Prevalence and antimicrobial resistance of Salmonella serotypes isolated from retail chicken meat and giblets in Iran

Introduction: Salmonella is one of the major foodborne pathogens responsible for outbreaks of foodborne illness in humans worldwide. Methodology: A total of 560 samples of chicken meat and giblets were collected from retail markets for Salmonella identification, serotyping, and antimicrobial resistance testing. Results: Salmonella was detected in 19.8% of samples. Among the five serotypes identified, S. Thompson was the predominant type (48.7%). High antimicrobial resistance rates were observed to nalidixic acid (92.8%), tetracycline (81%), trimethoprim (68.4%), sulfamethoxazole / trimethoprim (61.2%), streptomycin (56.7%), and kanamycin (36.9%). Although resistance to chloramphenicol (3.6%), amoxicillinclavulanic acid (5.4%), and ampicillin (11.7%) was detected, none of the isolates were resistant to ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, colistin, gentamicin, nor imipenem. Conclusions: Restrictions on the irrational use of antibiotics in humans and animals are suggested for the reduction of resistant strains.


Introduction
Salmonella is one of the major foodborne pathogens worldwide [1,2] and is responsible for outbreaks of foodborne illness in humans via crosscontamination and consumption of undercooked meats [3,4].It has been estimated that Salmonella has caused approximately one million cases of foodborne illness and 378 deaths per year in the United States [5].Salmonella spp. is also the most commonly reported cause of foodborne outbreaks (39.2%) in the European Union (EU), with 2,201 outbreaks in 2007, of which 142 occurred in France [6].High percentages (approximately 70%-80%) of foodborne bacterial outbreaks in China were caused by Salmonella [7].Although other food products, such as eggs and red meats, can be contaminated with Salmonella spp., poultry products have been recognized to be the main source of this pathogen in humans [8].Contamination of poultry meat can occur at different stages, including production, slaughter, processing, handling, and storage [9].Antimicrobial agents are widely use in veterinary medicine, not only for prevention and treatment of diseases, but also as growth-promoting substances.However, the widespread use of antibiotics promotes the development of antimicrobial-resistant bacteria, potentially worldwide [10].The prevalence of Salmonella serotypes and their antimicrobial resistance in chicken meat and giblets have been reported in many investigations around the world [11][12][13][14][15][16][17][18][19][20].Despite considerable of progress in human public health, Salmonella still remains a significant foodborne pathogen in the food chain.Based on the importance of this pathogen in human public health, this study was carried out to determine the prevalence and antimicrobial resistance of Salmonella strains isolated from retail chicken meat and giblets in Alborz Province, Iran.

Methodology
A total of 560 samples of retail chicken meat (200) and giblets, which included liver (120), gizzard (120), and heart (120), were purchased from retail markets in Alborz Province between October 2013 and March 2014.All samples were transferred under cold conditions (using icepacks) to the laboratory for microbial examination.For Salmonella identification, 25g of each meat sample was homogenized for 2 minutes with 225 mL of buffered peptone water (Merck, Darmstadt, Germany) and then incubated for 24 hours at 37°C.After incubation, 0.1 mL of the broth was transferred into 10 mL of selenite cystine broth (Merck) and incubated at 42°C for 24 hours.The enrichment samples were then subcultured on to Salmonella-Shigella agar and brilliant green agar (both Merck) and incubated for 24 hours at 37°C.Presumptive Salmonella isolates were identified by using triple sugar iron agar, lysine iron agar, and urea agar (all Merck) at 37°C.

Serotyping
Salmonella isolates were further serotyped by direct agglutination method using antisera against O and H antigens (BioRad, Marnes-la-Coquette, France) and the Kauffman-White classification schema [21].

