, ducks, geese, and pigeons) have been sampled from commercial markets, and potential Salmonella contamination has also been isolated and identified via serotype analysis. Furthermore, resistance of isolated Salmonella toward 14 commonly used antibiotics has also been conducted. Ninety-two Salmonella strains were isolated from 236 commercial poultry samples. The detection rates of Salmonella in pigeon, goose, duck, and chicken were 28.89, 44.44, 39.34, and 38.30%, respectively. The detection rate of Salmonella exhibits considerable variation across different years. The serotype composition of Salmonella in poultry demonstrates annual variability, undergoing significant changes from year to year. The majority serotypes of Salmonella have been revealed as S. Typhimurium, S. Enteritidis, and S. Agona. Relatively higher drug resistance was discovered with nalidixic acid, tetracycline, ampicillin and chloramphenicol, with drug resistance rate as 58.70, 53.25, 44.57, and 38.04%, respectively. Low drug resistance was revealed with cefotaxime, and completely sensitive to imipenem. Significant difference in drug resistance was noted in the Salmonella isolated from meats. Different serotypes of Salmonella strains have also revealed as difference in drug resistance. A total of 15.22% of Salmonella strains were nonresistant to any tested drugs. Multidrug-resistant strains accounted for 36.96% of isolated strains. The highest number of resistant antibiotics can reach 12 kinds of different antibiotics, Salmonella resistance is exhibiting a consistent upward trend overall. AMP-TET or CHL-CFZ drug resistance pattern suggested that the strain was multidrug resistant. The contamination of Salmonella in raw poultry meat samples in Jiading District, Shanghai is serious, and the drug resistance is increasing. The measures taken for epidemic prevention and control have a certain impact on the contamination of Salmonella in poultry meat. Therefore, monitoring and control should be strengthened.

INTRODUCTION

Salmonella is a significant zoonotic pathogen that can cause gastrointestinal diseases in humans and is one of the primary foodborne pathogens. Consumption of contaminated food, including poultry meat, can lead to Salmonella enteritis and septicemia, resulting in symptoms such as acute diarrhea, abdominal pain, fever, and vomiting. Globally, it is estimated that Salmonella infection affects 93.8 million people each year, with 80.3 million of these cases being foodborne infections. Additionally, Salmonella infection leads to 155,000 deaths each year (Majowicz et al., 2010).

In China, Salmonella is the primary bacterial foodborne illness. In fact, Salmonella was responsible for 29.76% of outbreaks of foodborne illness caused by pathogenic microbe contamination in 2021 (Li et al., 2022). Therefore, Salmonella poses a significant threat to human health and remains an important public health problem.

Salmonella is commonly found in livestock and poultry, with poultry products serving as a vital medium for Salmonella infection in humans. This makes them one of the main sources of Salmonella infection in humans. Consuming or handling contaminated poultry can result in bacterial infections, leading to food poisoning. As people's living standards continue to improve, the consumption of poultry meat is constantly increasing, leading to an increased risk of Salmonella infection. The contamination of Salmonella in poultry meat has had a negative impact on public health, resulting in severe disease burdens.

The issue of Salmonella resistance is becoming increasingly severe, with antibiotics widely used even in poultry farming, resulting in increased resistance of Salmonella and an expanded range of resistance. The proportion of resistant strains is continually increasing, leading to a rise in infections by these strains through the food chain. This poses a threat to public health and safety, bringing more challenges to treatment and prevention. Therefore, understanding the pathogenic characteristics and drug resistance of Salmonella in poultry meat is crucial in reducing the harm of drug-resistant Salmonella in poultry meat to the population.

MATERIALS AND METHODS

Sample Source

From 2019 to 2021, a total of 236 samples of fresh poultry meat were collected from 4 different types of locations in Jiading District at the beginning of each month. Each type of location included major online shopping warehouses, supermarkets, restaurant, and farm produce markets, with 6 to 8 pieces of each of the 4 types of fresh poultry meat collected from each location. The sampling locations and stalls were randomly selected to ensure representativeness, and no repeated sampling was carried out at the same location to avoid duplication. After sample collection, the samples were transported in transport boxes with ice packs and temporarily stored in a 4°C refrigerator upon arrival at the laboratory. Testing was conducted on the same day as the samples were collected to minimize delays and ensure timely analysis.

