Olgunlaştırılmış (Klasik) Beyaz Peynir Üretiminin Farklı Aşamalarından İzole Edilen Enterococcus spp.’nin Virülens Faktörlerinin ve Antibiyotik Dirençliliğinin Belirlenmesi

Öz

Bu çalışmada, daha önce klasik beyaz peynir üretiminin farklı aşamalarından identifiye edilen Enterococcus spp. izolatlarının virülens genleri (asa1, gelE, cylA, ace, esp, hyl ve efaA) ve vankomisin direnç genlerinin (vanA, vanB, vanC2/C3) varlığı ile bazı antibiyotiklere dirençliliklerinin belirlenmesi amaçlandı. Ayrıca jelatinaz, β-hemolitik ve DNase aktiviteleri ile biyofilm oluşumları fenotipik olarak ise incelendi. İzolatların %95,9’unda efaA, %89’unda asa1, %68,4’ünde ace, %52,1’inde esp, %78,1’inde gelE, %16,4’ünde cylA ve %23,3’ünde hyl virülens genleri tespit edildi. Ayrıca %31,5’inde vanA, %8,2’sinde vanB ve %23,3’ünde vanC2/C3 direnç genleri belirlendi. Fenotipik olarak ise izolatların sırasıyla %23,2'sinde ve %16,4'ünde β-hemolitik aktivite ve DNase aktivitesi tespit edilirken, jelatinaz aktivitesi ve biyofilm oluşumu tespit edilememiştir. Bunun yanı sıra izolatların %73,9’unda streptomycin ve %43,8’inde erythromycin direnci saptandı. Sonuç olarak, Enterococcus spp.’nin virülans faktörleri ve antibiyotik direnci açısından halk sağlığı ve gıda güvenliği için risk oluşturabileceği kanaatine varıldı. Bu nedenle starter kültür olarak seçilecek suşların virülens faktörleri ve antibiyotiklere dirençlilik bakımından değerlendirildikten sonra kullanılmaları önerildi.

Anahtar Kelimeler

• Antibiyotik Direnci
• Enterococcus spp.
• Klasik Beyaz Peynir
• Virülens Faktör.

Determination of Virulence Factors and Antibiotic Resistances of Enterococcus spp. Identified from Different Stages of Ripened (Classical) White Cheese Production

Abstract

The objective of this research was to determine the presence of virulence genes (asa1, gelE, cylA, ace, esp, hyl and efaA), and vancomycin resistance genes (vanA, vanB, vanC2/C3) and the resistance to some antibiotics of Enterococcus spp. isolates previously identified from different stages of ripened white cheese production. In addition, gelatinase, β-hemolytic and DNase activity, and biofilm formations were examined phenotypically. In this study, efaA in 95.9%, asa1 in 89%, ace in 68.5%, esp in 52.1%, gelE in 78.1%, cylA in 16.4% and hyl in 23.3% of isolates were detected. Also, vanA in 31.5%, vanB in 8.2%, and in vanC2/C3 23.3% resistance genes were determined. β-hemolytic and DNase activity were detected in 23.2% and 16.4% of the isolates, while gelatinase activity and biofilm formation could not be detected phenotypically. Moreover, streptomycin and erythromycin resistances were found in 73.9% and in %43.8 of isolates. As a result, it was concluded that Enterococcus spp. may pose a risk for public health and food safety in terms of their virulence factors and antibiotic resistance. For this reason, it was suggested that the strains to be selected as starter cultures should be used after evaluating their virulence factors and resistance to antibiotics. 

Keywords

• Antibiotic Resistance
• Enterococcus spp.
• Ripened White Cheese
• Virulence Factor.

