Antimicrobial resistance , class 1 integrons , and horizontal transfer in Salmonella isolated from retail food in Henan , China

Introduction: Salmonellosis remains one of the most frequently occurring foodborne diseases worldwide, especially in developing countries. The increasing prevalence of multidrug resistance among Salmonella isolates from food has been an emerging problem in China. Methodology: In this study, a total of 638 food samples including raw meat, seafood, vegetables, and cooked meat were collected in Henan province of China between July 2007 and August 2008 to determine the prevalence of Salmonella. These isolates were subjected to serotyping, antimicrobial susceptibility, presence of class 1 integrons, and horizontal transfer of integrons. Results: The overall percentage of Salmonella prevalence was 9.7% (n = 62). Among these isolates, S. Anatum and S. Senftenberg were most common, and high rates of antimicrobial resistance were observed to sulfamethoxazole (90.3%), trimethoprim/sulfamethoxazole (87.1%), streptomycin (29.0%), and ciprofloxacin (25.8%). Class 1 integrons were detected in 16.1% of these isolates, and contained gene cassettes dfrA12-aadA2, dfrA1-aadA1, and dfrA1. Three Salmonella isolates could transfer their integrons and resistance genes to Escherichia coli by conjugation. Conclusions: Our findings indicate that the mobile DNA elements could play an important role in the dissemination of resistance determinants among those Salmonella isolates.


Introduction
Salmonella, including more than 2,500 different serovars, represents a major cause of foodborne diseases throughout the world [1,2].In most cases, the infections are associated with ingestion of contaminated food products, particularly those of animal origin such as poultry, eggs, beef, and pork [3,4].Vegetables and fruits also have been reported to be carriers in Salmonella transmission, and contamination can occur at multiple steps along the food chain [5].Although there is a lack of official surveillance data for Salmonella in China, it is estimated that 22.2% of foodborne diseases are caused by Salmonella [6].
The increasing prevalence of multidrug resistance among Salmonella and resistance to clinically important antimicrobial agents has also been an emerging problem in China and other countries [7,8].
The spread of antimicrobial resistance potential in Salmonella is mainly attributed to integrons, which are genetic elements capable of capturing and transferring resistance genes among bacteria [9].The class 1 integron is the most common type of integron identified in multidrug-resistant (MDR) Salmonella, and it plays an important role in the dissemination of resistance genes among pathogens [10].
To the best of our knowledge, there are several studies about antimicrobial resistance of foodborne Salmonella isolates in China [7,11,12]; however, similar research in Henan province is limited.Therefore, the objective of this study was to investigate the prevalence, serovars, antimicrobial resistance, and class 1 integrons of Salmonella in retail food in Henan province, China.We also examined the integron-positive isolates for the ability to transfer antimicrobial resistance genes via conjugation.

Isolation and identification of Salmonella
Between July 2007 and August 2008, a total of 638 food samples including pork (n = 92), beef (n = 89), chicken (whole and parts, n = 95), mutton (n = 91), seafood (fish and shrimp, n = 73), vegetables (n = 98), and cooked meat products (sauced meats, roasted meats, and sausages, n = 100) were collected monthly from supermarkets and open-air markets in six cities in Henan province.The sample size was determined using the Sample Size Calculator (http://www.surveysystem.com/sscalc.htm) in order to ensure the reliability of the results.Henan, located in the central part of China, is a major agricultural center with about 104.9 million inhabitants.Due to its popularity in Henan as well as other provinces in China, meat -including raw meat and cooked meat products -accounted for the largest proportion of samples collected in this study.Isolation of Salmonella was performed using standard procedures described in the National Standard of the People's Republic of China (GB/T 4789. .Briefly, a rinse was performed by adding 25 g of food sample to 225 mL of buffer peptone water (BPW; Huankai Ltd., Guangzhou, Guangdong, China) in sterile lateral filter bags with thorough mixing by a homogenizer (BagMixer lab blender 400; Interscience, Saint-Nom-La-Breteche, France) and incubated at 37°C for 18 hours as pre-enrichment for Salmonella.Then 1 mL of the pre-enriched sample was inoculated into 10 mL of selenite cystine broth (SC; Huankai) and tatrathionate broth base (TTB; Huankai).Samples were incubated for 24 hours at 37°C (SC) and at 42°C (TTB), respectively.A loop of inoculum was streaked onto bismuth sulfite agar (BS; Huankai) and hektoen enteric agar (HE; Huankai) and incubated for 24 hours at 37°C.The presumptive isolates were picked from each plate and identified using the API 20E bacterial identification system (BioMerieux, Marcy l'Etoile, France).

Serotyping
Salmonella isolates were serotyped by the slide agglutination method using O and H antisera (Difco, Detroit, USA), according to the manufacturer's instructions.

Conjugation experiments
Conjugation was performed by the filter mating method as described previously [16].Briefly, donor and recipient cells (1:1) were mixed in Luria-Bertani broth (LB; Huankai).The mixture was then collected on a sterilized 0.45 µm-pore-size filter and incubated on a blood agar (BA) plate at 37°C overnight.The mating mixture was washed from the filter and spread onto BA plates containing rifampicin (512 µg/mL) and streptomycin (50 µg/mL).Transconjugants were confirmed to be E. coli by the API test and the class 1 integron was detected by PCR using primers shown above.

