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Characterization of mechanisms of resistance to antibiotics and molecular typing of Escherichia coli isolates originating from cows and pigs

dc.contributor.advisorKrnjaić, Dejan
dc.contributor.advisorVelhner, Maja
dc.contributor.otherStanimirović, Zoran
dc.contributor.otherRadojičić, Marina
dc.contributor.otherGrego, Edita
dc.creatorTodorović, Dalibor
dc.date.accessioned2019-07-31T14:32:17Z
dc.date.available2019-07-31T14:32:17Z
dc.date.available2020-07-03T09:30:07Z
dc.date.issued2018-11-13
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=6850
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/11279
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:19962/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=51496463
dc.description.abstractU istraživanju su korišćeni izolati Escherichia coli iz mleka krava sa kliničkim mastitisom i od svinja s ciljem njihove molekularne tipizacije, ispitivanja rezistencije na antibiotike, karakterizacije gena za rezistenciju i identifikacije mobilnih genetičkih elemenata. Ispitano je ukupno sedam izolata iz mleka krava i 15 izolata od svinja. Svi izolati su poticali sa različitih farmi koje su se nalazile u Autonomnoj Pokrajini Vojvodini, izuzev jednog izolata E. coli od svinje koji je bio iz Mačvanskog upravnog okruga. Metodama elektroforeze u pulsnom polju (PFGE) i nasumične amplifikacije polimorfne DNK (RAPD), utvrđeno je da izolati E. coli nisu genetički srodni. Urađena je filogenetska tipizacija izolata metodom multipleks PCR sa tri prajmera za identifikaciju dva gena i jednog DNK fragmenta E. coli koji daju različite PCR produkte tako da se na osnovu njihove kombinacije izolati klasifikuju u četiri filogenetske grupe (A, B1, D i B2). Utvrđeno je da 17 izolata E. coli pripada filogenetskoj grupi A, tri izolata pripada grupi B1 i dva grupi D. Rezistencija na tri i više antibiotika je nađena kod svih 22 izolata E. coli na osnovu čega se smatra da su izolati multirezistentni. Rezistencija na fluorohinolone koji spadaju u grupu značajnih antibiotika u humanoj medicini, nađena je kod 13 izolata E. coli. Kod izolata E. coli 1M-13, 2M-13, 3M-13 i 2S-13, nađene su četiri mutacije na genima koji kodiraju topoizomeraze i to na gyrA genu nađene su mutacije Ser83→Leu i Asp87→Asn, na genu parC identifikovana je mutacija Ser80→Ile, a na parE genu Ser458→Ala. Kod izolata 7S-13 nađene su identične mutacije osim na parE genu gde je utvrđena supstitucija na Ser448→Thr. Kod četiri izolata E. coli 3S-13, 6S-13, 14S-14 i 15S-14, nađene su identične mutacije na gyrA i parC genima kao kod prethodnih izolata osim što je kod izolata broj 6S-13 na parC genu identifikovana izmena aminokiselina Glu84→Lys. Kod izolata 4M-13, 1S-13, 5S-13, 12S-13, gde je MIK na ciprofloksacin bio od 0.5 do 0.125 mg/L, nađena je samo jedna mutacija na gyrA genu i to Ser83→Leu, osim kod izolata 5S-13 gde je identifikovana mutacija Asp87→Asn (MIK na CIP 0.062 mg/L). Plazmidom prenosiva rezistencija na hinolone identifikovana je kod jednog izolata E. coli iz mleka (3M-13) i kod jednog izolata E. coli od svinje (7S-13) kodirana aac6’Ib-cr genom, dok je kod jednog izolata E. coli od svinje (16S-14) identifikovan qnrS gen. Rezistencija na prošireni spektar cefalosporina nađena je kod po jednog izolata E. coli iz mleka krave (7M-14) i od svinje (7S-13) i identifikovan je gen blaCTX-M-1. Kod najvećeg broja izolata (17 izolata E. coli) identifikovani su geni koji kodiraju enzime koji inaktivišu streptomicin, odnosno strA i strB geni...sr