References

1. Adifon DE, Tuncer Y. Peynirden izole edilen yüksek seviyede aminoglikozid dirençli enterokoklarda virülens faktörlerin fenotipik ve genotipik yöntemler ile araştırılması. GIDA. 2019; 44(4):719-732 doi: 10.15237/gida.GD19075
2. Ahmed MO, Baptiste KE. Vancomycin-resistant enterococci: A review of antimicrobial resistance mechanisms and perspectives of human and animal health. Microb Drug Resist. 2018; 24:590-606.
3. Anderson AC, Jonas D, Huber I, Karygianni L, Wölber J, Hellwig W, Arweiler N, Vach K, Wittmer A, Al-Ahmad A. Enterococcus faecalis from food, clinical specimens, and oral sites: Prevalence of virulence factors in association with biofilm formation. Front. Microbiol. 2016; 6:1534. doi:10.3389/fmicb.2015.01534.
4. Arias CA, Murray BE. The rise of the enterococcus: Beyond vancomycin resistance. Nat. Rev. Microbiol. 2012; 10:266-278.
5. Bauer RW, Kirby MDK, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966; 45:493-496.
6. Ben Omar N, Castro A, Lucas R, Abriouel H, Yousif NMK, Franz CMAP, Holzapfel WH, Pérez-Pulido R, Martínez-Cañamero M, Gálvez A. Functional and safety aspects of enterococci isolated from different Spanish foods. Systematic and Applied Microbiology. 2004; 27:118-130.
7. Câmara SPA, Dapkevicius A, Silva CCG, Malcata FX, Enes Dapkevicius MLN. Artisanal Pico cheese as reservoir of Enterococcus species possessing virulence and antibiotic resistance properties: Implications for food safety. Food Biotechnology. 2020; 34(1): 25-41.
8. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-Resistant Enterococci. Clin Microbiol Rev. 2000; 13:686-707.