Results
A total of 62 Salmonella isolates were isolated from various food samples examined in this study.The overall isolation rate of Salmonella in the retail foods was 9.7%.The distribution of the isolates from a variety of food products is presented in Table 1.The Salmonella isolates were found most frequently in beef (16.8%), followed by pork (15.2%).Sixteen different serovars were identified among the 62 isolates, and one isolate from pork sample was untypable (Table 2).The top five serovars were S. Anatum (n = 14, 22.6%), S. Senftenberg (n = 11, 17.7%), S. Derby (n = 8, 12.9%), S. Enteritidis (n = 5, 8.1%), and S. Choleraesuis (n = 4, 6.4%).
Class 1 integrons were detected in 10 Salmonella isolates, only one of which was negative for the resistance gene cassette (Table 4).The remaining nine intI1-positive isolates contained three groups of resistance gene cassette, consisting of dfrA12-aadA2 (2.0 k, n = 5), dfrA1-aadA1 (1.5 k, n = 3), and dfrA1 (1.2 k, n = 1).Three Salmonella isolates could transfer their integrons and resistance genes to E. coli by conjugation.The results indicated the transconjugants obtained had a plasmid carrying the class 1 integron and the transfer frequency was in the range of 2.4×10 -6 to 8.0×10 -5 transconjugant per recipient cell.

Discussion
In the current study, we examined Salmonella isolates recovered from retail food purchased in Henan province.The prevalence of Salmonella in food products of other provinces in China was 3.5% in Jiangsu, 20.9% in Hebei, and 36.8% in Shaanxi [7,11,17].Our results not only provide information about the prevalence of foodborne Salmonella in Henan, but also provide a better understanding of the differences in contamination by Salmonella among the different provinces in China.Previous studies showed that the prevalence of Salmonella in chicken was highest [11], while in our study, the isolates from beef samples constituted the largest proportion.However, it was difficult to compare the data on the prevalence of Salmonella in different studies, because the prevalence may be affected by diversity in sampling methods, sampling seasons, and isolation procedures.Moreover, we also found that the level of Salmonella contamination in cooked meat products was much higher (9.0%).Given that these products are ready-toeat foods that are consumed without further cooking or processing, they may be an increasing cause of enteric diseases [18].
Among these isolates, S. Anatum and S. Senftenberg were most common; however, S. Senftenberg was not found in the previous report about the occurrence of Salmonella serovars isolated from food in Henan province [19].In the same area, the distribution of serovars may be varied due to the different food types collected.S. Derby and S. Enteritidis were commonly identified serovars in the present study.Furthermore, the two serovars were also frequently observed in clinical Salmonella in Henan [20], suggesting an association between Salmonellacontaminated food and salmonellosis.In China, the predominant serovar of foodborne Salmonella was S. Derby, while S. Typhimurium was the main serovar isolated from humans [21].The difference in dominant serovars between foodborne and clinical isolates may be due to differences in pathogenicity and resistance profiles of the two serovars [22].Compared to the previous studies conducted in Henan as well as other provinces in China, Salmonella isolates recovered from retail food in our study showed a higher resistance to trimethoprim/sulfamethoxazole and ciprofloxacin [11,19].Almost all the isolates were resistant to at least one antimicrobial, and nearly 35.0% were MDR isolates.The high prevalence of antimicrobial resistance from retail food was also reported by other studies [12,23].The use of antimicrobials in food animals for disease treatment and growth promotion may potentially lead to the emergence of antimicrobial-resistant pathogens [24].The increasing prevalence of resistant Salmonella in China and other countries presents an enormous challenge to the treatment of Salmonella infections in humans and animals [9,11,20,25].
All intI1-positive isolates were MDR strains, which supported the hypothesis of an association between the presence of class 1 integrons and emerging MDR in Salmonella [10].Our results showed that three Salmonella isolates could transfer their integrons and resistance genes to E. coli by conjugation.Therefore, it is concluded that the class 1 integron was located on a conjugative plasmid in these isolates.Previous reports have also suggested that most of the resistance determinants and class 1 integrons in Salmonella isolates were encoded in a transferable plasmid, which might be transferred to the same or different bacterial species by conjugation [23].

Conclusion
Our study illustrates a potential public health risk of Salmonella in Henan because of its presence in various food items, particularly in raw meats and cooked meat products.Efforts that include further implementation of hazard analysis of critical control point programs in food production are needed to reduce the incidence of Salmonella in food.The high rate of MDR Salmonella isolates and the presence of integrons in this study suggest that effective measures should be taken to facilitate the reasonable use of antimicrobials in both human and veterinary medicine.The monitoring of antimicrobial resistance among foodborne Salmonella is important because the resistance determinants could be spread from food products to humans by transferable elements.

Table 1 .
Prevalence of Salmonella in food samples collected in Henan province of China

Table 2 .
Distribution of Salmonella serovars in retail food

Table 3 .
Antimicrobial resistance of Salmonella isolated from retail food