Antimicrobial Susceptibilities of Salmonella spp. Isolated from Chicken Meat Samples

F. Kaynak Onurda?1， B. Er2， S. ?zgen1， B. Demirhan2， U. Abbaso?lu1 and A. Bayhan ?ktem2

1. Department of Pharmaceutical Microbiology， Faculty of Pharmacy， Gazi University， Ankara 06330， Turkey

2. Department of Food Analysis， Faculty of Pharmacy， Gazi University， Ankara 06330， Turkey

Received: April 28， 2011 / Published: July 20， 2011.

Abstract: The aim of this study was to determine presence of Salmonella spp. in chicken meat samples collected from Ankara， Turkey and determine the susceptibility of the Salmonella isolates to some antimicrobial agents. For this purpose， 127 chicken samples were collected from local markets. Investigation of Salmonella was done according to horizontal method， the guidelines of the method recommended by International Standards Organization (ISO). Antimicrobial susceptibilities of Salmonella spp. was performed with microdilution method according to the guidelines of CLSI M100-S18. Ampicillin， gentamicin sulphate， ofloxacin， levofloxacin， ciprofloxacin， enrofloxacin， tetracycline， ceftriaxon， amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole were used in the study. Salmonella spp. was isolated from 5 (3.94%) of the 127 chicken meat samples. Although our findings are not within Turkish Food Codex (TFC) values， lower number of samples that involve Salmonella spp. indicates an improvement in the hygienic conditions in Turkey. Among 5 isolates， one isolate was sensitive to all antimicrobial agents tested. 2 isolates exhibited multidrug resistance. Successfully， all the isolates were sensitive to quinolones as a good result in spite of the reported reduced susceptibility from different regions of the world. However， this study should be improved with more chicken samples and Salmonella spp. isolate numbers to support these results.

Key words: Salmonella， chicken meat， susceptibility， antimicrobial， minimum inhibitory concentration.

1. Introduction

Diseases resulted from food borne pathogens is an important threat for public health， in many parts of the earth [1] and infections caused by Salmonella are among the most zoonotic diseases worldwide [2]. Contaminated chicken meat is a risk of human gastroenteritis because of the possibility of containing Salmonella spp. [3].

In developing countries， chicken meat is consumed as much as red meat. Because of the low price of chicken meat， the change in the nutrition habits and the lower content of fat in chicken meat， consumption of chicken meat is increasing in Turkey [4].

Salmonella often causes a self limiting gastroenteritis and antimicrobial agents are not essential for the treatment. However， Salmonella can cause severe infections in children， the elderly and immunocomprimised patients. For severe Salmonellosis， treatment is crucial. Although ampicillin， chloramphenicol， and trimethoprim-sulfamethoxazole (TMP-SXT) were frequently used for treatment， today fluoroquinolones are the first choice [5， 6]. The common use of antimicrobial agents for therapeutic and non-therapeutic purposes have mentioned to be a reason for occurence of antibiotic-resistance because of selecting the resistant bacteria [7] and so， subtherapeutic use of these antimicrobial agents in animals is considered to be an impact for gaining resistance among environmental bacteria [8]. An increase in reduced susceptibility of Salmonella strains to antimicrobial agents and recently fluoroquinolones such as ciprofloxacin is a public health problem [9-14].

In our study， it is aimed to determine presence of Salmonella spp. in chicken meat samples collected from Ankara region and determine the susceptibility of the Salmonella isolates to some antimicrobial agents.

2. Materials and Methods

2.1 Samples Collection

In this study， total of 127 chicken samples have been analysed from local markets in different districts of Ankara， Turkey. All samples were carried to the laboratory in cold chain. 2.2 Analysis of Samples

The detection of Salmonella in chicken meats was investigated by the horizontal method recommended by ISO [15].

In isolation process， Buffered Peptone Water were used as pre-enrichment medium， Selenite Cystine Broth (SC)， Rappaport-Vassiliadis (RV) select as enrichment broth and Salmonella-Shigella agar (SS Agar)， and Xylose Lysine Deoxycholate agar (XLD Agar) were used as selective agar.

