Yenidoğan buzağı septisemilerinden elde edilen Escherichia coli izolatlarının enterotoksijenik virülans genleri, serotip ve antibiyotik duyarlılıklarının belirlenmesi

Yıl 2025, Cilt: 36 Sayı: 1, 27 - 37, 24.07.2025

Serhan Akgöz , Timur Gülhan

https://doi.org/10.35864/evmd.1607193

Öz

Bu çalışma, neonatal septisemili buzağılardan E. coli izolasyonu, izolatlardaki antibiyotik direncinin tespiti, sık görülen serotip ve virülens faktörlerinin belirlenmesi amacıyla yürütüldü. Bu amaçla septisemili buzağılarda PCR ile analiz edilen ve doğrulanan izolatlarda önemli serotip genleri, virülens genleri ve 8 farklı antibiyotiğe ait direnç genleri araştırıldı. Fenotipik antibiyotik direnç analizine göre izolatların % 94,7'si streptomisin, % 93,7'si tetrasiklin, % 89,5'i ampisilin, % 87,4'ü trimetoprim-sülfametoksazol, % 87,4'ü kloramfenikol, % 74,7'si enrofloksasin, % 62,1'i gentamisin ve % 24,2'si sefalotine dirençli bulundu. İzolatlarda % 88,4 tet(B), % 74,7 sul3, % 67,4 sul1, % 61 cmlA, % 56,8 qnr(B), % 54,7 tet(A), % 53,7 sul2, % 38,9 CITM, % 36,8 aadA1, % 28,4 cat1, % 26,3 qnr(S), % 12,6 aac[3]-IV, % 5,3 tet(C) ve % 1.05 qnr(A) genleri belirlendi. Serogenotipleme sonucunda izolatların % 33,7'sinde O101, % 6,3'ünde O26, % 2,1'inde ise O145 serotip genlerinin varlığı tespit edildi. Virülans genlerinin belirlenmesi amacıyla yapılan PCR çalışmaları sonucunda; İzolatların % 23.2'sinin East1, % 22.1'inin F5, % 20'sinin STa, % 17.9'unun F41 ve % 3.2'sinin LT genine sahip olduğu belirlendi. Böylece örnekleme bölgesinde neonatal septisemili buzağılarda ETEC varlığı, yaygınlığı, izolatların patotip/virotip dağılımı ortaya konuldu ve hastalığın tedavisinde kullanılabilecek en uygun antibiyotik seçeneği belirlendi.

Anahtar Kelimeler

Antibiyotik direnci , Buzağı , E. coli , Serotip , Virülens

Etik Beyan

Bu araştırma için etik kurul onayı gerekmemektedir.

Destekleyen Kurum

Bu çalışma Ondokuz Mayıs Üniversitesi tarafından maddi olarak desteklenmiştir (proje numarası: PYO.VET.1904.21.031).

Proje Numarası

PYO.VET.1904.21.031

Teşekkür

Bu makale, birinci yazarın (Serhan AKGÖZ) Prof. Dr. Timur GÜLHAN danışmanlığında ve Ondokuz Mayıs Üniversitesi tarafından PYO.VET.1904.21.031 proje numarasıyla desteklenen ‘‘Neonatal Septisemili Buzağı Kökenli Escherichia Coli İzolatlarında Enterotoksijenik Virülans Genlerinin Tanımlanması’’ başlıklı doktora tezinden yapılmıştır. Ayrıca 26-28 Ekim 2022 tarihleri ​​arasında Şanlıurfa, Türkiye’de düzenlenen “XV. Ulusal Veteriner Mikrobiyoloji Kongresi”nde konferans özet bildirisi olarak sunulmuştur.

