Antimicrobial Resistance and Virulence Genes of Enterococci Isolated from Water Buffalo's Subclinical Mastitis

Abstract

This study aimed to investigate the antimicrobial resistance and virulence genes of enterococci isolated from water buffalo’s subclinical mastitis cases. The antimicrobial susceptibilities of the isolates were determined by the disc diffusion method. Identification at the species level of enterococci, virulence [aggregation substance (asa1), gelatinase (gelE), cytolysin (cylA), enterococcal surface protein (esp), and hyaluronidase (hyl)] and resistance genes [macrolide (ermA, ermB, mefA/E) and tetracycline (tetK, tetL, tetM, tetO, and tetS)] were investigated by polymerase chain reaction (PCR). Overall, Enterococcus spp. was recovered from 65 of 200 (32.5%) mastitic milk samples, comprising E. faecium (n=26), E. durans (n=22), E. faecalis (n=12), and E. hirae (n=5). Most isolates (56.9%) were susceptible to all tested antibiotics. The rest of the isolates showed various rate of resistance against rifampicin (23.1%), tetracycline (21.5%), quinupristin-dalfopristin (10.8%), ciprofloxacin (7.7%), erythromycin (6.2%), and chloramphenicol (3.1%). Out of 65 enterococci, only 16 (24.6%) were detected to have virulence genes, of which 12 were positive for gelE, seven were positive for esp, two were positive for asa1, and one was positive for hlyA. The gene cylA was not detected in any isolate tested. Resistance to tetracycline was mainly associated with tetM. Two erythromycin-resistant isolates were positive for ermB, and one was positive for mefA/E. This study was the first to report species distribution, antimicrobial susceptibility, and virulence traits of enterococci isolated from subclinical mastitis of water buffaloes in Çorum Province, Türkiye.

Keywords

• Water buffalo.
• Antimicrobial resistance
• Enterococci
• Subclinical mastitis
• Virulence
• Water bufalo

Subklinik Manda Mastitislerinden İzole Edilen Enterokok Türlerinde Antimikrobiyal Direnç ve Virulans Genleri

Abstract

Bu çalışmada subklinik manda mastitis vakalarından izole edilen enterokokların antimikrobiyal direnç ve virülans genlerinin araştırılması amaçlanmıştır. İzolatların antimikrobiyal duyarlılıkları disk difüzyon yöntemi ile belirlendi. Enterokokların, tür düzeyinde identifikasyonu, virülans (asa1, gelE, cylA, esp ve hyl) ve direnç genleri [makrolid (ermA, ermB, mefA/E) ve tetrasiklin (tetK, tetL, tetM, tetO ve tetS)] polimeraz zincir reaksiyonu (PZR) ile araştırıldı. İncelenen 200 mastitisli süt örneğinin 65'inden (%32,5) Enterococcus spp. izole edildi ve izole edilen türler E. faecium (n=26), E. durans (n=22), E. faecalis (n=12) ve E. hirae (n=5) olarak identifiye edildi. İzolatların %56,9’u test edilen tüm antibiyotiklere duyarlı bulundu. İzolatların geri kalanı rifampisin (%23,1), tetrasiklin (%21.5), kuinupristin-dalfopristin (%10,8), siprofloksasin (%7,7), eritromisin (%6,2) ve kloramfenikol (%3,1)'e karşı çeşitli direnç oranları gösterdi. İzole edilen 65 Enterococcus spp.’nin sadece 16'sının (%24,6) virülans genlerine sahip olduğu tespit edildi. Virulens genlerine sahip izolatların 12'si gelE, yedisi esp, ikisi asa1 ve biri de hlyA yönünden pozitif bulundu. cylA geni incelenen hiçbir izolatta saptanmadı. Tetrasikline direncin esas olarak tetM ile ilişkili olduğu saptanırken; eritromisine dirençli iki izolat ermB ve bir izolat ise mefA/E geni yönünden pozitif bulundu. Bu çalışma, Türkiye’nin Çorum ilinde yetiştirilen mandalarda saptanan subklinik mastitisli süt örneklerinden izole edilen enterokokların tür dağılımı, antimikrobiyal duyarlılık ve virülans özelliklerini bildiren ilk çalışmadır.

