Fenotipske i genotipske karakteristike Escherichia coli izolovanih iz fecesa pasa

Abstract

Šiga toksin produkujuće Escherichia coli (STEC) čine jednu od šest grupa dijarejagenih E. coli. Na svjetskom nivou, infekcija izazvana STEC najčešći je uzrok akutne renalne insuficijencije kod djece i starijih osoba. Kako su psi prepoznati kao rezervoari STEC, blizak kontakt ljudi i pasa predstavlja rizik za zoonotsku transmisiju ovih bakterija. U Republici Srbiji, osim ograničenog broja istraživanja o prisustvu STEC kod domaćih životinja i ljudi nema dostupnih literaturnih podataka o istraživanjima ove grupe E. coli kod pasa. Stoga, cilj ove doktorske disertacije bio je da se dokaže prisustvo i utvrdi prevalencija STEC u populaciji pasa s teritorije grada Novog Sada, izvrši tipizacija i molekulrna karakterizacija sojeva E. coli izolovanih iz fecesa pasa i utvrdi prisustvo rezistentnih sojeva E. coli. U ovo istraživanje bio je uključen 101 pas s teritorije Novog Sada, a kao materijal korišćeni su uzorci fecesa pasa. U cilju izolacije E. coli iz fecesa pasa primijenjeni su standardni bakteriološki metodi izolacije i biohemijske identifikacije, a potvrda pripadnosti vrsti izvršena je korišćenjem matricom potpomognute laserske desorpcije/jonizacije-vrijeme preleta masene spektrometrije (MALDI-TOF MS). Za utvrđivanje prisustva STEC kod pasa primijenjeni su metodi lateks aglutinacije za serogrupu O157, direktne aglutinacije za serogrupe “velike šestorke” O26, O45, O103, O111, O121 i O145, test verocitotoksičnosti (VCA), imunoenzimski test (ELISA), kao i lančana reakcija polimeraze (PCR). Antibiotska rezistencija utvrđena je disk difuzionim metodom prema standardima EUCAST i CLSI. U ovom istraživanju, po prvi put u Republici Srbiji, dokazano je prisustvo STEC u populaciji pasa. Primjenom VCA metoda ustanovljena je prevalencija od 1,98%, dok je primjenom ELISA i PCR metoda ustanovljena prevalencija od 5,94% odnosno 4,95%. Dva izolata E. coli pripadala su nekoj od serogrupa “velike šestorke” STEC, dok prisustvo sojeva iz serogrupe O157 nije utvrđeno. U ispitivanoj populaciji pasa utvrđeno je prisustvo sojeva E. coli rezistentnih na jedan ili više korišćenih antibiotika, kao i jednog multirezistentnog izolata. Najviše izolata E. coli pokazalo je rezistenciju na ampicilin (22,5%), dok nijedan izolat nije pokazao rezistenciju na gentamcin. Pozitivna povezanost utvrđena je između stila života psa i prisustva STEC. Naime, značajno veća prevalencija STEC, u odnosu na vlasničke pse, zabilježena je kod pasa lutalica, što je vjerovatno posljedica neograničene slobode kretanja, a time i češćeg kontakta s kontaminiranom hranom i vodom kao izvorom STEC. Nalaz STEC i izolata E. coli rezistentnih na antibiotike kod pasa od značaja je kako s aspekta male kliničke prakse tako i s aspekta javnog zdravlja u Republici Srbiji. Datum

Shiga toxin-producing Escherichia coli (STEC) strains constitute one of six groups of diarrheagenic E. coli. Infection caused by STEC is the most common cause of acute renal failure in young children and elderly people worldwide. As dogs are recognized as a reservoir for STEC, the close contact between humans and dogs poses a risk for zoonotic transmission of these bacteria. Except scarce studies of STEC in humans and domestic animals, there are no available data about this E. coli group in dogs in the Republic of Serbia. Therefore the aim of this doctoral dissertation was to determine the presence and prevalence of STEC in the dog populaton in the area of Novi Sad, to perform typing and molecular characterization of E. coli strains isolated from faeces of dogs, as well to determine the presence of antibiotic resistant E. coli strains. In total 101 dogs from the area of Novi Sad was included in this research. As the material fecal samples of dogs were used. In order to isolate E. coli strains from faeces standard methods of bacterial isolation and biochemical identification were used, and for the confirmation of species identity matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was used. In order to determine the presence of STEC in dogs latex agglutination for the serogroup O157, direct agglutination for “big six” serogroups (O26, O45, O103, O111, O121 and O145), Vero cell cytotoxicity assay (VCA), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were used. Antibiotic resistance was determined by disc diffusion method according to the EUCAST and CLSI guidelines. In this research, for the first time in the Republic of Serbia, the presence of STEC has been proven in the dog population. By VCA method the prevalence of 1,98% was determined, while by ELISA and PCR the determined prevalence was 5,94% and 4,95% respectively. Two E. coli isolates belonged to some of the STEC “big six” serogroups, while none of the isolates belonged to O157 serogroup. Furthermore, in the dog population included in this research, the presence of strains resistant to one or more of used antibiotics was determined, as well as the presence of one multiresistant strain. Most of the E coli isolates showed resistance to ampicillin (22,5%), while every of the isolates showed susceptibility to gentamicin. In this research significant association between lifestyle of dogs and STEC harbouring. That is to say, significantly higher STEC prevalence was noticed in stray dogs in comparison to pet dogs, what is likely due to unlimited freedom of movement and thereby more frequent contact with contaminated water and food as sources of STEC. Finding of STEC and E. coli strains resistant to antibiotics in dog population is of importance not just in small animal practice, but also in terms of public health in the Republic of Serbia.