Mastitisli Sığırlardan İzole Edilen Staphylococcus aureus İzolatlarının Virülens Gen Profillerinin Belirlenmesi

Year 2021, , 144 - 152, 12.12.2021

Ayşe Nalça , Timur Gülhan

https://doi.org/10.31196/huvfd.978277

Abstract

Bu çalışmada anabilim dalı kültür koleksiyonunda bulunan subklinik mastitisli sığır sütlerinden izole edilen 140 adet stafilokok izolatından Staphylococcus aureus izolatlarının moleküler identifikasyonu ve bazı önemli virülens genlerinin tespiti amaçlandı. Bu amaçla 140 stafilokok izolatı termonükleaz (nuc) gen varlığı yönünden PZR ile incelendi. nuc geni (279 bp) tespit edilen 42 izolat S. aureus olarak identifiye edildi. Ayrıca izolatlarda lökotoksin geni (pvl), stafilokokal klasik enterotoksin genleri (sea, seb, sec), eksfolyatif toksin genleri (eta, etb), hemolizin genleri (hla, hlb), adezin faktör genleri (fnbA, fnbB, clfa) ve toksik şok sendrom toksin geni (tst) olmak üzere 12 farklı virülens geninin varlığı 3 farklı multipleks PZR
kullanılarak araştırıldı. İzolatların 30 (%71.4)’u clfa, 24 (%57.1)’ü hla, 14 (%33.3)’ü hlb, 10 (%23.8)’u fnbB, 6 (%14.2)’sıfnbA, 6 (%14.2)’sıetb, 5 (%11.9)’i sec, 5 (%11.9)’i tst, 3 (%7.1)’ü sea, 2 (%4.7)’si pvl ve 1 (%2.3)’i eta geni açısından pozitif bulundu. İzolatların hiçbirinde seb geni saptanamadı. İzolatların38’inde bir ya da daha fazla virülens geni, 29’unda ise iki ya da daha fazla virülens geni belirlendi. 4 izolatta ise incelenen virülens genlerin hiçbiri saptanamadı. İncelenen izolatlarda en fazla tespit edilen virülens genlerin clfa ve hla olduğu görüldü. Sonuç olarak sınırlı dahi olsa sığır mastitislerinin patogenezinde rol oynayan virülens gen patternleri ortaya konuldu. Çalışma sonuçlarının sığır mastitislerinin etiyolojisinde önemli rol oynayan
S. aureus’a ait virülens faktörlerinin karakterizasyon çalışmalarına katkı sağlayacağı kanaatine varıldı.

Keywords

Mastitis, Sığır, S. aureus, Virülens gen profili

Supporting Institution

Ondokuz Mayıs Üniversitesi Bilimsel Araştırma Projeleri Başkanlığı

Project Number

PYO.VET.1904.20.015

Thanks

Bu çalışma, Ondokuz Mayıs Üniversitesi Bilimsel Araştırma Projeleri Başkanlığı tarafından PYO.VET.1904.20.015 nolu proje kapsamında maddi olarak desteklenmiş ve aynı isimli yüksek lisans tezinden özetlenmiştir.

Determination of Virulence Gene Profiles of Staphylococcus aureus Isolated from Cattle with Mastitis

Abstract

This study aimed to determine the molecular identification of Staphylococcus aureus isolates and some important virulence genes from 140 staphylococci samples isolated from cow milk with mastitis in the culture collection of the department. For this purpose, the presence of the thermonuclease (nuc) gene in 140 S. aureus isolates was examined by PCR. Forty‐two isolates that were positive for the nuc gene (279 bp) were identified as S. aureus. In addition, the isolates were examined for the presence of 12 different virulence genes, including leukotoxin gene (pvl), staphylococcal classical enterotoxin genes (sea, seb, sec), exfoliative toxin genes (eta, etb), hemolysin genes (hla, hlb), adhesin factor genes (fnbA, fnbB, clfa), and toxic shock syndrome toxin gene (tst), using three different multiplex PCR. Of the isolates were found to be positive 30 (71.4%) for clfa, 24 (57.1%) hla, 14 (33.3%) hlb, 10 (23.8%) fnbB, 6 (14.2%) fnbA, 6 (14.2%) etb, 5 (11.9%) sec, 5 (11.9%) tst, 3 (7.1%) sea, 2 (4.7%) pvl, and 1 (2.3%) eta genes. The seb gene was not detected in any of the isolates. At the same time one or more virulence genes were determined in 38 of 42 isolates, two or more virulence genes in 29 of the isolates. None of the virulence genes examined in 4 isolates could be detected. It was seen that the most detected virulence genes in the analyzed isolates were clfa and hla. As a result, virulence gene patterns that play a role in the pathogenesis of bovine mastitis, even if limited, were revealed. It was concluded that the results would contribute to the characterization studies of virulence factors belonging to S. aureus, which play an important role in the etiology of bovine mastitis.