Yenidoğan buzağı septisemilerinden elde edilen Escherichia coli izolatlarının enterotoksijenik virülans genleri, serotip ve antibiyotik duyarlılıklarının belirlenmesi

Öz

This study was conducted for the isolation of E. coli from calves with neonatal septicemia, the determination of antibiotic resistance, common serotypes and virulence factors. For this purpose, important serotype genes, virulence genes and resistance genes of 8 different antibiotics in calves with septicemia in confirmed isolates analyzed by PCR. According to the phenotypic antibiotic resistance analysis, the isolates were 94,7% streptomycin, 93,7% tetracycline, 89,5% ampicillin, 87,4% trimethoprim-sulfamethoxazole, 87,4% chloromphenicol, 74,7% enrofloxacin, 62,1% gentamicin and 24,2% was found to be resistant to cephalothin. In isolates, 88.4% tet(B), 74.7% sul3, 67.4% sul1, 61% cmlA, 56.8% qnr(B), 54.7% tet(A), 53.7% sul2, 38.9% CITM, 36.8% aadA1, 28.4% cat1, 26.3% qnr(S), 12.6% aac[3]-IV, 5.3% tet(C) and 1.05% qnr(A) genes were identified. According to the presence of O101, O26 and O145 serotype genes, the isolates were found to be 33.7%, 6.3% and 2.1%, respectively, as a result of serogenotyping. As a result of PCR studies carried out to determine virulence genes; It was determined that 23.2% isolates had East1, 22.1% isolates F5, 20% isolates STa, 17.9% F41 and 3.2% isolates LT. Thus, the presence, prevalence of ETEC in calves with neonatal septicemia in the sampling region, the pathotype/virotype distribution of the isolates were determined and the most appropriate antibiotic option that could be used in the treatment of disease.

Anahtar Kelimeler

Antibiotic resistance , Calf , E. coli , Virulence , Serotype

Etik Beyan

Ethics committee approval is not required for this research.

Destekleyen Kurum

This study was financially supported by Ondokuz Mayis University (project number: PYO.VET.1904.21.031).

Proje Numarası

PYO.VET.1904.21.031

Teşekkür

This article is extracted from first author’s Ph.D. dissertation entitled ‘‘Identification Of Enterotoxigenic Virulence Genes In Calf Originated Escherichia Coli Isolates With Neonatal Septicemia’’, supervised by Prof. Dr. Timur GÜLHAN and supported by Ondokuz Mayıs University with the project number PYO.VET.1904.21.031. Also presented as conference abstract paper at “XV. National Congress Of Veterinary Microbiology”, 26-28 October 2022, Şanlıurfa, Türkiye.

