11 Uludag Univ. J. Fac. Vet. Med. 31 (2012), 2: 11-16 Detection of Antibiotic Resistance in Staphylococcus aureus Strains Isolated from Various Foods Figen ÇETİNKAYA1 Tülay ELAL MUS1 Geliş Tarihi: 26.11.2012 Kabul Tarihi: 26.12.2012 Abstract: This study was performed to determine the antibiotic resistance patterns of 27 Staphylococcus aureus strains, isolated from different foodstuffs, to a range of antibiotics including methicillin, penicillin, ampicillin, cefotaxime, teicoplanin, gentamicin, kanamycin, erythromycin, tetracycline, ciprofloxacin, clindamycin, chlo- ramphenicol, quinupristin/dalfopristin and linezolid. Antibiotic susceptibility testing revealed that 88.9% (24/27) of the strains was resistant to at least one of the antibiotics. Most of S. aureus strains showed the resistance to penicillin (62.9%) and ampicillin (59.3%). For other antibiotics, the resistance rates were 22.2% for erythromy- cin, 11.1% for cefotaxime, kanamycin and tetracycline, and 7.4% for methicillin and clindamycin. Moreover, 18 of the strains had resistance to multiple antibiotics. So that, 12 strains exhibited resistance to two antibiotics, four strains to three antibiotics, one strain to four antibiotics, and one strain to six antibiotics. However, all the strains were found to be susceptible to teicoplanin, gentamicin, chloramphenicol and linezolid. Key Words: Staphylococcus aureus, food, contamination, antibiotic resistance. Çeşitli Gıdalardan İzole Edilen Staphylococcus aureus Suşlarında Antibiyotik Dirençliliğinin Belirlenmesi Özet: Bu çalışma, çeşitli gıda maddelerinden izole edilen 27 adet S. aureus suşunun, metisilin, penisilin, ampisi- lin, sefotaksim, teikoplanin, gentamisin, kanamisin, eritromisin, tetrasiklin, siprofloksasin, klindamisin, kloram- fenikol, kinopristin/dalfopristin ve linezolid gibi antibiyotiklere dirençlilik özelliklerini belirlemek amacıyla gerçekleştirildi. Antibiyotik duyarlılık testi, suşlardan %88.9’unun (24/27) en az bir antibiyotiğe dirençli oldu- ğunu ortaya koydu. S. aureus suşlarının çoğunluğu penisilin (%62.9) ve ampisiline (%59.3) dirençlilik gösterdi. Diğer antibiyotikler bakımından dirençlilik oranları, eritromisin için %22.2, sefotaksim, kanamisin ve tetrasiklin için %11.1, metisilin ve klindamisin için %7.4 idi. Üstelik, 18 suş birden fazla antibiyotiğe karşı dirençliliğe sahipti. Öyleki, suşlardan 12’si iki antibiyotiğe, 4’ü üç antibiyotiğe, biri dört antibiyotiğe ve bir suş da altı anti- biyotiğe direnç gösterdi. Bununla birlikte, tüm suşların teikoplanin, gentamisin, kloramfenikol ve linezolid’e duyarlı olduğu bulundu. Anahtar Kelimeler: Staphylococcus aureus, gıda, kontaminasyon, antibiyotik dirençliliği. 1 Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Uludag University, Bursa, Turkey, fcetinkaya@uludag.edu.tr 1 2 Introduction Materials and Methods Staphylococci are ubiquitous Gram- Bacterial strains and culture conditions positive bacteria that represent part of the nor- A total of 27 S. aureus strains that had mal bacterial microflora of the skin and mucosal 21 been isolated from various foodstuffs were used surfaces of humans and animals . Staphylococ- in the present study. The origins of the strains cus aureus is widely recognised as the causative used are shown in Table 1. Working stock cul- pathogen of various infections, ranging from tures were kept frozen at -20°C in a 20% glyc- infections of the skin and soft tissues, to septi- erol supplemented with Brain-Heart Infusion caemia and endocarditis9. (BHI, Oxoid CM1135) broth. The cultures were Although S. aureus strains can harbour activated in BHI broth at 37°C before use. different virulence genes22, the most notable virulence factors associated with this microor- Table 1. CLSI disk diffusion breakpoints for ganisms are the heat-stable enterotoxins (SEs), S. aureus (CLSI, 2011) and diame- that cause