Main Culture Media and Reagents

Buffered peptone water (BPW), tetrathionate broth (TTB) enrichment broth, selenite cysteine (SC) enrichment broth, xylose lysine deoxycholate (XLD), and related biochemical identification tubes were purchased from Haibo Biotechnology Co., Ltd. (Qingdao, China). Salmonella chromogenic culture media were purchased from Shanghai Koma and Shanghai Shenqi companies (Shanghai, China). Salmonella diagnostic serum was purchased from Ningbo Tianrun Biopharmaceutical Co., Ltd. (Ningbo, China). The antibiotic susceptibility test plates were purchased from Zhuhai Coal Chemical Medical Technology Co., Ltd. (Zhuhai, China).

Detection Methods

Salmonella isolation, identification, and serum typing were conducted in accordance with the standard operating procedures for foodborne pathogen testing outlined in GB 4789.4-2016 “Food Safety National Standard—Microbiology Examination of Food—Salmonella Testing.” To isolate, identify, and serum-type Salmonella, 25 g of raw poultry meat was homogenized with 225 mL of buffered peptone water (BPW) and then incubated for 18 h at 36°C. Subsequently, 1 mL of the enriched broth was inoculated into 10 mL of tetrathionate broth (TTB) and 10 mL of selenite cystine (SC), which were incubated at 42°C and 36°C for 24 h, respectively. The inoculum was streaked onto bismuth sulfite (BS) agar, xylose lysine deoxycholate (XLD) agar, and Salmonella chromogenic agar (SCA), and incubated at 36°C for 24 to 48 h. Suspicious colonies were picked and subjected to biochemical identification.

The minimum inhibitory concentration (MIC) for antimicrobial susceptibility testing was determined using the microbroth dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI2017). Fourteen antibiotics were tested, including sulfamethoxazole/trimethoprim (SXT), nalidixic acid (NAL), tetracycline (TET), ciprofloxacin (CIP), ampicillin (AMP), ampicillin/sublactam (AMS), gentamicin (GEN), chloramphenicol (CHL), cefazolin (CFZ), cefotaxime (CTX), ceftazidime (CAZ), cefixime (CFX), imipenem (IPM), and erythromycin (ERY). ATCC 25922 was used as the quality control strain. The broth microdilution method was used to determine the MIC values. Strains that showed resistance to 3 or more antibiotics were considered multidrug resistant (MDR).

Statistical Methods

The data were analyzed and summarized using SPSS 19.0 software. A significance level of α = 0.05 was used, and P < 0.05 was considered statistically significant. The statistical methods included the chi-square test.

RESULTS AND DISCUSSION

Detection of Salmonella in Different Poultry Meats

From 2019 to 2021, a total of 236 samples of 4 commercially available poultry meat types, namely goose, pigeon, chicken, and duck, were analyzed. Out of the 89 samples that tested positive for Salmonella, a total of 92 strains were identified. Three strains were detected in 1 sample of duck meat, and 2 strains were found in 1 sample of goose meat. Table 1 shows the detection rates for the other categories. Salmonella positive rates in poultry samples from online store warehouses, supermarkets, farm produce markets, and restaurants were 30.00% (12/40), 31.11% (14/45), 41.60% (52/12), and 42.31% (11/26), respectively. While no statistically significant differences were found between Salmonella detection rates from various poultry sources (χ² = 2.886, P = 0.410 >0.05), a general trend of higher positive rates in areas of higher human density can be observed. Further research is required to determine if human density is correlated with Salmonella positivity.

Table 1.

Detection results of Salmonella in different kinds of poultry products in different years.