For detection of Salmonella spp.， a sample of 25 g was weighed under sterile conditions and transferred into 225 mL buffered peptone water. After incubation at 37 °C for 18 ± 2 hours， a volume of 0.1 mL of the preenriched samples were used to inoculate 10 mL of the Rappaport-Vassiliadis medium (RV) and 10 mL of the Selenite-Cystine medium (SC) and they were incubated at 42 °C and 37 °C for 24 ± 3 hours， respectively. After incubation a loopfull of the cultures were transferred to Salmonella-Shigella agar (SS Agar) and Xylose Lysine Deoxycholate agar (XLD Agar) and incubated at 37 °C for 24-27 hours. The typical and atypical colonies that were isolated from the samples were passaged to McConkey agar and the pure colonies that were isolated from McConkey agar plates were used in biochemical tests.

2.3 Antimicrobial Susceptibility Testing

Antimicrobial susceptibilities of Salmonella spp. was performed with microdilution method according to the guidelines of CLSI M100-S18 [16].

Standard strain of Escherichia coli ATCC 25922 and Salmonella isolates were included in the study. Standard powders of ampicillin， gentamicin sulphate， ofloxacin， levofloxacin， ciprofloxacin， enrofloxacin， tetracycline， ceftriaxon， amoxicillin/clavulanic acid and trimethoprim/sulfamethoxazole were used in the study.

Standard antibiotic solutions were dissolved in appropriate solvents recommended by CLSI guidelines[16].

Salmonella isolates and E. coli ATCC 25922 were subcultured in Mueller Hinton Agar (MHA) plates and incubated overnight at 37 °C.

Cation-Adjusted Mueller Hinton Broth (CAMHB) was added each well of the microplates. Stock solutions of the tested antibiotics were diluted two-fold in the wells. The bacterial suspensions used for inoculation were prepared at 105 CFU mL-1 by diluting fresh cultures at McFarland 0.5 density (107 CFU mL-1). Suspensions of the bacteria at 105 CFU mL-1 concentrations were inoculated to the twofold-diluted solution of the compounds. There were 104 CFU mL-1 bacteria in the wells after inoculations. 10 μL bacteria inoculum was added to each well of the microplates. Microplates were incubated at 37 °C overnight. After incubation， the lowest concentration of the compounds that completely inhibits macroscopic growth was determined and reported as minimum inhibitory concentrations (MICs).

3. Results and Discussion

Salmonella spp. was isolated from 5 (3.94%) of the 127 samples that were collected from local markets.

Antimicrobial susceptibility of the isolated Salmonella spp. was determined with microdilution method and MIC values are presented in Table 1.

The numbers ranging from 1 to 127 were given to all chicken samples. Existence of Salmonella spp. was observed in 43， 45， 47， 70 and 78 numbered chicken samples. According to MIC breakpoints for Enterobacteriaceae according to CLSI M100-S18(Table 2)， Salmonella isolated from chicken meat was found to be sensitive to all quinolon antibiotics tested in our study.

In our country， several investigators have studied Salmonella spp. presence in chicken meat samples. Usca et al. [17] studied with 50 chicken meat samples and in 30% of the samples， they isolated Salmonella spp. Fidanc? et al. [18] isolated 16 Salmonella spp. from 120 chicken meat samples investigated (13%). Kaynak Onurda? et al [4] studied with 51 chicken meat samples and isolated 17 Salmonella spp. (33.3%). K?l?n? et al [19] isolated 61 Salmonella spp. from 578 chicken samples (10.5%). Telli et al. [20] investigated 200 samples and isolated Salmonella spp. from 13 samples (6.5%). Efe et al. [21] studied with 50 samples of chicken meat which were bought for the Turkish Military Forces， frozen at -18 °C and reported that in 26% of the chicken skin， 18% of the chicken thigh and 16% of the chicken thorax samples， Salmonella spp. were detected [21]. In our study， we isolated 5 Salmonella spp. from 127 chicken meat samples(3.94%).

In foreign countries different ratios in Salmonella spp. were detected in chicken meat samples. Hue et al.[22] reported 12 Salmonella positive samples of the 219 chicken meat samples in France. Kozacinski et al.[23] isolated Salmonella from 10.60% of the 66 chicken meat samples in Croatia. Minami et al. [24] determined Salmonella spp. in 17 of the 34 chicken meat samples in Thailand. These ratios are also higher than our study results.

Table 1 MIC values of antimicrobial agents on Salmonella strains.

Table 2 MIC breakpoints for Enterobacteriaceae according to CLSI M100-S18.

According to Turkish Food Codex (TFC) in 25 gr of the chicken meat samples Salmonella should not be isolated [25]. So， although the percentage of Salmonella spp. presence in our study is lower than the other studies， this finding is not within the TFC values. Furthermore， this indicates an improvement in the hygienic conditions in Turkey.