9. Ch’ng, JH, Chong KKL, Lam LN, Wong JJ, Kline K A. Biofilm-associated infection by enterococci. Nat. Rev. Microbiol. 2019; 17:82–94. doi: 10.1038/s41579-018-0107-z
10. Chajęcka-Wierzchowska W, Zandernowska A, Łaniewska-Trokenheim Ł. Virulence factors of Enterococcus spp. presented in food. LWT-Food Sci Technol. 2017; 75:670-676.
11. CLSI M100-ED31:2021. Clinical Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, 31st Edition https://www.treata.academy/wp-content/uploads/2021/03/CLSI-31-2021.pdf . Access Date: 22.11.2021.
12. Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, Baldassarri L. Survey for virulence determinants among Enterococcus faecalis isolated from different sources. Journal of Medical Microbiology. 2004; 53:13-20.
13. De Paula PLM, De Moraes ML, Schueler J, De Souza NAA, Furlaneto MC, Maia LF, Katsuda MS. Enterococcus faecium in artisanal ripening cheese: technological and safety aspect. Research, Society and Development. 2020; 9(11):e299119452.
14. De Vuyst L, Foulquie Moreno MR, Revets H. Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. Int J Food Microbiol. 2003; 84:299-318.
15. Deasy BM, Rea MC, Fitzgerald GF, Cogan TM, Beresford TP. A rapid PCR-based method to distinguish between Lactococcus and Enterococcus. Syst. Appl. Microbiol. 2000; 23:510-522.
16. Domingos-Lopes MFP, Stanton C, Ross PR, Dapkevicius MLE, Silva CCG. Genetic diversity, safety, and technological characterization of lactic acid bacteria isolated from artisanal Pico cheese. J Food Microbiol, 2017; 63:178-190.
17. Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol. 2001; 67:1628-1635.
18. Elsayed S, Hamilton N, Boyd D, Mulvey M. Improved primer design for multiplex PCR analysis of vancomycin-resistant Enterococcus spp. J Clin Microbiol. 2001; 39:2367-2368.
19. Foulquie Moreno MR, Sarantinopoulos P, Tsakalidou E, De Vuyst L. The role and application of enterococci in food and health. Int. J. Food Microbiol. 2006; 106:1–24.
20. Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol. 1989; 42:872-874.
21. Fuka MM, Maksimovic AZ, Tanuwidjaja I, Hulak N, Schloter M. Characterization of enterococcal community isolated from an artisan Istrian raw milk cheese: Biotechnological and safety aspects. Food Technol. Biotechnol. 2017; 55:368–380. doi: 10.17113/ftb.55.03.17.5118.
22. Gaglio R, Couto N, Marques C, de Fatima Silva Lopes M, Moschetti G, Pomba C, Settanni L. Evaluation of antimicrobial resistance and virulence of enterococci from equipment surfaces, raw materials, and traditional cheeses. Int J Food Microbiol. 2016; 236:107-114.
23. García-Solache M, Rice LB. The Enterococcus: a Model of Adaptability to Its Environment. Clin Microbiol Rev. 2019; 32(2).
24. Gaspar FB, Crespo MTB, Lopes MFS. Proposal for a reliable enterococcal cytolysin production assay avoiding apparent incongruence between phenotype and genotype. Journal of Medical Microbiology. 2009; 58:1122-1124.
25. Giraffa G. Enterococci from foods. FEMS Microbiol Rev. 2002; 26:163-171.
26. Giraffa G. Functionality of enterococci in dairy products. Int. J. Food Microbiol. 2003; 88:215–222.
27. Hammad AM, Aly SS, Hassan HA, Abbas NH, Eltahan A, Khalifa E, Shimamoto T. Occurrence, phenotypic and molecular characteristics of vancomycin-resistant enterococci isolated from retail raw milk in Egypt. Foodborne Pathogens and Disease. 2021; ahead of print. doi:10.1089/fpd.2021.0054
28. Hegstad K, Mikalsen T, Coque TM, Werner G, Sundsfjord A. Mobile genetic elements and their contribution to the emergence of antimicrobial-resistant Enterococcus faecalis and Enterococcus faecium. Clin Microbiol Infect. 2010; 16(6):541-54. doi: 10.1111/j.1469-0691.2010.03226.x.
29. Jackson CR, Fedorka-Cray PJ, Barrett JB. Use of a genus- and species-specific multiplex PCR for identification of Enterococci. J Clin Microbiol. 2004; 42(8):3558-3565.
30. Jurkovič D, Križková L, Dušinský R, Belicová A, Sojka M, Krajčovič J, Ebringer L. Identification and characterization of enterococci from bryndza cheese. Letters in Applied Microbiology. 2006; 42(6):553-559. 10.1111/j.1472-765X.2006.01918.x
31. Kariyama R, Mitsuhata R, Chow JW, Clewell DB, Kumon H. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J Clin Microbiol. 2000; 38:3092-3095.
32. Lopes MS, Simões AP, Tenreiro R, Marques FJJ, Crespo MTB. Activity and expression of a virulence factor, gelatinase, in dairy enterococci. Int J Food Microbiol. 2006; 112: 208–214.
33. Margalho, LP, Feliciano MD, Silva CE, Abreu JS, Piran MVF, Sant'Ana AS. Brazilian artisanal cheeses are rich and diverse sources of nonstarter lactic acid bacteria regarding technological, biopreservative, and safety properties-Insights through multivariate analysis. Journal of Dairy Science. 2020; 103:7908-7926. https://doi.org/10.3168/jds.2020-18194.
34. Ogier JC, Serror P. Safety assessment of dairy microorganisms: the Enterococcus genus. Int. J. Food Microbiol. 2008; 126:291–301. 10.1016/j.ijfoodmicro.2007.08.017
35. Perin LM, Miranda RO, Todorov SD, Franco BDGM, Nero LA. Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk. Int. J. Food Microbiol. 2014; 185:121–126. 10.1016/j.ijfoodmicro.2014.06.001
36. Riboldi GP, Frazzon J, Azevedo PA, Frazzon APG. Antimicrobial resistance profile of Enterococcus spp. isolated from food in Southern Brazil. Braz. J. Microbiol. 2009; 40:125-128.
37. Sanlibaba P, Senturk E. Prevalence, characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey. International Journal of Food Properties. 2018; 21(1):1955-1963. DOI: 10.1080/10942912.2018.1489413
38. Semedo T, Santos MA, Lopes MFS, Marques JJF, Crespo MTB, Tenreiro R. Virulence factors in food, clinical and reference enterococci: a common trait in the genus? Syst. Appl. Microbiol. 2003; 26:13-22.
39. Settanni L, Di Grigoli A, Tornambé G, Bellina V, Francesca N, Moschetti G, Bonanno A. Persistence of wild Streptococcus thermophilus strains on wooden vat and during the manufacture of a Caciocavallo type cheese. Int. J. Food Microbiol. 2012; 155:73–81.
40. Templer SP, Baumgartner A. Enterococci from Appenzeller and Schabziger raw milk cheeses: Antibiotic resistance, virulence factors and persistence of particular strains in the products. J. Food Prot. 2007; 70:450–455. doi: 10.4315/0362-028X-70.2.450
41. Trivedi K, Cupakova S, Karpiskova R. Virulence factors and antibiotic resistance in enterococci isolated from food-stuffs. Vet Med-Czech. 2011; 56:352-357.
42. Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H. Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium. J Clin Microbiol. 2004; 42(10):4473-4479. doi: 10.1128/JCM.42.10.4473-44 79.2004