Keywords

• Antimikrobiyal direnç
• Enterococcus spp
• Manda
• Subklinik mastitis
• Virulens

Supporting Institution

Herhangi bir kurumdan destek alınmamıştır.

References

1. 1. Turkish Statistical Institute (TSI, 2018): Animal Production Statistics. https://data.tuik.gov.tr/Bulten/Index?p=Hayvansal-Uretim-Istatistikleri-2019-33873, Erişim tarihi: 05.04.2022.
2. 2. Atasever S, Erdem H, 2008: Manda Yetiştiriciliği ve Türkiye'deki Geleceği. J Fac Agric, OMU, 23, 59-64.
3. 3. Şahin A, Yıldırım A, 2015: Mandalarda mastitis olgusu. TURJAF, 3, 1-8.
4. 4. Sarıözkan S, 2011: The importance of water buffalo breeding in Turkey. Review. Kafkas Univ Vet Fak Derg, 17 (1), 163-166.
5. 5. Singha S, Ericsson CD, Chowdhury S, Nath SC, Paul OB, Hoque MA, Boqvist S, Persson Y, Rahman MM, 2021: Occurrence and aetiology of subclinical mastitis in water buffalo in Bangladesh. J Dairy Res, 88(3), 314-320.
6. 6. Yang F, Zhang S, Shang X, Wang X, Yan Z, Li H, Li J, 2019: Antimicrobial resistance and virulence genes of Enterococcus faecalis isolated from subclinical bovine mastitis cases in China. J Dairy Sci, 102 (1), 140-144.
7. 7. Saini V, McClure JT, Léger D, Keefe GP, Scholl DT, Morck DW, Barkema HW, 2019: Antimicrobial resistance profiles of common mastitis pathogens on Canadian dairy farms. J Dairy Sci, 95 (8), 4319-4332.
8. 8. Vankerckhoven V, Van Autgaerden T, Vael C, Lammens C, Chapelle S, Rossi R, Jabes D, Goossens H (2004): 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 of Enterococcus faecium. J Clin Microbiol, 42 (10), 4473–4479.

