Hrana životinjskog podrijetla predstavlja mogući izvor patogenih sojeva Escherichia coli opasnih za ljude. Iako su većina sojeva crijevni komenzali, pojedini mogu uzrokovati crijevne (intestinalne) i izvancrijevne (ekstraintestinalne) infekcije. Njihova patogenost povezana je s prisutnošću gena za čimbenike virulencije, pripadnošću filogrupi te s biokemijskim svojstvima kod pojedinih serovarova.
U ovom istraživanju pretraženo je 100 izolata bakterije E. coli izdvojenih iz uzoraka mesa i obrisaka trupova različitih vrsta životinja. Izolatima su određene biokemijske karakteristike VITEK2 sustavom, a molekularnim metodama određena je prisutnost gena za čimbenike virulencije i pripadnost filogrupi.
U izolatima je dokazana prisutnost patogrupa: EAEC (EAST1), ETEC (STII), EPEC (eae), ExPEC (cnf1, cnf2) i EHEC/VTEC (vtx1, vtx2). Najučestalija patogrupa je EAEC (20%), dokazana u izolatima podrijetlom od mesa peradi, divljači, svinja i goveda. Patogrupe EPEC (9%) i ExPEC (6%) također su dokazane u potonjima, dok su patogrupe ETEC (5%) i VTEC (2%) dokazane u izolatima podrijetlom od divljači i svinja. Najviše gena za čimbenike virulencije ustanovljeno je u izolatima podrijetlom od divljači.
Biokemijskom karakterizacijom izolata ustanovljena je povezanost između prisustva eae gena i aktivnosti alkalinizacije sukcinata. Osim toga, također je primjetna povezanost između prisustva cnf1 gena i aktivnosti enzima arilamidaze prema tirozinu.
Pomoću lančane reakcije polimerazom (engl. polymerase chain reaction – PCR) izolati su svrstani u filogrupe. Najučestalije filogrupe bile su A (38%) i B1 (36%) u koje je svrstana većina pretraživanih izolata. Od ostalih filogrupa, s manjom učestalošću, bile su zastupljene filogrupe B2 (4%), C (3%), D (9%), E (4%) i F (6%). Rezultati statističke analize prikazuju povezanost između podrijetla izolata i pripadnosti filogrupi, što ukazuje na sklonost održavanja filogrupa unutar određenih domaćina (životinjskih vrsta). Također, prikupljeni podaci prikazuju nova saznanja o filogenetskoj strukturi E. coli u domaćih i divljih životinja na području Republike Hrvatske.
Prikazani rezultati ukazuju kako hrana različitog životinjskog podrijetla predstavlja potencijalni izvor crijevnih i izvancrijevnih patogenih E. coli.
|Abstract (english)|| |
INTRODUCTION: Escherichia coli (E. coli) is a Gram negative, aerobic and facultatively anaerobic, non-sporogenic, rod-shaped bacterium, member of the Enterobacteriaceae family. While most strains are intestinal commensal bacteria, some can cause intestinal and extraintestinal infections. Their pathogenicity is linked to the presence of virulence genes, phylo-group and in some strains to biochemical characteristics. Pathogenic and commensal (nonpathogenic) E. coli can be differentiated in several ways. According to virulence genes and clinical symptoms pathogenic E. coli are divided in seven pathogenic groups: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroadherent E. coli (EAEC) or enteroaggregative E. coli (EaggEC), enteroinvasive E. coli (EIEC), diffusely adherent E. coli (DAEC) and extraintestinal pathogenic E. coli (ExPEC). Pathogenic and nonpathogenic E. coli are divided in four main phylo-groups (A, B1, B2 and D), but according to the latest research there are now eight phylo-groups (A, B1, B2, C, D, E, F and I). Commensal and strains that cause intestinal disease mainly belong to A and B1 phylo-group, while extraintestinal strains mostly belong to B2 phylo-group and D in lower incidence. Food of animal origin presents a possible source of pathogenic Escherichia coli that pose a danger for human health. Aim of this study is to determine biochemical proprerties, presence of virulence genes and phylo-group affiliation of E. coli strains isolated from foods of different animal origin and carcass swabs from the area of the Republic of Croatia. The properties of the strains will be compared and will provide an insight to the presence of potentially pathogenic strains and their characteristics.
