DETERMINATION OF STAPHYLOCOCCUS AUREUS PREVALENCE AND ANTIBIOTIC RESISTANCE PROFILES AND RESISTANCE GENES IN TULUM CHEESE

Abstract

In this study, it was aimed to determine the prevalence of Staphylococcus aureus in tulum cheese samples sold in Isparta province and to determine the antibiotic resistance profiles and resistance genes in S. aureus isolates. A total of 141 presumptive S. aureus colonies were isolated from 75 Tulum cheese samples. As a result of Gram staining and catalase test, it was determined that 100 of the isolates had Gram positive cocci morphology and catalase positive feature. Whether these isolates are members of the S. aureus species was investigated by polymerase chain reaction (PCR) using primer pairs specific to the thermostable nuclease gene (nuc) in S. aureus. As a result of PCR experiments, nuc gene-specific 458 bp amplicons were obtained in 15 isolates. The frequency of S. aureus in Tulum cheese samples was calculated as 13.33% (10/75). As a result of disk diffusion test of 15 S. aureus isolates, penicillin G was found to be the most resistant antibiotic. It has been determined that the most common antibiotic resistance gene in isolates is blaZ. Apart from the blaZ gene, the presence of mecA, msrA and msrB genes was determined in isolates. The presence of S. aureus in tulum cheese samples, albeit at a low rate, is a concern for consumer health.

Keywords

• Tulum Cheese
• Staphylococcus aureus
• Antibiotic Resistance
• Antibiotic Resistance Gene
• Polymerase Chain Reaction

Project Number

FYL-2018-6913

TULUM PEYNİRİNDE STAPHYLOCOCCUS AUREUS YAYGINLIĞI VE ANTİBİYOTİK DİRENÇ PROFİLLERİNİN VE DİRENÇ GENLERİNİN BELİRLENMESİ

Abstract

Bu çalışmada, Isparta ilinde satışı yapılan Tulum peyniri örneklerinde Staphylococcus aureus yayğınlığının tespiti ve S. aureus izolatlarında antibiyotik direnç profillerinin ve direnç genlerinin belirlenmesi amaçlanmıştır. 75 Tulum peyniri örneğinden toplam 141 muhtemel S. aureus kolonisi izole edilmiştir. Gram boyama ve katalaz testi sonucu izolatların 100’ünün Gram pozitif kok morfolojisinde ve katalaz pozitif özellik gösterdiği tespit edilmiştir. Bu izolatların S. aureus türü üyesi olup olmadığı S. aureus’da termostabil nükleaz genine (nuc) özgü primer çiftleri kullanılarak polimeraz zincir reaksiyonu (PZR) ile araştırılmıştır. PZR denemeleri sonucu 15 izolatta nuc genine özgü 458 bç büyüklüğünde amplikonlar elde edilmiştir. Tulum peyniri örneklerinde S. aureus bulunma sıklığı % 13.33 (10/75) olarak hesaplanmıştır. 15 S. aureus izolatının disk difüzyon testi sonucu en dirençli olduğu antibiyotiğin penisilin G olduğu tespit edilmiştir. İzolatlarda en sık rastlanan antibiyotik direnç geninin blaZ olduğu belirlenmiştir. blaZ geni dışında izolatlarda mecA, msrA ve msrB geni varlığı tespit edilmiştir. Düşük oranda da olsa tulum peyniri örneklerinde S. aureus varlığına rastlanılması tüketici sağlığı açısından endişe uyandırıcıdır.

Keywords

• Tulum Peyniri
• Staphylococcus aureus
• Antibiyotik Direnç
• Antibiyotik Direnç Geni
• Polimeraz Zincir Reaksiyonu

Supporting Institution

Süleyman Demirel Üniversitesi

Project Number

FYL-2018-6913

Thanks

Bu çalışma Süleyman Demirel Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi tarafından FYL-2018-6913 nolu proje kapsamında desteklenmiştir.