Kaynakça

• References Akyüz E, Naseri A, Erkılıç EE, Makav M, Uzlu E, Kırmızıgül AE, Gökçe G. (2017) Neonatal Buzağı İshalleri ve Sepsis. Kafkas Univ Vet Fak Derg. 10(2), 181-191.
• Al-Abri SS, Beeching NJ, Nye FJ. (2005) Traveller's diarrhoea. Lancet Infect Dis. 5 (6), 349-360. doi: 10.1016/s1473-3099(05)70139-0.
• Algammal AM, El-Kholy AW, Riad EM, Mohamed HE, Elhaig MM, Yousef SAA, Hozzein WN, Ghobashy MOI. (2020) Genes Encoding the Virulence and the Antimicrobial Resistance in Enterotoxigenic and Shiga-Toxigenic E. coli Isolated from Diarrheic Calves. Toxins (Basel). 12 (6). doi: 10.3390/toxins12060383.
• Astorga F, Navarrete-Talloni MJ, Miró MP, Bravo V, Toro M, Blondel CJ, Hervé-Claude LP. (2019) Antimicrobial resistance in E. coli isolated from dairy calves and bedding material. Heliyon. 5 (11), e02773. doi: 10.1016/j.heliyon.2019.e02773.
• Aydın F, Umur S, Gökçe G, Genç O, Güler MA. (2001) The Isolation and Identification of Bacteria and Parasites from Diarrhoeic Calves in Kars District. Kafkas Univ Vet Fak Derg. 7(1), 7-14.
• Bartels CJ, Holzhauer M, Jorritsma R, Swart WA, Lam TJ. (2010) Prevalence, prediction and risk factors of enteropathogens in normal and non-normal faeces of young Dutch dairy calves. Prev Vet Med. 93 (2-3), 162-169. doi: 10.1016/j.prevetmed.2009.09.020.
• Cattoir V, Poirel L, Rotimi V, Soussy C-J, Nordmann P. (2007) Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. Journal of Antimicrobial Chemotherapy. 60 (2), 394-397. doi: 10.1093/jac/dkm204.
• Cengiz S, Adıgüzel MC. (2020) Determination of virulence factors and antimicrobial resistance of E. coli isolated from calf diarrhea, part of eastern Turkey. Ankara Üniv Vet Fak Derg. 67 (4), 365-371. doi: 10.33988/auvfd.640990.
• Chanawong A, M'Zali FH, Heritage J, Lulitanond A, Hawkey PM. (2000) Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP). FEMS Microbiol Lett. 184 (1), 85-89. doi: 10.1111/j.1574-6968.2000.tb08995.x.
• Çitil M, Gökçe E. (2013) Neonatal Septisemi. Turk Klin J Vet Sci. 4(1), 62-70.
• de Verdier K, Nyman A, Greko C, Bengtsson B. (2012) Antimicrobial resistance and virulence factors in Escherichia coli from Swedish dairy calves. Acta Vet Scand. 54 (1), 2. doi: 10.1186/1751-0147-54-2.
• Eid HM, Algammal AM, Elfeil WK, Youssef FM, Harb SM, Abd-Allah EM. (2019) Prevalence, molecular typing, and antimicrobial resistance of bacterial pathogens isolated from ducks. Vet World. 12 (5), 677-683. doi: 10.14202/vetworld.2019.677-683.
• El Ayis AA, Elgaddal AA, Almofti YA. (2015) Isolation, Identification and Enterotoxin Detection of Escherichia Coli Isolated from Calf Diarrhea and their Virulence Characteristics. J App Ind Sci. 3 (4), 141-149.
• El-Razik K, Abdelrahman K, Ahmed Y, Gomaa AM, Eldebaky H. (2010) Direct Identification of Major Pathogens of the Bubaline Subclinical Mastitis in Egypt using PCR. Am J Sci. 6(10), 652-660.
• Engelen F, Thiry D, Devleesschauwer B, Heyndrickx M, Mainil J, De Zutter L, Cox E. (2021) Pathogenic potential of Escherichia coli O157 and O26 isolated from young Belgian dairy calves by recto-anal mucosal swab culturing. J Appl Microbiol. 131 (2), 964-972. doi: 10.1111/jam.14909.
• Farfan AE, Ariza SC, Vargas FA, Vargas LV. (2016) Virulence mechanisms of enteropathogenic Escherichia coli. Chilean J Infectol, 33, 438–450. doi: 10.4067/S0716-10182016000400009.