9. 9. Layton BA, Walters SP, Lam LH, Boehm AB, 2010: Enterococcus species distribution among human and animal hosts using multiplex PCR. J Appl Microbiol, 109 (2), 539-547.
10. 10. Clinical and Laboratory Standards Institute (CLSI): Performance standards for antimicrobial susceptibility testing; twenty fifth informational supplement. CLSI Document M100-S31, 2021.
11. 11. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL, 2012: Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect, 18 (3), 268-28.
12. 12. Malhotra-Kumar S, Lammens C, Piessens J, Goossens H (2005): Multiplex PCR for simultaneous detection of macrolide and tetracycline resistance determinants in streptococci. Antimicrob Agents Chemother, 49 (11), 4798–4800.
13. 13. Gürler H, Fındık A, Gültiken N, Ay SS, Çiftçi A, Koldaş E, Arslan S, Fındık M (2015): Investigation on the etiology of subclinical mastitis in Jersey and hybrid Jersey dairy cows. Acta Vet-Beograd, 65 (3), 358-370.
14. 14. Kuyucuoğlu Y, 2011: Antibiotic resistance of enterococci isolated from bovine subclinical mastitis. Eurasian J Vet Sci, 27, 231-234.
15. 15. Herkmen TB, Türkyılmaz S, 2016: Mastitisli sığırlardan izole edilen Enterococcus faecium izolatlarında gelE, esp ve efaAfm genlerinin varlığının incelenmesi. Kocatepe Vet J, 9 (2), 54-60.
16. 16. Nam HM, Lim SK, Moon JS, Kang HM, Kim JM, Jang KC, Kim JM, Kang MI, Joo YS, Jung SC, 2010: Antimicrobial resistance of enterococci isolated from mastitic bovine milk samples in Korea. Zoonoses Public Health, 57 (7-8), e59-64.
17. 17. Cameron M, Saab M, Heider L, McClure JT, Rodriguez-Lecompte JC, Sanchez J, 2016: Antimicrobial susceptibility patterns of environmental streptococci recovered from bovine milk samples in the maritime provinces of Canada. Front Vet Sci, 3, 79.
18. 18. Cervinkova D, Vlkova H, Borodacova I, Makovcova J, Babak V, Lorencova A, Vrtkova I, Marosevic D, Jaglic Z, 2013: Prevalence of mastitis pathogens in milk from clinically healthy cows. Vet Med, 58 (11), 567–575.
19. 19. Różańska H, Lewtak-Piłat A, Kubajka M, Weiner M, 2019: Occurrence of enterococci in mastitic cow's milk and their antimicrobial resistance. J Vet Res, 63 (1), 93-97.
20. 20. Kateete DP, Kabugo U, Baluku H, Nyakarahuka L, Kyobe S, Okee M, Najjuka CF, Joloba ML, 2013: Prevalence and antimicrobial susceptibility patterns of bacteria from milkmen and cows with clinical mastitis in and around Kampala, Uganda. PLoS One, 8 (5), e63413.
21. 21. Klimienė I, Ružauskas M, Špakauskas V, Mockeliūnas R, Pereckienė A, Butrimaitė-Ambrozevičienė Č, 2011: Prevalence of Gram positive bacteria in cow mastitis and their susceptibility to beta-lactam antibiotics. Vet Med Zoot, 56 (78), 65-67.
22. 22. Aslam M, Diarra MS, Checkley S, Bohaychuk V, Masson L, 2012: Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada. Int J Food Microbiol, 156 (3), 222-230.
23. 23. Yılmaz EŞ, Aslantaş Ö, Pehlivanlar Önen S, Türkyılmaz S, Kürekci C, 2016: Prevalence, antimicrobial resistance and virulence traits in enterococci from food of animal origin in Turkey. LWT - Food Sci Technol, 66, 20-26.
24. 24. Kürekci C, Önen SP, Yipel M, Aslantaş Ö, Gündoğdu A, 2016: Characterisation of phenotypic and genotypic antibiotic resistance profile of enterococci from cheeses in Turkey. Korean J Food Sci Anim Resour, 36 (3), 352-388. 25. Boyar Y, Aslantaş Ö, Türkyılmaz S, 2017: Antimicrobial resistance and virulence characteristics in enterococcus isolates from dogs. Kafkas Univ Vet Fak Derg, 23 (4), 655-660. 26. Kim YB, Seo KW, Jeon HY, Lim SK, Sung HW, Lee Yj, 2019: Molecular characterization of erythromycin and tetracycline-resistant Enterococcus faecalis isolated from retail chicken meats. Poult Sci, 98 (2), 977-983.
25. 27. Aslantaş Ö, 2019: Molecular and phenotypic characterization of enterococci isolated from broiler flocks in Turkey. Trop Anim Health Prod, 51 (5): 1073-1082.
26. 28. Mundy LM, Sahm DF, Gilmore M, 2000: Relationships between enterococcal relationships between enterococcal virulence and antimicrobial resistance. Clin Microbiol Rev, 13 (4), 513-522.
27. 29. Chajęcka-Wierzchowska W, Zadernowska A, Łaniewska-Trokenheim Ł, 2017: Virulence factors of Enterococcus spp. presented in food. LWT - Food Sci Technol, 75, 670-676.
28. 30. Sava IG, Heikens E, Kropec A, Theilacker C, Willems R, Huebner J, 2010: Enterococcal surface protein contributes to persistence in the host but is not a target of opsonic and protective antibodies in Enterococcus faecium infection. J Med Microbiol, 59, 1001-1004.