	Positive samples % (No. of positive Samples/No. of samples)
Category	2019	2020	2021	Total
Goose	53.85 (7/13)	0.00 (0/12)	81.82 (9/11)	44.44 (16/36)
Pigeon	0.00 (0/10)	0.00 (0/20)	86.67 (13/15)	28.89 (13/45)
Chicken	22.58 (7/31)	32.26 (10/31)	59.38 (19/32)	38.30 (36/94)
Duck	27.78 (5/18)	14.29 (3/21)	72.73 (16/22)	39.34 (24/61)
Total	26.63 (19/72)	15.48 (13/84)	71.25 (57/80)	37.71 (89/236)

From 2019 to 2021, the average detection rate of Salmonella in samples of chicken, goose, duck, and pigeon meat in Jiading District, Shanghai was 37.71%. According to research, the detection rates of Salmonella in poultry from Jiangsu, Henan, and Fujian provinces in China range from 7.5 to 44.3% (Sun et al., 2021). In Japan, the detection rates of Salmonella in poultry from retail stores and processing plants reached 55.9%, while in Benin City, Nigeria, retail poultry samples showed a 41.2% Salmonella positive rate (Igbinosa et al., 2022). In comparison, the Salmonella contamination rate in fresh poultry samples from Jiading District is at a medium level.

The highest detection rate of Salmonella was found in goose meat at 44.44%, followed by duck meat and chicken meat, while the lowest detection rate was found in pigeon meat at 28.88%. Although there are some differences in the detection rate of Salmonella among different types of poultry meat, they are not significant (χ² = 2.269, P = 0.519 >0.05). It is necessary to conduct further monitoring of Salmonella in different types of poultry meat and investigate the risk of cross-contamination between the 4 types of fresh poultry meat.

Among the 3 yr, the lowest detection rate of Salmonella in poultry meat was in 2020, while the highest detection rate occurred in 2021. There are statistically significant differences in the detection rates of Salmonella in poultry meat between different years (χ² = 59.918, P < 0.05).

Over the course of 3 yr, there has been a general upward trend in the detection rate of Salmonella in poultry meat. The highest detection rate of Salmonella in poultry meat was observed in 2021, while the lowest detection rate occurred in 2019. China had a COVID-19 outbreak at the end of 2019, after the outbreak of the novel coronavirus, proactive measures were implemented in China to control the epidemic, including strict control of human movement and environmental disinfection in various premises. As a result, the contamination rate of Salmonella in poultry products significantly decreased. In 2020, China successfully controlled the COVID-19 outbreak, with only sporadic cases occurring domestically. Increased mobility of individuals and accelerated recovery of various commercial activities led to a surge in the detection of Salmonella in poultry meat. The shifts in the Salmonella detection rates in poultry from 2019 to 2021 indicate that epidemic prevention and control measures impacted contamination levels of Salmonella in poultry.

Serotype Distribution

A total of 92 Salmonella strains were isolated from market poultry, belonging to 7 groups (10 subgroups) and 29 serotypes. From 2019 to 2022, there were 9, 8, and 21 different serotypes of Salmonella in poultry meat, respectively. The most common group was Group B, accounting for 42.22% (37/92), followed by Group C at 29.35% (27/92), Group D at 19.57% (18/92), and Group E at 6.52% (6/92). From 2019 to 2021, the predominant serotypes of Salmonella in live poultry meat in Jiading District were Salmonella Typhimurium, S. Enteritidis, and S. Agona, accounting for 17.39% (16/92), 15.22% (14/92), and 11.96% (11/92) of the total samples, respectively.

From 2019 to 2021, there were significant variations in the serotypes of Salmonella over the 3-yr period. In 2019, the top 4 serotypes based on their respective proportions were S. Enteritidis (26.32%), S. Typhimurium (26.32%), S. Agona (10.53%), and S. Chartres (10.53%). In 2020, the dominant serotypes were S. Agona (30.77%), S. Mbandaka (15.38%), S. Typhimurium (15.38%), and S. Enteritidis (7.69%). For the yr 2021, the prominent serotypes were S. Typhimurium (15.00%), S. Enteritidis (13.33%), S. Corvallis (11.67%), and S. Kentucky (10.00%). Over the 3-yr period, there were significant variations in the serotypes of Salmonella in poultry meat, with inconsistencies observed each year.