After susceptibility testing， among 5 isolates， one isolate was determined to be sensitive to all antimicrobial agents tested and all isolates were sensitive to quinolones. 2 isolates exhibited resistance to at least three antimicrobial agents (multidrug resistance). MIC values of the antimicrobial agents are shown in Table 1.

Drug resistance in food borne pathogens may be a result of antimicrobial drug use in food animals and because of this， zoonotic microorganisms has become a public health issue with the possibility of transmitting the resistance genes to humans via food chain [1].

Fashae et al. [6] determined that Salmonella isolates from chickens were more commonly resistant to tetracycline， sulfamethoxazole， nalidixic acid， and trimethoprim and reported that this resistance may be attributed to indiscriminate use of antibiotics at recommended doses or at subtherapeutic doses as feed additives to promote growth， and as chemotherapeutic agents to control epizootics on the farms. They have also reported that among the isolated Salmonella spp. from fecal samples， none of the chicken isolates showed high levels of resistance to ciprofloxacin (MIC≥ 2 μg/mL); however， 87% of the isolates exhibited reduced susceptibility to ciprofloxacin [6].

In our study one of the isolates (20%) was determined to be resistant to ampicillin. K?l?n? et al and Fidanc? et al [18] have determined 20% and 75% of the isolated Salmonella strains to be resistant to ampicillin， while Oliveira et al. [26] from Brasil and Carraminana et al [27] from Spain have reported this ratio as 1.1% and 0% of the isolated Salmonella spp. respectively. From China， Yan et al. [28] also reported a high ratio(47.4%) of ampicillin resistance of Salmonella in retail foods. Our results are thought to be within K?l?n? et al.[19] in spite of the lower isolate number.

Soltan et al. [29] found that 69% of the isolates were resistant to tetracycline while， Oliviera et al. [26] and Carraminana et al. [27] determined a resistance ratio of 15.4% and 21.8% to tetracycline， respectively. Yan et al. [28] determined that 47.4% of the Salmonella spp. isolated from retail foods was resistant to tetracycline. Oral et al. [30] from Turkey， reported that none of the isolates were resistant to oxytetracycline. In our study all of the isolates were sensitive to tetracycline. This results indicates that resistance ratios may differ from one region to another so although the number of isolated Salmonella spp. is lower than the other studies， our study findings is thought to be in accordance with Oral et al. [30]. Different from these results， Fidanc? et al. [18] and Y?ld?r?m et al. [31] reported high ratios(75% and 67.6% respectively) of tetracycline resistance.

Oral et al. [30] determined 21.2% resistance in Salmonella isolates for gentamicin， while in our study 2 of the 5 Salmonella isolates (40%) were determined to be resistant to gentamicin. However， Fidanc? et al.[18] determined that 15 of the isolated 16 Salmonella spp. were resistant to gentamicin. In China， Yan et al.[28] reported a ratio of 31.6% gentamicin resistance in Salmonella spp. In Brasil， Oliviera et al. [26] and in Spain Carraminana et al. [27] reported lower ratios(5.5% and 0.7% respectively) than these results.

Yan et al. [28] determined antimicrobial resistance of Salmonella spp. with a ratio of 10.5%， 10.5%， and 57.9% for ceftriaxon， amoxicillin/clavulanic acid and TMP-SXT respectively. Carraminana et al. [27] reported that 2 of the 133 Salmonella spp. isolates were resistant to amoxicillin/clavulanic acid; however， surprisingly none of the isolates were determined to be resistant to TMP-SXT. In our study， one of the isolates(20%) was resistant and one of the isolates was intermediate susceptible to ceftriaxon. Three of our Salmonella isolates were resistant to amoxicillin/clavulanic acid and two of our isolates were resistant to TMP-SXT.

Although the ratios differ from one region to another， all over the world， resistance to other antimicrobial agents makes quinolones to be the first choice antibiotics for Salmonella infections. However， in many studies reduced susceptibility to ciprofloxacin have been reported [6， 28].

4. Conclusion

In this study， for this purpose， susceptibility of Salmonella to quinolones that are known to be used in chickens for growth promotion has also been studied and successfully， all the isolates were sensitive to quinolones as a good result. However， this study should be improved with more chicken samples and Salmonella spp. isolate numbers to support these results.

Acknowledgment

This study was supported by a grant from Gazi University Research Foundation (Project No: 02/2010-24).

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