• Feuerstein A, Scuda N, Klose C, Hoffmann A, Melchner A, Boll K, Rettinger A, Fell S, Straubinger RK, Riehm JM. (2021) Antimicrobial Resistance, Serologic and Molecular Characterization of E. coli Isolated from Calves with Severe or Fatal Enteritis in Bavaria, Germany. Antibiotics (Basel). 11 (1), 23. doi: 10.3390/antibiotics11010023.
• Foster DM, Smith GW. (2009) Pathophysiology of diarrhea in calves. Vet Clin North Am Food Anim Pract. 25 (1), 13-36. doi: 10.1016/j.cvfa.2008.10.013.
• Gharieb R, Fawzi E, Elsohaby I. (2019) Antibiogram, virulotyping and genetic diversity of Escherichia coli and Salmonella serovars isolated from diarrheic calves and calf handlers. Comp Immunol Microbiol Infect Dis. 67, 101367. doi: 10.1016/j.cimid.2019.101367.
• González Pasayo RA, Sanz ME, Padola NL, Moreira AR. (2019) Phenotypic and genotypic characterization of enterotoxigenic Escherichia coli isolated from diarrheic calves in Argentina. Open Vet J. 9 (1): 65-73. doi: 10.4314/ovj.v9i1.12.
• Gow SP, Waldner CL, Harel J, Boerlin P. (2008) Associations between antimicrobial resistance genes in fecal generic Escherichia coli isolates from cow-calf herds in western Canada. Appl Environ Microbiol. 74 (12), 3658-3666. doi: 10.1128/aem.02505-07.
• Gow SP, Waldner CL. (2009) Antimicrobial resistance and virulence factors stx1, stx2, and eae in generic Escherichia coli isolates from calves in western Canadian cow-calf herds. Microb Drug Resist. 15 (1), 61-67. doi: 10.1089/mdr.2009.0860.
• Guerra B, Junker E, Schroeter A, Helmuth R, Guth BE, Beutin L. (2006) Phenotypic and genotypic characterization of antimicrobial resistance in Escherichia coli O111 isolates. J Antimicrob Chemother. 57 (6), 1210-1214. doi: 10.1093/jac/dkl127.
• Güler L, Gündüz K, Ok U. (2008) Virulence factors and antimicrobial susceptibility of Escherichia coli isolated from calves in Turkey. Zoonoses Public Health. 55 (5), 249-257. doi: 10.1111/j.1863-2378.2008.01121.x.
• Gülhan T, İlhan Z, Aksakal A, Solmaz H, Ekin İH. (2009) Hayvan Orijinli Escherichia coli Suşlarının Enterotoksin Tiplerinin (LT, ST) Belirlenmesi. YYU Vet Fak Derg. 20(2), 27-31.
• Hakim AS, Omara ST, Syame SM, Fouad EA. (2017) Serotyping, antibiotic susceptibility, and virulence genes screening of Escherichia coli isolates obtained from diarrheic buffalo calves in Egyptian farms. Vet World. 10 (7), 769-773. doi: 10.14202/vetworld.2017.769-773.
• Haque MA, Hossain MT, Islam MS, Islam MZ, Islam P, Shaha SN, Sikder MH, Rafiq K. (2022) Isolation of multidrug-resistant Escherichia coli and Salmonella spp. from sulfonamide-treated diarrheic calves. Vet World. 15 (12), 2870-2876. doi: 10.14202/vetworld.2022.2870-2876.
• Hasan M, Hussein M, Yousif AA. (2018) Confirmatory Detection of Escherichia coli O157:H7 in Diarrheic and Non-Diarrheic Calves by using Real Time PCR with Studying the Antimicrobial Susceptibility of these Bacteria. J Glob Pharma Technol. 10, 90-96. doi: 10.5958/0974-360X.2019.00046.5.
• Jia Y, Mao W, Liu B, Zhang S, Cao J, Xu X. (2022) Study on the drug resistance and pathogenicity of Escherichia coli isolated from calf diarrhea and the distribution of virulence genes and antimicrobial resistance genes. Front Microbiol. 13, 992111. doi: 10.3389/fmicb.2022.992111.