Results and Discussion
Of 560 examined samples, 111 (19.8%) were contaminated with Salmonella spp.Table 1 shows the prevalence of Salmonella spp.isolated from examined samples.The incidence of Salmonella spp. in chicken products obtained by other authors varied between 0 and 100% [23,24].In this study, the overall prevalence of Salmonella in chicken meat and giblets was 19.8% (111/560).The contamination rate of chicken meat samples (29%) is in agreement with results reported in Belgium [25], the United Kingdom [26], Iran [27],China [28], Turkey [29], Iraq [30], and the Russian Federation [31], but lower than results found in Iran [20,32] and many other countries, such as China, Mexico, and Poland [16,18,[33][34][35].The incidence of Salmonella spp. was found to be 21.6% in chicken liver samples, 14.1% in heart samples, and 8.3% in gizzard samples.In a previous study in Iran, the incidence of Salmonella spp.contamination in chicken liver, heart, and gizzard was found to be 18%, 6%, and 4%, respectively [36], which was lower than that found in the present study.However, contamination levels higher than those in this study were reported from Egypt and Ethiopia at incidence rates of 40% and 48%, respectively, in chicken livers, and 34.5% and 23.7%, respectively, in chicken hearts [37,11].The reasons for the higher incidence of Salmonella contamination in chicken meat than in giblets in the present study can be due to the defeathering process, which may spread microorganisms between carcasses [37].All previous studies showed that Salmonella was widely distributed in retail meat globally, which increased salmonellosis following consumption of contaminated meat or crosscontamination in households.This risk may be higher if chicken meat or giblets are consumed undercooked [38,25].Data on the prevalence of Salmonella in different studies were difficult to compare because the observed prevalence may be biased by diversity in sampling methods, sampling seasons, and techniques [28].However, variations observed between the reported Salmonella prevalence in previous investigations around the world may be due to several factors, including the initial salmonellosis in live birds, sanitation within the slaughterhouse, possible contamination during poultry processing steps (e.g., the amount of cross-contamination of chicken carcasses by contact with intestinal tracts during slaughter or processing), and differences among isolation methods applied to detect Salmonella [36].Among the 111 positive isolates found in the present study, five different serotypes (S.Thompson, S. Enteritidis, S. Typhimurium, S. Newport, and S. Hadar) were completely identified in 107 samples.S. Thompson (48.7%) was the serotype most frequently detected, followed by S. Enteritidis (22.5%), S. Typhimurium (12.6%), S. Newport (7.2%), and S. Hadar (5.4%).Four isolates (3.6%) were not completely serotyped (Table 2).S. Thompson was the predominant serotype in uncooked chicken and beef in Tehran.It is one of the most important Salmonella serotypes that can cause outbreaks and infection in poultry [32].In the United States, the Centers for Disease Control and Prevention (CDC) has reported that S. Enteritidis, S. Typhimurium, and S. Newport are the most prevalent serotypes reported by public health laboratories [39].All these serotypes are common isolates from chicken meat.Salmonella serotypes detected in this study are in agreement with CDC report.Previous studies have reported some of the serotypes that were identified in this study [13,14,16,20,32,36].This distribution of Salmonella serotypes could vary from country to country.Among the Salmonella serotypes, S. Enteritidisand, S. Typhimuriumare the predominant serotypes found in human salmonellosis in many developed countries, including Japan, Australia, New Zealand, and many countries in Europe [40].There are reports of human salmonellosis caused by consumption of poultry meat contaminated by S. Typhimurium [41].Although S. Typhimurium is the most common agent of human foodborne disease, in the last few decades, S. Enteritidis has become more common [42].
The increase in the prevalence of resistant microorganisms is an important problem for the treatment and prevention of infectious diseases in both humans and animals [2].Antimicrobial resistance in Salmonella serotypes has been a global problem, with rates increasing from between 20% and 30% in the early 1990s to as high as 70% in some countries at the turn of the century [43,44].The results of antimicrobial resistance tests of 111 Salmonella isolates to 16 antimicrobials are shown in Table 3.
These findings confirm that in Iran, poultry is a major reservoir of multidrug-resistant Salmonella, and suggest that it is difficult to achieve successful antimicrobial therapy for human salmonellosis caused by strains of poultry origin.Resistance to two or more antibiotics was found in 26 isolates of S. Thompson, followed by 17, 8, 8, 6, and 4 isolates of S. Enteritdis, S. Typhimurium, S. Newport, S. Hadar, and not completely serotyped serotypes, respectively (Table 4).The high prevalence of antimicrobial resistance identified in the present study can be explained by the widespread use of common antimicrobials as prophylactics, growth promoter agents, or in veterinary medicine.Furthermore, unlimited access to these agents without a prescription as well as low rates of antibiotic sensitivity tests for the selection of suitable drugs in Iran may be additional reasons for the high level of resistance.

Conclusions
The results of this study suggest that improving sanitation in poultry slaughterhouses could decrease Salmonella contamination and, as a result, human disease.Increasingly antibiotic-resistant strains constitute a public health hazard through transmission of these strains to humans via food products.Therefore, conducting antibiotic sensitivity tests and establishing a regular monitoring system for identification of resistance prevalence in food is necessary to reduce the spread of resistant strains.

Table 1 .
Prevalence of Salmonella isolated from chicken meat and giblets

Table 2 .
Distribution of Salmonella serotypes in chicken meat and giblets *Not completely serotyped

Table 3 .
Prevalence of antimicrobial resistance of Salmonella serotypes isolated from retail chicken meat and giblets