the sporadic food-poisoning syn- ters of the inhibition zone obtained drome or foodborne outbreaks, and the toxic for 27 S. aureus strains in disk dif- shock syndrome toxin 1 (TSST-1), which di- fusion testing minishes the immune response of a colonized host16. It is relevant to consider that the trans- Tablo 1. S. aureus için CLSI disk difüzyon mission of S. aureus is possible either by direct sınır değerleri (CLSI, 2011) ve 27 S. contact with animals or through contaminated aureus suşu için disk difüzyon tes- 6 tinde alınan inhibisyon zon çapları food . Human handling of food products as well as infection/colonization of livestock or farm The range of CLSI workers have been described as mechanisms for Antimicrobial Disk zone diame- breakpoints (mm) the contamination of food with this bacterium3. agents content ters (mm) in this study S I R Among the factors contributing to the Methicillin 5 µg 6-38 ≥14 10-13 ≥9 virulence of this pathogen, antibiotic resistance Penicillin 10 U 9-46 ≥29 - ≥28 also plays an important role22. Antimicrobial Ampicillin 10 µg 8-48 ≥29 - ≥28 resistance is an increasingly serious public Cefotaxime health concern in the worldwide7. The develop- 30 µg 6-37 ≥23 15-22 ≥14 ment of resistance both in human and animal Teicoplanin 30 µg 15-24 ≥14 11-13 ≥10 bacterial pathogens has been associated with the Gentamicin 10 µg 18-26 ≥15 13-14 ≥12 extensive therapeutic use of antimicrobials or Kanamycin 30 µg 11-28 ≥18 14-17 ≥13 with their administration as growth promoters in Erythromycin 15 µg 6-30 ≥23 14-22 ≥13 food animal production1,13. Increasing antimi- Tetracycline 30 µg 10-31 ≥19 15-18 ≥14 crobial resistance and multiple resistance have Ciprofloxacin 5 µg 20-35 ≥21 16-20 ≥15 resulted in increasing difficulties in the treat- Clindamycin 2 µg 9-32 ≥21 15-20 ≥14 ment of bacterial infections5. Among antibiotic Chloram- 30 µg 20-31 ≥18 13-17 ≥12 resistant staphylococci, multidrug-resistant S. phenicol aureus strains are of great public concern since Quinupristin/ 15 µg 18-29 ≥19 16-18 ≥15 resistances make more difficult the treatment of dalfopristin infections21. Worldwide, methicillin-resistant S. Linezolid 30 µg 24-35 ≥21 - ≥20 aureus (MRSA) is a significant and increasing S, susceptible; I, intermediate; R, resistant. cause of nosocomial infections. Cases of illness following the consumption of MRSA- Antibiotic susceptibility testing contaminated food have been reported10,11. The antibiotic susceptibility of the strains The purpose of this work was to deter- was evaluated by the disk diffusion method mine the resistance profiles to various antimi- according to the recommendations of the Clini- crobial agents of 27 S. aureus strains isolated cal and Laboratory Standards Institute 2. Single from food samples including milk and dairy colony of S. aureus picked from fresh cultures products (cheese, kefir), meat products (raw on Tryptone Soya Agar (TSA, Oxoid CM131), meatball, sausage) and seafood (fish, shrimp, incubated aerobically 37°C for 24 h, were inoc- mussel). ulated into Mueller-Hinton Broth (MHB, Oxoid CM405). When broth culture reached the 0.5 13 Table 2. Antibiotic resistance profiles of S. aureus strains isolated from a variety of food Tablo 2. Gıdalardan izole edilen S. aureus suşlarının antibiyotik dirençlilik profilleri Strain no. Source Coagulase activity ME P AMP CTX TEC CN K E TE CIP DA C QD LIN 1 Goat milk Negative S S S S S S S S R S S S S S 2 Goat milk Negative S S S S S S S S S S S S S S 3 Shrimp Positive S R R S S S S S S S S S S S 4 Raw meatball Positive S S S S S S S R S S S S S S 5 Kefir Negative S S S S S S S S S S S S S S 6 Kefir Negative S S S S S S S S R S S S S S 7 Kefir Negative S R R S S S S R S S S S S S 8 Shrimp Positive S R R S S S S I S S S S S S 9 Shrimp Positive S R R S S S S S S S S S S S 10 Fish Positive S R R S S S S S S S S S S S 11 