References

1. Abolghait, S.K., Fathi, A.G., Youssef, F.M., Algammal, A.M., 2020. Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from Chicken Meat and Giblets Often Produces Staphylococcal Enterotoxin B (SEB) in Non-Refrigerated Raw Chicken Livers. International Journal of Food Microbiology, 328, 108669.
2. Al-Ashmawy, M.A., Sallam K.I., Abd-Elghany, S.M., Elhadidy, M., Tamura, T., 2016. Prevalence, Molecular Characterization, and Antimicrobial Susceptibility of Methicillin-Resistant Staphylococcus aureus Isolated from Milk and Dairy Products. Foodborne Pathogens and Disease, 13(3), 156-162.
3. Anonymous, 2020. UK standarts for microbiology investigations. Identification of Staphylococcus species, Micrococcus species and Rothia species. UK SMI ID: 7, Issue no: 4, Issue date: 26.05.20, England.
4. Baran, A., Erdoğan, A., Turgut, T., Adiguzel, M.C., 2017. A Review on the Presence of Staphylococcus aureus in Cheese. Turkish Journal of Nature and Science, 6, 100-105.
5. Bayston, R., 2006. Coagulase-Negative Methicillin-Resistant Staphylococcus aureus. Journal of Hospital Infection, 62(1), 127.
6. Bingöl, K.K., Özmen Toğay, S., 2017. Urfa Peynirlerinden İzole Edilen Staphylococcus aureus Suşlarında Enterotoksin Üretim Potansiyeli ve Metisilin Dirençliliği. Akademik Gıda, 15(1), 29-35.
7. Bitrus, A.A., Peter, O.M., Abbas, M.A., Goni. M.D., 2018. Staphylococcus aureus: A Review of Antimicrobial Resistance Mechanisms. Veterinary Sciences: Research and Reviews, 4(2), 43-54.
8. Black, R.E., Williams, S.M., Jones, L.E., Goulding, A., 2002. Children Who Avoid Drinking Cow Milk Have Low Dietary Calcium Intakes and Poor Bone Health. The American Journal of Clinical Nutrition, 76(3), 675-680.