MATERIAL AND METHODS: A total of 100 E. coli isolates were analysed in this study. The strains were isolated from meat (poultry, game, pigs and cattle) and carcass swabs (pigs and cattle). Biochemical characteristics of the strains were determined using VITEK2 system. Using molecular methods, the strains were tested for the presence of virulence genes and assigned to a phylogroup. A group of 17 specific virulence genes (ipaH, aggR, aaiC, lt, stp, sth, STI, STII, bfp, vtx1, vtx2, eae, saa, hlyA, EAST1, cnf1, cnf2) was tested for determination of pathogroups in foods of different animal origin.
RESULTS: Using specific virulence genes the presence of pathogroups EAEC (EAST1), ETEC (STII), EPEC (eae), ExPEC (cnf1, cnf2) and EHEC/VTEC (vtx1, vtx2) was confirmed. Pathogroup EAEC was detected in strains isolated from samples of different animal origin (poultry, game, pigs and cattle) with the highest prevalence (20%). Pathogroups EPEC (9%) and ExPEC (6%) were also detected in strains isolated from the latter, while ETEC (5%) and VTEC (2%) were detected in strains isolated from game and pigs. Some strains isolated from poultry, game and pigs had more than one virulence gene detected in one strain, which shows that a lot of virulence genes are located on mobile genetic elements and that assigning a strain of E. coli to a specific pathogroup is very complicated. Most of the virulence genes (EAST1, STII, eae, cnf1, cnf2, stx1, hlyA) were detected in strains isolated from game and a connection between the source of the isolate and the presence of virulence genes was confirmed statistically (p=0.004). Contamination of the meat and the high percentage of virulence genes could be explained with inadequate shot placement, eviscertation of the animal on the field and improper storage of the meat. Biochemical characteristics of the strains were very similar. Comparing them with the presence of virulence genes, this study determined a link between the presence of eae virulence gene and succinate alkalinization which was confirmed statistically. Except from the above, a link between the presence of cnf1 virulence gene and the enzyme tyrosine arylamidase was also confirmed. This study analyzed the occurence and distribution of phylo-gruops in 100 E. coli strains isolated from poultry, game, pigs and cattle. Using polymerase chain reaction (PCR) the isolates were assigned to a phylo-group. Most of the strains were assigned to A (38%) and B1 (36%) phylo-group and other strains were represented in a lower incidence: B2 (4%), C (3%), D (9%), E (4%) and F (6%). Results of statistical analysis demonstrate that phylo-groups are associated with the source of the strain (p=0.039) and the results provide a knowledge in phylogenetic structure of E. coli isolated from domestic animals in the Republic of Croatia.
CONCLUSIONS: In this study the most prevalent pathogenic group of E. coli was EAST (20%). The following are EPEC (9%), ExPEC (6%), ETEC (5%) and VTEC (2%). Listed data shows a distribution and presence of pathogenic goups in food of different animal origin. Most virulence genes were present in meat from game which confirms that game is a significant source of pathogenic E. coli.
Most strains were assigned to A and B1 phylo-groups, which confirms that most commensal and intestinal pathogenic E. coli belong in these phylo-groups. Statistical analysis shows a link between the origin of the sample and phylo-group affiliation, which confirms that phylo-groups have a tandency towards specific hosts. For most strains researched in this study, biochemical characteristics were very similar and had no relevance. Their comparison with the presence of virulence genes shows a link between the presence of eae virulence gene and succinate alkalinization and cnf1 and tyrosine arylamidase, but for further conclusions more strains of this virulence characteristics have to be tested. Results presented in this research indicate that foods of different animal origin represents a source of intestinal and extraintestinal pathogenic E. coli.