Fish Positive S R R S S S S S S S S S S S 12 Shrimp Positive S R R S S S S I S S S S I S 13 Mussel Negative S R R S S S S S S S S S S S 14 Raw meatball Positive S S S S S S S R S I S S S S 15 Cheese Negative S R R S S S S R S S S S S S 16 Fish Positive S R R S S S S S S S S S S S 17 Cheese Positive S R R S S S S S S S S S S S 18 Shrimp Positive S R R S S S S S S S S S S S 19 Fish Positive S R R S S S S S S S S S S S 20 Cheese Positive S S S R S S S I S S R S S S 22 Raw meatball Positive S R R S S S S R S S S S S S 23 Raw meatball Positive S R R S S S S R S S S S S S 24 Kefir Positive I S S S S S R I I S S S S S 34 Sausage Positive R R S R S S R S R S R S S S 35 Raw meatball Positive R R R R S S S S S S S S S S 36 Raw meatball Positive S S S S S S S S S S S S I S 37 Raw meatball Positive I S S S S S R S S S S S S S ME, methicillin; P, penicillin G; AMP, ampicillin; CTX, cefotaxime; TEC, teicoplanin; CN, gentamicin; K, kanamycin; E, erythromycin; TE, tetracycline; CIP, ciprofloxacin; DA, clindamycin; C, chloramphenicol; QD, quinupristin/dalfopristin; LIN, linezolid; R, resistant,, I, intermediate susceptible, S, susceptible McFarland standard turbidity at 30ºC, each sus- Results pension was spread on the surface of Mueller Hinton Agar (MHA, Oxoid CM337) plates by a Susceptibility testing of the S. aureus sterile ecuvion. Antibiotic disks were placed strains isolated from different foodstuffs to 14 aseptically on the agar surface and plates were antimicrobials was carried out with disk diffu- then incubated at 37ºC for 24 h. After incuba- sion method according to CLSI guidelines. Ta- tion, the results were recorded by measuring the ble 2 summarizes the antibiotic susceptibility inhibition zones, and each organism was classi- profiles of the strains. All of 27 strains were fied as resistant (R), intermediate (I) or suscep- found to be susceptible to teicoplanin, gentami- tible (S) according to the guidelines of CLSI. cin, chloramphenicol and linezolid. The highest Staphylococcus aureus ATCC 25923 was used resistance percentage was obtained for penicil- as a reference strain. Table 1 shows CLSI lin (62.9%), followed by ampicillin (59.3%), breakpoints for disk diffusion susceptibility erythromycin (22.2%), kanamycin (11.1%), testing (zone diameter, mm) of S. aureus. tetracycline (11.1%), cefotaxime (11.1%), The following antimicrobial susceptibility methicillin (7.4%) and clindamycin (7.4%.). test disks were used: methicillin 5 µg (Bioanal- Table 3 shows the numbers and incidence of ysis, Turkey), penicillin 10 U, ampicillin 10 µg, resistant S. aureus strains to antibiotics tested. cefotaxime 30 µg, teicoplanin 30 µg, gentami- Additionally, resistance to multiple antibiotics cin 10 µg, kanamycin 30 µg, erythromycin 15 was observed among the strains. µg, tetracycline 30 µg, ciprofloxacin 5 µg, clindamycin 2 µg, chloramphenicol 30 µg, qui- Discussion nupristin/dalfopristin 15 µg, linezolid 30 µg Due to the intensive use of antibiotics in (Oxoid, Basingstoke, UK). public health and animal husbandry and the risk 14 of transfer of antibiotic resistance determinants, result disagreed with Peles et al.15 and Vazquez- the incidence of antibiotic resistances in food Sanchez et al.24 who reported that all of S. aure- associated pathogenic bacteria including S. au- us strains from bovine milk in Hungary and reus has been an increasing problem during the fishery products in Spain, respectively, were last decades17. susceptible to erythromycin. In the present study, 88.9% (24/27) of In our study, 11.1% (3/27) of S.aureus strains showed antimicrobial resistance proper- strains exhibited resistance pattern to tetracy- ties to at least one of the antibiotics tested (Ta- cline, which is below (from 16.7% to ble 2). However, all 27 S. aureus strains were 82.4%)4,18,23,24 or above (from 0.7% to 6.0%)16,22 susceptible to