9. Brakstad, O.G., Aasbakk, K., Maeland, J.A., 1992. Detection of Staphylococcus aureus by Polymerase Chain Reaction Amplification of the nuc Gene. Journal of Clinical Microbiology, 30(7), 1654-1660.
10. Cancilla, M., Powell, J.B., Hillier, A.J., Davidson, B.E., 1992. Rapid Genomic Fingerprinting of Lactococcus lactis Strains by Arbitrarily Primed Polymerase Chain Reaction with Phosphorus-32 and Fluorescent Labels. Applied and Environmental Microbiology, 58(5), 1772-1775.
11. Cariolato, D., Andrighetto, C., Lombardi, A., 2008. Occurrence of Virulence Factors and Antibiotic Resistances in Enterococcus faecalis and Enterococcus faecium Collected from Dairy and Human Samples in North Italy. Food Control, 19(9), 886-892.
12. CLSI, 2016. Performance Standards for Antimicrobial Susceptibility Testing; 26th Informational Supplement M100-S-26. Pennsylvania: Wayne, United States of America.
13. Cremonesi, P., Perez, G., Pisoni, G., Moroni, P., Morandi, S., Luzzana, M., Brasca, M., Castiglioni, B., 2007. Detection of Enterotoxigenic Staphylococcus aureus Isolates in Raw Milk Cheese. Letters in Applied Microbiology, 45(6), 586-591.
14. Depardieu, F., Perichon, B., Courvalin, P., 2004. Detection of van Alphabet and Identification of Enterococci and Staphylococci at the Species Level by Multiplex PCR. Journal of Clinical Microbiology, 42(12), 5857-5860.
15. Dutka-Malen, S., Evers, S., Courvalin, P., 1995. Detection of Glycopeptide Resistance Genotypes and Identification to the Species Level of Clinically Relevant Enterococci by PCR. Journal of Clinical Microbiology, 33(1), 24-27.
16. El Feghaly, R.E., Stamm, J.E., Fritz, S.A., Burnham, C.D., 2012. Presence of the blaZ Beta-Lactamase Gene in Isolates of Staphylococcus aureus that Appear Penicillin Susceptible by Conventional Phenotypic Methods. Diagnostic Microbiology and Infectous Disease, 74(4), 388-393.
17. Ferreira, A.M., Martins, K.B., da Silva, V.R., Mondelli, A.L., da Cunha, M. de L.R. de S., 2017. Correlation of Phenotypic Tests with the Presence of The blaZ Gene for Detection of Beta-Lactamase. Brazilian Journal of Microbiology, 48(1), 159–166. doi:10.1016/j.bjm.2016.10.011
18. Foster, T.J., 2017. Antibiotic Resistance in Staphylococcus aureus. Current Status and Future Prospects. FEMS Microbiology Reviews, 41, 430-449.
19. Frey, Y., Rodriguez, J.P., Thomann, A., Schwendener, S., Perreten, V., 2013. Genetic Characterization of Antimicrobial Resistance in Coagulase-Negative Staphylococci from Bovine Mastitis Milk. Journal of Dairy Science, 96, 2247-2257.
20. Gandra, E.A., Fernandez, M.A., Silva, J.A., Silva, W.P.D., 2011. Standardization of a Multiplex PCR for the Identification of Coagulase-Pozitive Staphylococcus. Food Science and Technology, 31(4), 946-949.
21. Güven, K., Mutlu, M.B., Gülbandılar, A., Cakır, P., 2010. Occurrence and Characterization of Staphylococcus aureus Isolated from Meat and Dairy Products Consumed in Turkey. Journal of Food Safety, 30(1), 196-212.
22. Hennekinne, J.A., De Buyser, M. L., Dragacci, S., 2012. Staphylococcus aureus and Its Food Poisoning Toxins: Characterization and Outbreak Investigation. FEMS Microbiology Reviews, 36(4), 815-836.
23. Janštová, B., Necidová, L., Skockova, A., Janštová, B., 2014. Staphylococcal Enterotoxin Production in Model Samples of Milk and Fresh Cheese. Journal of Food and Nutrition Research, 53(4), 389-392.
24. Kaase, M., Lenga, S, Friedrich, S., Szabados, F., Sakinc, T, Kleine, B, Gatermann, S.G., 2008. Comparison of Phenotypic Methods for Penicillinase Detection in Staphylococcus aureus. Clinical Microbiology and Infection. 14(6), 614-616.
25. Karmakar, A., Dua, P., Ghosh, C., 2016. Biochemical and Molecular Analysis of Staphylococcus aureus Clinical Isolates from Hospitalized Patients. Canadian Journal of Infectious Diseases and Medical Microbiology, 9041636.
26. Kayili, E., Sanlıbaba, P., 2020. Prevalence, Characterization and Antibiotic Resistance of Staphylococcus aureus Isolated from Traditional Cheeses in Turkey. International Journal of Food Properties, 23(1), 1441-1451.