Keywords

Bovine, Mastitis, S. aureus, Virulence gen profile

Project Number

PYO.VET.1904.20.015

References

• Artursson K, Söderlund R, Liu L, Monecke S, Schelin J, 2016: Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics. Vet Microbiol, 193, 156-161.
• Bar-Gal GK, Blum SE, Hadas L, Ehricht R, Monecke S, Leitner G, 2015: Host-specificity of Staphylococcus aureus causing intramammary infections in dairy animals assessed by genotyping and virulence genes. Vet Microbiol, 176. 143-154.
• Boynukara B, Gulhan T, Alisarli M, Gurturk K, Solmaz H, 2008: Classical enterotoxigenic characteristics of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Van, Turkey. Int J Food Microbiol, 125(2), 209-211.
• Fursova KK, Shchannikova MP, Loskutova IV, Shepelyakovskaya AO, Laman AG, Boutanaev AM, Sokolov SL, Artemeva OA, Nikanova DA, Zinovieva NA, Brovko FA, 2018: Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia. J Dairy Sci, 101, 4325-4331.
• Fursova, K, Sorokin A, Sokolov S, Dzhelyadin T, Shulcheva I, Shchannikova M, Nikanova D, Artemeva O, Zinovieva N, Brovko F, 2020: Virulence factors and phylogeny of Staphylococcus aureus associated with bovine mastitis in russia based on genome sequences. Front Vet Sci, 7(135), doi: 10.3389/fvets.2020.00135.
• Grispoldi L, Massetti L, Sechi P, Iulietto MF, Ceccarelli M, Karama M, Popescu PA, Pandolfi F, Cenci-Goga BT, 2019: Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows. J Dairy Sci, 102, 1059-1065.
• Kot B, Szweda P, Frankowska-Maciejewska A, Piechota M, Wolska K, 2016: Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland. J Dair Res, 83, 228-235.
• Kotzamanidis C, Vafeas G, Giantzi V, Anastasiadou S, Mygdalias S, Malousi A, Loukia E, Daniel S, Zdragas A, 2021: Staphylococcus aureus isolated from ruminants with mastitis in Northern Greece dairy herds: genetic relatedness and phenotypic and genotypic characterization. Toxins, 13(176), https://doi.org/10.3390/toxins13030176.
• Kreausukon K, Fetsch A, Kraushaar B, Alt K, Müller K, Krömker V, Zessin KH, Käsbohrer A, Tenhagen BA, 2012: Prevalence, antimicrobial resistance, and molecular characterization of methicillin-resistant Staphylococcus aureus from bulk tank milk of dairy herds. J Dairy Sci, 95, 4382-4388.
• Li X, Fang F, Zhao J, Lou N, Li C, Huang T, Li Y, 2018: Molecular characteristics and virulence gene profiles of Staphylococcus aureus causing bloodstream infection. Braz J Infect Dis,. 22, 487-494.
• Liu H, Li S, Meng L, Dong L, Zhao S, Lan X, Wang J, Zheng N, 2017: Prevalence, antimicrobial susceptibility, and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China. J Dairy Sci, 100, 8796-8803.
• Ote I, Taminiau B, Duprez JN, Dizier I, Mainil JG, 2011: Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis. Vet Microbiol, 153, 285-292.
• Pereyra EAL, Picech F, Renna MS, Baravalle C, Andreotti CS, Russi R, Calvinho LF, Diez C, Dallard BE, 2016: Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells. Vet Microbiol, 183, 69-77.
• Ronco T, Klaas IC, Stegger M, Svennesen L, Astrup LB, Farre M, Pedersen K, 2018: Genomic investigation of Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis. Vet Microbiol, 215, 35-42.
• Seyoum ET, Mekonene TK, Woldetsadik DA, Zewudie BM, Gebreyes WA, 2016: Enterotoxin gene profile of Staphylococcus aureus isolates recovered from bovine milk produced in central Ethiopia. J Infect Dev Cttries, 10(2), 138-142.
• Sezener MG, Fındık A, Ergüden VE, Akgöz S, Gülhan T, Çiftci A, 2019: Mastitis izolatı Staphylococcus aureus suşlarında antibiyotik dirençliliğinin ve bazı virülens genlerinin araştırılması. J Anatol Environ Animal Sci, 4(2), 182-187.
• Wang D, Zhang L, Zhou X, He Y, Yong C, Shen M, Szenci O, Han B, 2016: Antimicrobial susceptibility, virulence genes, and randomly amplified polymorphic DNA analysis of Staphylococcus aureus recovered from bovine mastitis in Ningxia, China. J Dairy Sci, 99, 9560-9569.
• Wang D, Zhang L, Yong C, Shen M, Ali T, Shahid M, Han K, Zhou X, Han B, 2017: Relationships among superantigen toxin gene profiles, genotypes, and pathogenic characteristics of Staphylococcus aureus. J Dairy Sci, 100(6), 4276-4286.
• Wang X, Wang X, Wang Y, Guo G, Usman T, Hao D, Tang X, Zhang Y, Yu Y, 2014: Antimicrobial resistance and toxin gene profiles of Staphylococcus aureus strains from Holstein milk. Lett Appl. Microbiol, 58, 527-534.
• Xu J, Tan X, Zhang X, Xiaoli X, Sun H, 2015: The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd. Microb Pathog, 88, 29-38.
• Yang FL, Li XS, Liang XW, Zhang XF, Qin GS, Yang BZ, 2012: Detection of virulence associated genes in Staphylococcus aureus isolated from bovine clinical mastitis milk samples in Guangxi. Trop Anim Health Prod, 44, 1821-1826.
• Yang F, Zhang S, Shang X, Li H, Zhang H, Cui D, Wang X, Wang L, Yan Z, Sun Y, 2020: Detection and molecular characterization of methicillin-resistant Staphylococcus aureus isolated from subclinical bovine mastitis cases in China. J Dairy Sci, 103, 840-845.
• Zhang L, Gao J, Barkema HW, Ali T, Liu G, Deng Y, Naushad S, Kastelic JP, Han B, 2018: Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China. Vet Res, 14(63), https://doi.org/10.1186/s12917-018-1374-7.
• Zuniga E, Melville PA, Saidenberg ABS, Laes MA, Gonsales FF, Salaberry SRS, Gregori F, Brandao PE, dos Santos FGB, Lincopan NE, Benites NR, 2015: Occurrence of genes coding for MSCRAMM and biofilm-associated protein Bap in Staphylococcus spp. isolated from bovine subclinical mastitis and relationship with somatic cell counts. Microb Pathog, 89, 1-6.