teicoplanin, gentamicin, chloram- the incidence rates of tetracycline resistance phenicol and linezolid. The sensitivity of S. reported by some other groups. aureus strains to teicoplanin, gentamicin and chloramphenicol was similar to the antibiotic sensitivity of S. aureus isolated from rabbit Table 3. Numbers and incidence of resistant meat by Rodriguez-Calleja et al.18. On the con- S.aureus strains isolated from food- trary, the resistance of S. aureus isolates was stuffs suggested to gentamicin by Pereira et al.16, by Tablo 3. Gıda maddelerinden izole edilen Ezzeldeen et al.4 and by Normanno et al.14 and dirençli S. aureus suşlarının sayıları to chloramphenicol by Vazquez-Sanchez et al.24 ve insidensi and by Umoh and Odoba23. Resistant strains Antibiotics As shown in Table 3, the overall antimi- Number Incidence (%) crobial susceptibility profiles revealed that the Methicillin 2 7.4 highest percentage of resistance among the Penicillin 17 62.9 strains was detected for penicillin (62.9%), fol- Ampicillin 16 59.3 Cefotaxime 3 11.1 lowed by ampicillin (59.3%) and erythromycin Teicoplanin 0 0 (22.2%). The lower resistance rates was ob- Gentamicin 0 0 served for kanamycin, tetracycline and cefotax- Kanamycin 3 11.1 ime (11.1%), methicillin and clindamycin Erythromycin 6 22.2 (7.4%). Several authors have reported resistance Tetracycline 3 11.1 of S. aureus strains obtained from different Ciprofloxacin 0 0 types of food to a variety of antibiotics. The Clindamycin 2 7.4 rates of resistance to penicillin in our work Chloramphenicol 0 0 compare with those found by Pereira et al.16 and Quinupristin/dalfopristin 0 0 Umoh and Odoba23, who, among S. aureus from Linezolid 0 0 different food origins, observed 73% and 56.7% of resistance to this antibiotic, respectively. Methicillin-resistant S. aureus (MRSA) Much lower prevalence rates of penicillin re- was first reported in the United Kingdom (UK) 12 sistance were reported for S. aureus strains in in 1961 , and MRSA infections had become a studies conducted by Shahraz et al.20 (18.7%), global health issue due to the severity of the 13 by Peles et al. (15) (30.5%), by Rodriguez- illness they may cause . MRSA, posing a po- Calleja et al.18 (26.9%) and by Spanu et al.22 tential threat to human health, in food samples (18%). However, Vazquez-Sanchez et al.24 analysed in several surveys from different parts found a high incidence of penicillin resistant S. of the world has been found, with following aureus (100%) in fishery products from retail rates reported: Iran 89% 20, Malaysia 20%19, 4 8 13 outlets in Galicia (Spain). We found that 59.3% Egypt 12.6% , Turkey 97.2% , Italy 3.7% . The of strains were resistant to ampicillin. Different present survey also showed that two (7.4%) out rates of ampicillin resistance were suggested for of 27 S.aureus strains had resistance to methicil- S. aureus strains obtained from various food lin. However, some early reports suggested the samples in Portugal (70%)16, in Nigeria (50%)23, absence of meticillin-resistant S. aureus in bo- 15 in Italy (18%)22 and in Italy (46.4%)14. vine milk , fishery products 24, raw ewe’s milk 22 In this investigation, it was found that six cheese , food samples implicated in suspected (22.2%) of the strains were resistant to erythro- food poisoning complaints and foodborne out-3 mycin. Other some workers have also indicated breaks . erythromycin resistance with incidence rates of According to Table 2, only two strains 20.3%20 and 32.0%14. On the other hand, our had intermediate resistance to quinupris- 15 tin/dalfopristin and one strain to ciprofloxacin. 7. Graham, J.P., Evans, S., Price, L.B., Silbergeld, All the remaining strains were shown to be sus- E.K., 2009. Fate of antimicrobial-resistant enter- ceptible to these antibiotic agents. 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