• Karakaya E, Aydin F, Kayman T, Abay S. (2022) Escherichia coli in different animal feces: phylotypes and virulence genes. World J Microbiol Biotechnol, 39(1), 14. doi: 10.1007/s11274-022-03451-w.
• Khachatryan AR, Hancock DD, Besser TE, Call DR. (2004) Role of calf-adapted Escherichia coli in maintenance of antimicrobial drug resistance in dairy calves. Appl Environ Microbiol. 70 (2), 752-757. doi: 10.1128/aem.70.2.752-757.200432.
• Khawaskar DP, Sinha DK, Lalrinzuala MV, Athira V, Kumar M, Chhakchhuak L, Mohanapriya K, Sophia I, Abhishek, Kumar ORV, Chaudhuri P, Singh BR, Thomas P. (2022) Pathotyping and antimicrobial susceptibility testing of Escherichia coli isolates from neonatal calves. Vet Res Commun. 46 (2), 353-362. doi: 10.1007/s11259-021-09857-5.
• Kozak G, Boerlin P, Janecko N, Reid-Smith R, Jardine C. (2009) Antimicrobial Resistance in Escherichia coli Isolates from Swine and Wild Small Mammals in the Proximity of Swine Farms and in Natural Environments in Ontario, Canada. J Appl Environ Microbiol. 75, 559-566. doi: 10.1128/AEM.01821-08.
• Lim JY, Yoon J, Hovde CJ. (2010) A brief overview of Escherichia coli O157:H7 and its plasmid O157. J Microbiol Biotechnol. 20(1), 5-14.
• Liu B, Wu F, Li D, Beutin L, Chen M, Cao B, Wang L. (2010) Development of a serogroup-specific DNA microarray for identification of Escherichia coli strains associated with bovine septicemia and diarrhea. Vet Microbiol. 142 (3-4), 373-378. doi: 10.1016/j.vetmic.2009.10.019.
• 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), 268-281. doi: 10.1111/j.1469-0691.2011.03570.x.
• Mainga AO, Cenci-Goga BT, Malahlela MN, Tshuma T, Kalake A, Karama M. (2018) Occurrence and characterization of seven major Shiga toxin-producing Escherichia coli serotypes from healthy cattle on cow-calf operations in South Africa. Zoonoses Public Health. 65 (7), 777-789. doi: 10.1111/zph.12491.
• Naderi Z, Ghanbarpour R, Sami M. (2016) Antimicrobial Resistance Characteristics and Phylogenetic Groups of Escherichia coli Isolated From Diarrheic Calves in Southeast of Iran. Journal of Enteric Pathogens. 4(4), 21-27.
• Nagy B, Fekete PZ. (2005) Enterotoxigenic Escherichia coli in veterinary medicine. Int J Med Microbiol. 295 (6-7), 443-454. doi: 10.1016/j.ijmm.2005.07.003.
• Pérez-Pérez FJ, Hanson ND. (2002) Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol. 40 (6), 2153-2162. doi: 10.1128/jcm.40.6.2153-2162.2002.
• Picco NY, Alustiza FE, Bellingeri RV, Grosso MC, Motta CE, Larriestra AJ, Vissio C, Tiranti KI, Terzolo HR, Moreira AR, Vivas AB. (2015) Molecular screening of pathogenic Escherichia coli strains isolated from dairy neonatal calves in Cordoba province, Argentina. Rev Argent Microbiol. 47 (2), 95-102. doi: 10.1016/j.ram.2015.01.006.
• Pourtaghi H, Sodagari H. (2016) Antimicrobial Resistance of Entrotoxigenic and Non-Entrotoxigenic Escherichia coli Isolated From Diarrheic Calves in Iran. Int J Enteric Pathog. 4(2), e34557. doi: 10.17795/ijep34557.
• Prieto A, López-Novo C, Díaz P, Díaz-Cao JM, López-Lorenzo G, Antón C, Remesar S, García-Dios D, López C, Panadero R, Díez-Baños P, Morrondo P, Fernández G. (2022) Antimicrobial Susceptibility of Enterotoxigenic Escherichia coli from Diarrhoeic Neonatal Calves in Spain. Animals. 12(3), 264.
