Incidence and antibiotic susceptibilities of Yersinia enterocolitica and other Yersinia species recovered from meat and chicken in Tehran , Iran

Department of Medical Laboratory Sciences, School of Paramedicine, Tehran University of Medical Sciences. Tehran, Iran. Zonotic research centre. Tehran University of Medical Sciences, Tehran, Iran. Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Antibiotic resistant research center, Tehran University of Medical Sciences, Tehran, Iran. National Research Department of Foodborne Diseases (NRDFD), Research Center of Gastroenterology and Liver Diseases, Shaheed Beheshti University of Medical Sciences, Tehran, Iran.

needs long period to grow (Fredriksson-Ahomaa and Hannu Korkeala, 2003).Y. enterocolitica is known as a psychotropic waterborne and foodborne enteropathogen.This microorganism can grow to large numbers at refrigeration temperatures, so meat, chicken, milk, cheese contaminated with that organism could become a significant health risk for consumers (Black and Jackson, 1978;Stern and Pierson, 1979;Soltan-Dallal et al., 2004;Hudson et al., 2008).This microorganism is primarily a gastrointestinal tract pathogen with a strong propensity for extra intestinal spread under defined host conditions (Soltan-Dallal and Moezardalan, 2004a).It causes a broad range of diseases from acute bowel disease to extra intestinal manifestation such as reactive arthritis , uveitis and sepsis (Jacobs et al., 1989;Chandler and Parisi, 1994).Systemic and extraintestinal infections and enterocolitis in immune-compromised patients require antibiotic therapy, and the agents used most commonly include chloramphenicol, gentamicin, tetracycline, cotrimoxazole and ciprofloxacin (Butler, 1990;Hoog, 1987).Although there is data concerning the incidence of Y. enterocolitica and related species in foods are reported in some countries (Fredriksson-Ahomaa and Hannu, 2003;Siriken, 2002;Dominique et al., 1981;Soltan and Moezardalan, 2004b), but compared with other bacterial enteropathogenes, there are a few studies about the antimicrobial susceptibility of Yersinia spp, which are isolated from staff foods and human.The aims of this study are to investigate the prevalence of Y. enterocolitica and other Yersinia species in meat and chicken samples in different seasons and to determine the antimicrobial susceptibility pattern of Yersinia entrocolitica and other Yersinia species isolated from meat and chicken in Tehran, Iran.

Sample collection
189 pieces of beef meat and 190 pieces chicken meat were purchased from 28 different local butcher's shops and supermarkets in Tehran, Iran, and were examined for the presence of Yersinia species between April 2007 and April 2008.

Cold enrichment, Bacterial isolation and identification
25 g of each sample was added to plastic bag containing 10 ml phosphate buffered saline (pH 7.2) and homogenized by Stomacher (400 circulator Seward-England) and incubated at 4°C for one, two, three and four weeks.At weekly intervals, a suspension aliquot was cultured linearly on Cefsulodin Irgasan Novobiocin Agar (CIN) (Merck-Germany) with Yersinia selective supplement.Cultured plates were incubated at 26°C, between 24 -48 h.The colonies were considered macroscopically and microscopically after incubation.Suspected colonies were examined to motility test by SIM medium both at 25 and 37°C.In addition, biochemical test including urease activity, KIA and Simon citrate were used for the bacterial identification (Johnson, 1998).

Antimicrobial susceptibility
Susceptibility testing of bacterial strains was achieved at 28˚C on Mueller-Hinton agar (Merck-Germany) by the agar diffusion method.

Data analysis
Statistical analysis of results was performed with SPSS/PC 12 software (SPSS Chicago, IL).A P value<0.05 was used for statistical significance.
Out of 375 pieces including 189 beef meat and 190 chicken meat samples, 16% (n=60) Yersinia species were isolated (Table 1).70% (n=42) of isolates were achieved from chicken meat and 30% (n=18) from beef meat.The prevalence of Y. enterocolitica with the most incidence was 80% (n=48), that were found in chicken meat and beef meat with rate of 62.5% (n=30) and 37.5% (n=18) respectively.The occurrence of Y. enterocolitica was slightly higher in chicken than meat.The frequency of the other Yersinia spp.including Y. frederiksenii, Y. intermedia and Y. kristensenii were 11.6% (n=7), 0.06% (n=4), and 0.01% (n=1).Except for Y. frederiksenii (n=1), none of Y. intermedia and Y. kristensen were not found in meat (Table 2).As we stated above, all of samples were cultured at the end of week for four weeks.The highest isolation rate was 33, 75 and 42% in 4 ▫ C/3-week enrichment for Y. entrocolitica, Y. intermedia and Y. frederiksenii respectively.The only isolated Y. kristensenii accrued in 2-week enrichment.The rates of Yersinia isolated in various seasons were 20, 16 and 13% in November, April and March respectively.The prevalence of Yersinia entrocolitica and other species in various month was mentioned in Table 3.
All isolates were subjected to antimicrobial resistance testing by the disc diffusion method.An overview of the antibiotic susceptibility of the strains is shown in Table 4.Our results indicated that among Y. entrocolitica, the sensitive rate for gentamicin and chloramphenicol was 97% (n=46) and for trimethoprim and ciprofloxacin was  In total, 15/8% (n=60) of this isolates exhibited a resistant phenotype, and most of these strains showed resistance to more than one antimicrobial test.34 (56%) isolates were resistant to two or more antibiotic test The highest co-resistance rate was seen for the ampicillin, cephalotin with rate of 28% (n=17), followed by 11% (n=7) for ampicilin, nalidixic acid and cephalotin.Four isolates (6%) were resistant to five antibiotics including ampicillin, nalidixic acid, cephalotin, tetracycline and streptomycin.