27. Kim, C. H., Khan, M., Morin, D. E., Hurley, W. L., Tripathy, D. N., Kehrli Jr., M., Oluoch, A. O., Kakoma, I., 2001. Optimization of the PCR for Detection of Staphylococcus aureus nuc Gene in Bovine Milk. Journal of Dairy Science, 84(1), 74-83.
28. Kot, B., Wierzchowska, K., Piechota, M., Grużewska, A., 2020. Antimicrobial Resistance Patterns in Methicillin Resistant Staphylococcus aureus from Patients Hospitalized during 2015–2017 in Hospitals in Poland. Medical Principles and Practice, 29, 61-68.
29. Kroning, I. S., Iglesias, M. A., Sehn, C. P., Gandra, T. K. V., Mata, M. M., da Silva, W. P., 2016. Staphylococcus aureus Isolated from Handmade Sweets: Biofilm Formation, Enterotoxigenicity and Antimicrobial Resistance. Food Microbiology, 58, 105-111.
30. Kümmel, J., Stessl, B., Gonano, M., Walcher, G., Bereuter, O., Fricker, M., Grunert, T., Wagner, M., Ehling-Schulz, M., 2016. Staphylococcus aureus Entrance into the Dairy Chain: Tracking S. aureus from Dairy Cow to Cheese. Frontiers in Microbiology, 7, 1603.
31. Licitra, G., 2013. Etymologia: Staphylococcus. Emerging Infectious Diseases, 19(9), 1553.
32. Lina G., Quaglia A., Reverdy M.E., Leclercq R., Vandenesch F., Etienne J., 1999. Distribution of Genes Encoding Resistance to Macrolides, Lincosamides, and Streptogramins Among Staphylococci. Antimicrob Agents and Chemotherapy, 43(5), 1062-1066.
33. Martineau, F., Picard F.J., Lansac, N., Ménard, C., Roy, P.H., Ouellette, M., Bergeron, M.G., 2000. Correlation Between the Resistance Genotype Determined by Multiplex PCR Assays and the Antibiotic Susceptibility Patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrobial Agents and Chemotherapy, 44(2), 231–238.
34. Mimica, M.J., Berezin, E.N., Carvalho, R.L.B., Mimica, I.M., Mimica, L.M.J., Sáfadi, M.A.P., Schneider, E., Caiaffa-Filho, H.H., 2007. Detection of Methicillin Resistance in Staphylococcus aureus Isolated from Pediatric Patients: Is the Cefoxitin Disk Diffusion Test Accurate Enough? The Brazilian Journal of Infectious Diseases, 11(4), 415-417.
35. Miranda, J. M., Mondragón, A., Vázquez, B. I., Fente, C. A., Cepeda, A., Franco, C. M., 2009. Microbiological Quality and Antimicrobial Resistance of Escherichia coli and Staphylococcus aureus Isolated from Conventional and Organic “Arzúa-Ulloa” Cheese. CyTA–Journal of Food, 7(2), 103-110.
36. Niu, H., Yu, H., Hu, T., Tian, G., Zhang, L., Guo, X., Hu, H., Wang, Z., 2016. The Prevalence of Aminoglycoside-Modifying Enzyme and Virulence Genes Among Enterococci with High-Level Aminoglycoside Resistance in Inner Mongolia, China. Brazilian Journal of Microbiology. 47(3), 691-696.
37. Olver, W. J., Carmichael, I. C., Ziglam, H. M., Morrison, D., 2005. Bacteraemia with Tube-Coagulase-Negative Methicillin-Resistant Staphylococcus aureus. Journal of Hospital Infection, 60(1), 87-88.
38. Ouoba, L.I.I., Lei, V., Jensen, L.B., 2008. Resistance of Potential Probiotic Lactic Acid Bacteria and Bifidobacteria of African and European Origin to Antimicrobials: Determination and Transferability of the Resistance Genes to Other Bacteria. International Journal of Food Microbiology, 121(2), 217-224.
39. Pal, M., Kerorsa, G.B., Marami, L.M., Kandi, V., 2020. Epidemiology, Pathogenicity, Animal Infections, Antibiotic Resistance, Public Health Significance, and Economic Impact of Staphylococcus aureus: A Comprehensive Review. American Journal of Public Health Research, 8(1), 14-21.
40. Pyzik, E., Marek, A., Pysniak, D.S., Chmiel, R.U., Jarosz, L.S., Podebska, I.J., 2019. Detection of Antibiotic Resistance and Classical Enterotoxin Genes in Coagulase Negative Staphylococci Isolated from Poultry in Poland. Journal of Veterinary Research, 63, 183-190.
41. Sadiq, A., Samad, M., Saddam, Basharat, N., Ali, S., Roohullah, Saad, Z., Khan, A.N., Ahmad, Y., Khan, A., Khan, J., 2020. Methicillin-resistant Staphylococcus aureus (MRSA) in slaughter houses and meat shops in capital territory of Pakistan during 2018-2019. Frontiers in Microbiology, 11, 1-12.