• Rai S, Kumar M, Jas R, Mandal GP, Samanta I, Rajendar M, Tripura S, Das SK, Mondal M, Mandal DK. (2023) Antibacterial effect of kitchen herbs against pathogenic multidrug-resistant E. coli isolates from calf diarrhoea. Trop Anim Health Prod. 55 (3), 211. doi: 10.1007/s11250-023-03628-x.
• Salaheen S, Kim SW, Cao H, Wolfgang DR, Hovingh E, Karns JS, Haley BJ, Van Kessel JAS. (2019) Antimicrobial Resistance Among Escherichia coli Isolated from Veal Calf Operations in Pennsylvania. Foodborne Pathog Dis. 16 (1), 74-80. doi: 10.1089/fpd.2018.2530.
• Shahrani M, Dehkordi FS, Momtaz H. (2014) Characterization of Escherichia coli virulence genes, pathotypes and antibiotic resistance properties in diarrheic calves in Iran. Biol Res. 47 (1), 28. doi: 10.1186/0717-6287-47-28.
• Skarżyńska M, Zaja CM, Bomba A, Bocian Ł, Kozdruń W, Polak M, Wia Cek J, Wasyl D. (2021) Antimicrobial Resistance Glides in the Sky-Free-Living Birds as a Reservoir of Resistant Escherichia coli With Zoonotic Potential. Front Microbiol. 12, 656223. doi: 10.3389/fmicb.2021.656223.
• Sobhy NM, Yousef SGA, Aboubakr HA, Nisar M, Nagaraja KV, Mor SK, Valeris-Chacin RJ, Goyal SM. (2020) Virulence factors and antibiograms of Escherichia coli isolated from diarrheic calves of Egyptian cattle and water buffaloes. PLoS One. 15 (5), e0232890. doi: 10.1371/journal.pone.0232890.
• Taghadosi R, Shakibaie MR, Alizade H, Hosseini-Nave H, Askari A, Ghanbarpour R. (2018) Serogroups, subtypes and virulence factors of shiga toxin-producing Escherichia coli isolated from human, calves and goats in Kerman, Iran. Gastroenterol Hepatol Bed Bench. 11 (1), 60-67.
• Tyson GH, McDermott PF, Li C, Chen Y, Tadesse DA, Mukherjee S, Bodeis-Jones S, Kabera C, Gaines SA, Loneragan GH, Edrington TS, Torrence M, Harhay DM, Zhao S. (2015) WGS accurately predicts antimicrobial resistance in Escherichia coli. J Antimicrob Chemother. 70 (10), 2763-2769. doi: 10.1093/jac/dkv186.
• Umpiérrez A, Acquistapace S, Fernández S, Oliver M, Acuña P, Reolón E, Zunino P. (2016) Prevalence of Escherichia coli adhesion-related genes in neonatal calf diarrhea in Uruguay. J Infect Dev Ctries. 10 (5), 472-477. doi: 10.3855/jidc.7102.
• Wernli D, Haustein T, Conly J, Carmeli Y, Kickbusch I, Harbarth S. (2011) A call for action: the application of The International Health Regulations to the global threat of antimicrobial resistance. PLoS Med. 8(4),e1001022. doi: 10.1371/journal.pmed.1001022.
• Yadegari Z, Nikbakht Brujeni G, Ghorbanpour R, Moosakhani F, Lotfollahzadeh S. (2019) Molecular characterization of enterotoxigenic Escherichia coli isolated from neonatal calves diarrhea. Vet Res Forum. 10 (1), 73-78. doi: 10.30466/vrf.2019.34313.
• Zajacova ZS, Konstantinova L, Alexa P. (2012) Detection of virulence factors of Escherichia coli focused on prevalence of EAST1 toxin in stool of diarrheic and non-diarrheic piglets and presence of adhesion involving virulence factors in astA positive strains. Vet Microbiol. 154 (3-4), 369-375. doi: 10.1016/j.vetmic.2011.07.029.
• Zhao H, Sun R, Yu P, Alvarez PJJ. (2020) High levels of antibiotic resistance genes and opportunistic pathogenic bacteria indicators in urban wild bird feces. Environ Pollut. 266 (Pt 2), 115200. doi: 10.1016/j.envpol.2020.115200.