Overall, B-group Salmonella is the most common contaminant in commercially available poultry, followed by C-group and D-group Salmonella. The main types include S. Typhimurium, S. Enteritidis, and S. Agona. S. Typhimurium and S. Enteritidis are the dominant serotypes of Salmonella in domestic poultry, with major serotype composition varying across different regions. In Jiading District, S. Agona is also a dominant strain, which differs from other regions. The dominant serotypes of Salmonella in poultry across China vary greatly and are related to regional environment, feeding, transportation, and storage methods, indicating the complexity of Salmonella. Prevention and control strategies for poultry Salmonella must be tailored to individual regions. Diarrhea patients in this district and other districts of Shanghai also commonly have S. Typhimurium and S. Enteritidis (Yu et al., 2021). The dominant serotypes of Salmonella in poultry are consistent with those in humans, suggesting a possible correlation between the 2. Strengthening the prevention and control of Salmonella in commercially available poultry can help reduce the population's incidence of Salmonella infection.

Drug Sensitivity Testing

Improper and excessive use of antibiotics in poultry breeding has led to the emergence of MDR strains, posing a serious threat to food safety and human health. In Jiading District's poultry market, the drug resistance rates of Salmonella can be found in Table 2. Except for complete resistance to ERY, the highest resistance rate of Salmonella was found for NAL at 58.70% (54/92), followed by TET at 53.26% (49/92). Among beta-lactam antibiotics, only AMP showed high resistance at 44.57% (41/92). The resistance rate of chloramphenicol is 38.04% (35/92), other drugs had resistance rates less than 30%. CFX showed the lowest resistance rate at 2.17% (2/92), and all strains were sensitive to IPM. Other studies have shown that Salmonella in poultry is highly resistant to TET and sulfonamide drugs, with resistance rates ranging from 60 to 90% in many areas (Igbinosa et al., 2022). CIP resistance rates vary widely across regions, ranging from less of 5 to 50% (Kalaba et al., 2021). Compared to other areas, Jiading District has relatively lower resistance rates to these drugs. It is crucial to continue monitoring changes in drug resistance rates to prevent increases.

Table 2.

Resistance rates of Salmonella in various poultry products and the 3 major serotypes to 14 antibiotics.

		Pigeon				Chicken				Duck				Goose
Types of antibiotics	Antibiotic	R	I	S	No. (%) of resistant	R	I	S	No. (%) of resistant	R	I	S	No. (%) of resistant	R	I	S	No. (%) of resistant
Amphenicols	CHL	6	0	7	46.15	18	0	18	50.00	5	0	21	19.23	6	6	11	35.29
Tetracyclines	TET	8	0	5	61.54	22	0	14	61.11	10	0	16	38.46	9	9	8	52.94
Quinolones	CIP	3	7	3	23.08	6	17	13	16.67	3	10	13	11.54	2	2	8	11.76
	NAL	9	1	3	69.23	19	5	12	52.78	16	1	9	61.54	10	10	5	58.82
Sulfonamides	SXT	4	0	9	30.77	12	0	24	33.33	2	0	24	7.69	3	3	14	17.65
Macrolides	ERY	13	0	0	100.00	36	0	0	100.00	26	0	0	100.00	17	17	0	100.00
Aminoglycosides	GEN	4	0	9	30.77	9	2	25	25.00	3	0	23	11.54	2	2	15	11.76
β-Lactams	AMP	6	0	7	46.15	18	0	18	50.00	10	0	16	38.46	7	7	10	41.18
	AMS	3	2	8	23.08	12	6	18	33.33	2	5	19	7.69	3	3	10	17.65
	CAZ	2	0	11	15.38	9	0	27	25.00	4	1	21	15.38	2	2	15	11.76
	CFX	0	1	12	0.00	2	2	32	5.56	0	2	24	0.00	0	0	17	0.00
	CFZ	4	3	6	30.77	14	1	21	38.89	6	5	15	23.08	2	2	10	11.76
	CTX	3	0	10	23.08	13	0	23	36.11	6	0	20	23.08	2	2	15	11.76
	IPM	0	0	13	0.00	0	0	36	0.00	0	0	26	0.00	0	0	17	0.00