42. Sasidharan, S., Prema, B., Latha, L. Y., 2011. Antimicrobial Drug Resistance of Staphylococcus aureus in Dairy Products. Asian Pacific Journal of Tropical Biomedicine, 1(2), 130-132.
43. Serwecińska, L., 2020. Antimicrobials and Antibiotic-Resistant Bacteria: A Risk to the Environment and to Public Health. Water, 12, 3313.
44. Sulieman, A.M.E., Ali, R.A.M., Razig, K.A.A., 2012. Production and Effect of Storage in the Chemical Composition of Mozzarella Cheese. International Journal of Food Science and Nutrition Engineering, 2(3), 21-26.
45. Tekinşen, K.K., Uçar, G., 2007. Konya Yöresinde Üretilen Mahalli Tulum Peynirleri. Akademik Gıda, 4(6), 33-37.
46. Temiz, A., 1994. Genel Mikrobiyoloji Uygulama Teknikleri. Şafak Matbaacılık Ltd. Şti., Ankara, Türkiye, 266 s. ISBN:975-95834-0-2
47. Tomar, O., Akarca, G., Beykaya, M., Çağlar, A., 2018. Some Characteristics of Erzincan Tulum Cheese Produced Using Different Probiotic Cultures and Packaging Material. Kafkas Universitesi Veteriner Fakültesi Dergisi, 24(5), 647-654.
48. Tong, S.Y.C., Davis, J.S., Eichenberger, E., Holland, T.L., Fowler, V.G. Jr. , 2015. Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management. Clinical Microbiology Reviews, 28(3), 603-661.
49. Turner, N.A., Sharma-Kuinkel, B.K., Maskarinec, S.A., Eichenberger, E.M., Shah, P.P., Carugati, M., Holland, T.L., Fowler, V.G. 2019. Methicillin-Resistant Staphylococcus aureus: An Overview of Basic and Clinical Research. Nature Reviews Microbiology, 17, 203-218.
50. Vakulenko, S.B., Donabedian, S.M., Voskresenskiy, A.M., Zervos, M.J., Lerner, S.A., Chow, J.W., 2003. Multiplex PCR for Detection of Aminoglycoside Resistance Genes in Enterococci. Antimicrobial Agents and Chemotherapy, 47(4), 1423-1426.
51. Vandenesch, F., Lebeau, C., Bes, M., McDevitt, D., Greenland, T., Novick, R. P., Etienne, J., 1994. Coagulase Deficiency in Clinical Isolates of Staphylococcus aureus Involves Both Transcriptional and Post-Transcriptional Defects. Journal of Medical Microbiology, 40(5), 344-349.
52. Vestergaard, M., Frees, D., Ingmer, H., 2019. Antibiotic resistance and the MRSA problem. In Gram Positive Pathogens, ASM Press, Washington DC, 747-765.
53. Vitale, M., Gaglio, S., Galluzzo, P., Cascone, G., Piraino, C., Di Marco Lo Presti, V., Alduina, R., 2018. Antibiotic Resistance Profiling, Analysis of Virulence Aspects and Molecular Genotyping of Staphylococcus aureus Isolated in Sicily, Italy. Foodborne Pathogens and Disease, 15(3), 177-185.
54. Wu, S., Huang, J., Wu, Q., Zhang, J., Zhang, F., Yang, X., Wu, H., Zeng, H., Chen, M., Ding, Y., Wang, J., Lei, T., Zhang, S. Xue L., 2018. Staphylococcus aureus Isolated from Retail Meat and Meat Products in China: Incidence, Antibiotic Resistance and Genetic Diversity. Frontiers in Microbiology, 9, 1-14.
55. Wu, S., Zhang, F., Huang, J., Wu, Q., Zhang, Y., Xue, L., Wang, J., Ding, Y., 2019. Phenotypic and Genotypic Characterization of PVL-Positive Staphylococcus aureus Isolated from Retail Foods in China. International Journal of Food Microbiology, 304, 119-126.
56. Yang, F., Wang, O., Wang, X., Wang, L., Xiao, M., Lı, X., Luo, J., Zhang, S., Lı., H., 2015. Prevalence of blaZ Gene and Other Virulence Genes in Penicillin-Resistant Staphylococcus aureus Isolated from Bovine Mastitis Cases in Gansu, China. Turkish Journal of Veterinary and Animal Sciences, 39: 634-636.
57. Yogurtcu, N.N., Tuncer, Y., 2013. Antibiotic Susceptibility Patterns of Enterococcus Strains Isolated from Turkish Tulum Cheese. International Journal of Dairy Technology, 66, 236-242.
58. Yücel, N., Anıl, Y., 2011. Çiğ Süt ve Peynir Örneklerinden Staphylococcus aureus ve Koagülaz Negatif Stafilokokların İdentifikasyonu ve Antibiyotik Duyarlılığı. Türk Hijyen ve Deneysel Biyoloji Dergisi, 68(2), 73-78.
59. Zhang, K., Sparling, J., Chow, B.L., Elsayed, S., Hussain, Z., Church, D.L., Gregson, D.B., Louie, T., Conly, J.M., 2004. New Quadriplex PCR Assay for Detection of Methicillin and Mupirocin Resistance, and Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci. Journal of Clinical Microbiology, 42(11), 4947-4955.