DISCUSSION
Yersinia spp. was reported from 9 to 99.5 percent in different sources such as meat, grandbeef, pork, environment, water and human (Kapperud, 1977;Ostroff, 1995;Soltan, 2001;Okwori et al., 2005).In our study, we analyzed two kinds of meat, chicken meat and beef meat, which are broadly consumed in Iran.Our results indicated Y. entrocolitica has a high incidence among of other Yersinia spp in meat and chicken.Similar findings been achieved in Mexico (Ramirez and Vazquez-Salinas;2000).The prevalence of Y. frederiksenii (11.6%) in present study was similar to results of Capita (Capita et al., 2002).Ibraham examined 50 beef samples for Yersinia spp. in Turkey and the isolation rate was 20% including of 9 (18%) Y. enterocolitica and 2 (4%) Y. frederiksenii (Ibraham and Mac Rae, 1991).Also in another study in Spain, Y. enterocolitica and Y. frederiksenii were detected in 22 (55%) and 6 (15%) samples, respectively (Capita et al., 2002).The various results between these different studies and our study probably can be due to several factors such as isolation methods, season of isolation and geographical location regarding to various temperatures and number of analyzed samples.These factors play an important role in isolation of the Yersinia spp.For instance, in this study the most of Yersinia spp.isolated in cold months such as November, April and December.It is known that the isolation rate of Y. enterocolitica is higher in cold climates (Warnken and Nunes, 1980).According present finding the highest rate of Yersinia spp, exception of Y. kristensenii, was isolated in 4 ▫ C/3-week enrichment (Jiang and Kang, 2000).
Although Y. entrocolitica has been known as a most principal cause of human infections, however there are some reports about coursing gastroenteritis in human by other species such as Y. intermedia, Y. kristensen and Y. frederiksenii (Brenner and Bercovier, 1980;Hamama and Marrakchi, 1992).Strains of these pathogenic biotypes contain marker associated with virulence and these are located on the chromosome (ail) and on the virulence plasmid (PYV) (Goverde et al., 1993;Khorramizadeh et al., 2007).In USA the Center for Diseases Control and Prevention (CDC, 1982), conducts investigations of outbreaks of yersiniosis to control them and to learn more about how to prevent these infections.According with others authors, it can be assumed that further clinical studies are needed to assess the epidemiological importance, the occurrence and the possible etiological relevance of Y. enterocolitica (Ramirez and Vazquez-Salinas, 2000;Hoffmann et al., 2002).
Therefore it seems the investigation of susceptibility pattern and antibiotic resistance can be necessary for treatment of strains clinically.Antimicrobial resistance in foodborne pathogens and therapeutical intervention has always been an important issue in public health (Soltan et al., 2010).Using of antimicrobial agent in veterinary as a growth promotion, treatment or prophylactic can develop the antibiotic resistance in food animals.It can be a reason for antibiotic resistance transfer to humans via the food chain (Mayrhofer and Paulsen, 2004;Ezekiel et al., 2011).According to our results, the majority of isolates were resistant to cephalothine that may be due to presence of Enzyme B. The first report about B lactamase production by Y. entrocolitica was in 1973 (Cornelis et al., 1973).Soon afterwards, the presence of two types of chromosomal β-lactamase was described in some clinical isolates of Y. enterocolitica, a non-inducible broad-spectrum β-lactamase, enzyme A, and an inducible cephalosporinase, enzyme B (Cornelis and Abraham, 1975).The presence of two types of chromosomal B-lactamase was described in some clinical isolates of Y. entrocolitica, Enzyme B as Inducible cephalosporinase is one of them (Jeannette et al., 2000;Pham and Bell, 2000) The antibiotic susceptibility of Y. enterocolitica to gentamicin and chloramphenicol, ciprofloxacin and streptomycin are similar to reports of Okwori et al. (2005), who documented sensitivity of Y. enterocolitica strains of animal origin to ciprofloxacin and floxavid.The results indicated most active pharmacologic agents were chloramphenicol, gentamicin and trimethoprim.Regarding to high sensitivity of Yersinia spp. to gentamicin and chloramphenicol, these agents should be effective in the treatment of Yersinia spp when clinically indicated.It is suggested from results that the presence of Yersinia in beef meat and chicken meat represent a health risk for consumers.The education of people who involve in production, processing and final preparation of animal products is required to avoid crosscontamination.

Table 1 .
Percentage number of contaminated samples.

Table 2 .
Percentage distribution of Yersinia spp in tested samples.

Table 3 .
Frequency distribution of Yersinia Spp in different months of year.

Table 4 .
Antibiotic resistance patterns of isolated Yersinia spp.