dc.description.abstractEscherichia coli isolates from milk samples of cows with clinical mastitis and from pigs were used for molecular typing, to determine antimicrobial resistance and to characterize resistance genes and mobile genetic elements. Seven isolates from cow milk and 15 isolates from pigs were included in the study. All of the isolates originated from farms located in the Autonomous Province of Vojvodina except for one isolate which originated from a pig raised in a farm located in the district of Mačva. Applying Pulsed-Field Gel Electrophoresis (PFGE) and Random Amplified Polymorphic DNA (RAPD) analysis it was established that all of the isolates were genetically distinguishable. The phylogenetic analysis was done by multiplex PCR method with three primers for identification of two genes and one DNA fragment. This method enables the classification of E. coli isolates into four phylogenetic groups (A, B1, D and B2) depending on the amplicon combination obtained. Seventeen E. coli isolates belong to phylogenetic group A, three isolates belong to group B1 and two isolates were classified in group D. All of the isolates (Nos 22) were multidrug resistant as resistance was found to >3 antibiotics. Resistance to fluoroquinolones which are important antibiotics in human medicine was found in 13 isolates. In all of these isolates single or multiple mutation were found in the topoisomerase genes. In isolates 1M-13, 2M-13, 3M-13, 2S-13, four amino acid substitutions were found i.e. Ser83→Leu i Asp87→Asn on gyrA gene, Ser80→Ile on parC gene and Ser458→Ala on parE gene. In isolate 7S-13 identical mutations were found on gyrA and parC genes but in parE gene amino acid substitution Ser448→Thr was found. In isolates 3S-13, 6S-13, 14S-14 and 15S-14, the same amino acid substitutions were found on gyrA and parC genes as well except for the isolate 6S-13, where on parC gene amino acid substitution Glu84→Lys was found. Single point mutation was found on gyrA gene in isolates 4M-13, 1S-13, 5S-13, 12S-13 with the minimal inhibitory concentration to ciprofloxacin of 0.5 to 0.125 mg/L. Isolates 4M-13, 1S-13 and 12S-13 had mutation Ser83→Leu, while isolate 5S-13 had mutation Asp87→Asn and MIC to CIP was 0.062 mg/L. Plasmid mediated resistance was identified in one isolate of E. coli from cow milk (3M-13) and one isolate from a pig (7S-13) and it was encoded by aac6’Ib-cr gene while in one isolate from a pig (16S-14) the qnrS gene was identified. The blaCTX-M-1 gene conferring resistance to extended-spectrum cephalosporins was found in one isolate from cow milk (7M-14) and one isolate from a pig (7S-13). The strA and strB pair of genes, encoding streptomycin inactivating enzymes were found in 17 isolates...en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Факултет ветеринарске медицинеsr
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Београдуsr
dc.subjectEscherichia colisr
dc.subjectEscherichia colien
dc.subjectsvinjesr
dc.subjectmleko kravasr
dc.subjectmastitisisr
dc.subjectPFGEsr
dc.subjectRAPDsr
dc.subjectgeni za rezistencijusr
dc.subjectplazmidisr
dc.subjectintegronisr
dc.subjectpigsen
dc.subjectcow milken
dc.subjectPFGEen
dc.subjectRAPDen
dc.subjectresistance genesen
dc.subjectplasmidsen
dc.subjectintegronsen
dc.titleKarakterizacija mehanizama rezistencije na antibiotikе i molekularna tipizacija izolata Escherichia coli poreklom od goveda i svinjasr
dc.title.alternativeCharacterization of mechanisms of resistance to antibiotics and molecular typing of Escherichia coli isolates originating from cows and pigsen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/19881/IzvestajKomisije20730.pdf
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/19880/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/19880/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/19881/IzvestajKomisije20730.pdf
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_11279


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