		Typhimurium				Enteritidis				Agona
Types of antibiotics	Antibiotic	R	I	S	No. (%) of resistant	R	I	S	No. (%) of resistant	R	I	S	No. (%) of resistant
Amphenicols	CHL	3	0	13	18.75	2	0	12	14.29	4	0	7	36.36
Tetracyclines	TET	8	0	8	50.00	3	0	11	21.43	7	0	4	63.64
Quinolones	CIP	3	10	3	18.75	1	3	10	7.14	1	7	3	9.09
	NAL	13	2	1	81.25	12	0	2	85.71	6	1	4	54.55
Sulfonamides	SXT	3	0	13	18.75	1	0	13	7.14	2	0	9	18.18
Macrolides	ERY	16	0	0	100.00	14	0	0	100.00	11	0	0	100.00
Aminoglycosides	GEN	2	0	14	12.50	1	0	13	7.14	2	0	9	18.18
β-Lactams	AMP	8	0	8	50.00	7	0	7	50.00	5	0	6	45.45
	AMS	4	3	9	25.00	1	5	8	7.14	3	1	7	27.27
	CAZ	2	0	14	12.50	2	0	12	14.29	3	0	8	27.27
	CFX	1	1	14	6.25	0	0	14	0.00	0	0	11	0.00
	CFZ	3	6	7	18.75	1	4	9	7.14	4	1	6	36.36
	CTX	3	0	13	18.75	1	0	13	7.14	4	0	7	36.36
	IPM	0	0	16	0.00	0	0	14	0.00	0	0	11	0.00

The overall resistance rate of Salmonella is generally higher in chicken and pigeon meat than in goose and duck meat. The 3 dominant Salmonella strains S. Enteritidis, S. Typhimurium, and S. Agona exhibit slightly different levels of antibiotic resistance, with S. Enteritidis having a lower overall resistance rate while S. Agona shows a higher one.

The erythromycin resistance gene in Salmonella is spread via plasmids. The overuse of erythromycin has led to the widespread carriage of resistance genes in Salmonella. A study demonstrated that the misuse of antibiotics in poultry on farms promotes the emergence and spread of antibiotic resistance in Salmonella (Xiong et al., 2018). The widespread use of antimicrobial drugs in poultry for prevention and treatment has continuously enhanced the antibiotic resistance of Salmonella, making poultry the main source of MDR Salmonella (Abd-Elghany et al., 2015). Strengthening the monitoring and regulation of antibiotics in poultry is necessary to reduce the further deterioration of Salmonella resistance.

Among the raw poultry samples, 15.22% of Salmonella strains showed no resistance, while 36.96% (34/92) were MDR. The proportion of strains resistant to more than 5 drugs was 30.43% (28/92), and the highest number of drug resistances reached 12. From 2019 to 2021, the proportions of MDR strains were 36.84% (7/19), 23.08% (3/13), and 46.67% (28/60), respectively. There was a downward trend in the proportion of Salmonella strains that were nonresistant to antibiotics, while the proportion of MDR strains showed an upward trend. Within the MDR strains, there was also an increasing trend in the number of antibiotic resistances exhibited. Other regions in China report the proportion of MDR Salmonella strains in poultry meat as over 50% (He et al., 2021). Compared to these studies, the incidence of multidrug resistance in raw poultry meat in Jiading District is comparatively lower, but still concerning, posing a significant threat to food safety and human health. Previous research has shown that strains resistant to AMP or TET may have the potential to become MDR strains (Proroga et al., 2018). This study found that the resistance patterns of AMP-TET or CHL-CFZ can be important indicators for determining whether a strain is MDR.

In Shanghai's Jiading district, Salmonella contamination in commercially available poultry meat varies and the resistance patterns are different in chicken, duck, goose, and pigeon meat. The contamination level, serotype distribution, and antibiotic resistance of Salmonella in poultry meat undergo changes every year. Simultaneously, the measures implemented by humans in epidemic prevention and control have a certain influence on the contamination and distribution of Salmonella in poultry meat. To reduce the food safety risks posed by Salmonella, relevant authorities can strengthen hygiene supervision, regulate the use of antibiotics, and take preventive measures by treating different types of poultry meat differently and developing corresponding measures according to local conditions.

DISCLOSURES

All authors declare no conflicts of interest.