Escherichia coli (E. coli) strains are mostly present as intestinal commensal bacteria in the gastro-intestinal tract of humans and other animals; however, some can cause intestinal and extraintestinal infections (11). The pathology and expression of clinical symptoms classify intestinal pathogenic E. coli into six pathogroups: enterotoxigenic E. coli (ETEC); enteropathogenic E. coli (EPEC); enterohaemorrhagic E. coli (EHEC); enteroaggregative E. coli (EAEC); enteroinvasive E. coli (EIEC); and diffusely adherent E. coli (DAEC) (6). Strains of the bacteria that cause extraintestinal infection are called extraintestinal pathogenic E. coli (ExPEC) (41).
Human infections occur through the consumption of contaminated food such as undercooked meat, drinking contaminated water, or direct person-to-person contact. In developing countries, the major causes of infantile diarrhoea are ETEC, EPEC and EAEC, while EHEC and EAEC are mainly associated with food poisoning in the developed world (6). The presence of intestinal and extraintestinal pathogenic E. coli in food suggests a public health impact, and agencies such as the European Food Safety Authority (EFSA) report foodborne outbreaks associated with verotoxigenic E. coli (VTEC) and other pathogenic E. coli. In the 5-year period of 2015–2019 there was an increase in reported VTEC cases in the EU. This can be due to the
enhanced general awareness of VTEC following reports of large outbreaks in the EU and worldwide. It was shown by the EFSA that VTEC is the fourth most frequent bacterial agent causing foodborne outbreaks in the EU, with 34 outbreaks, 208 cases, 30 hospitalisations and 1 death reported in 2020 (15).
One of the most important human pathogens is EHEC, and the most significant virulence genes within this pathogroup are verotoxins (vtx). Escherichia coli which harbours these genes is called verotoxigenic E. coli (VTEC). This pathogroup can be present in the intestines of many animal species, but the main reservoirs are ruminants, especially cattle (21). The presence of vtx genes is closely related to the pathogen’s induction of haemorrhagic diseases, such as haemolyticuremic syndrome (HUS) and bloody diarrhoea. The ability to cause disease also depends on the subtype (3). For EPEC, it is important to differentiate typical (tEPEC) from atypical (aEPEC) strains. There are two major diversities between tEPEC and aEPEC. The first includes the presence of the E. coli adherence factor plasmid that encodes the bundle-forming pili (bfp), present in tEPEC but not in aEPEC, and the second one is related to the host, because both humans and animals can equally be reservoirs for aEPEC and create a cycle of mutual infection, while only humans are believed to be the major reservoir for tEPEC (34, 37). As with tEPEC, humans were confirmed as the main reservoir for EIEC (37). Strains of EAEC are classified as typical EAEC (tEAEC) and atypical EAEC (aEAEC) based on the presence or absence of the aggR gene. Humans are the reservoir for tEAEC, while aEAEC has been isolated from different animal species, suggesting their role as its reservoir (37). Factors not regulated by aggR include the EAEC heat-stable toxin EAST-1, which is associated with EAEC causing diarrhoea (32). As for ETEC, heat-labile and heat-stable enterotoxins and colonisation factors are the pathogroups’ main characteristics (42), and humans are suggested as their main reservoir. However, young animals are susceptible to ETEC infections (17).
Depending on the pathogroup, the symptoms range from heavy diarrhoea and dehydration in ETEC and EPEC infection, with possible chronic diarrhoea, vomiting and slight fever in EPEC-caused cases (2, 36), to watery diarrhoea in EIEC infection and watery secretory diarrhoea with mucus in EAEC-caused diseases (10). In contrast to strains causing intestinal infection, ExPEC possesses a wide range of virulence genes that enable it to invade and colonise organs and generate infections outside of the gastro-intestinal tract (5), causing extraintestinal infections in humans and animals. These strains can be isolated from food of animal origin, such as chicken and pig meat (25).
In recent years, there have been many studies regarding the association between pathogroups and phylogenetic groups. Commensal and enteric strains are mainly associated with the A and B1 groups, while extraintestinal strains are mainly identified in the B2 group. Research demonstrates that ETEC, EHEC and EIEC affiliate to the A, B1, C, and E phylogenetic groups, while EPEC, EAEC and DAEC are present in all groups (14). Furthermore, the dispersion of phylogenetic groups within E. coli strains is closely associated with the host species, and although each group can acquire any virulence gene, they survive in clones ‘best adapted’ to the host species, making them their reservoir (1).
In Croatia, information regarding the molecular characteristics of E. coli strains is deficient. To rectify the deficiency, this study aimed to investigate the presence of virulence genes and phylogenetic group affiliation of E. coli isolated from different food samples and carcass swabs of different animals originating from Croatia. The collected data will be compared with the origin of the strain and provide an insight into the presence of potentially pathogenic strains and phylogenetic groups of E. coli and a better understanding of their dissemination in Croatia.
Material and Methods
Bacterial strains. A total of 100 E. coli strains were analysed in this study. The strains were isolated from samples of different animal origins, including poultry (n = 27), game (n = 14), pigs (n = 30), and cattle (n = 29), delivered as routine laboratory samples. Samples of poultry origin included 23 of broiler meat, 2 of mechanically separated meat and 2 of meat preparations. The strains isolated from game came from fresh meat from three red deer, four fallow deer, one moufflon, four roe deer and two wild boar. Pig strains were isolated from 23 carcass swabs, 1 sample of fresh meat and 6 of packaged minced meat. Cattle-origin strains were isolated from 15 carcass swabs, 5 from samples of fresh meat, 6 from packaged minced meat and 3 from meat preparations. Isolation was performed using the ISO 16649-2:2001 method (22) and an additional one. The second method was enrichment of samples in buffered peptone water (BPW) at 37°C for 18–24 h according to the European Union Reference Laboratory (EURL) Methods 05, 07 and 08 recommended for the detection of virulence genes in E. coli (16), together with ISO/TS 13136:2012 (23) which proposes the use of BPW as a broth for selective enrichment. Incubation was followed by plating on a Tryptone Bile Glucuronic Agar (TBX) plate (Oxoid, Oxford, UK) and a Sorbitol MacConkey Agar (SMAC) plate (Merck, Darmstadt, Germany) and further incubation at 37°C for 18–24 h. Characteristic colonies were plated on blood agar (Merck) and identified as E. coli using the VITEK2 system (Biomérieux, Marcyl’Étoile, France). After confirmation, the colonies were inoculated on Brain Heart Infusion (BHI) Broth (Merck) with 50% glycerol and stored at −80°C until further examination. DNA was isolated by heating the bacterial suspension at 95°C for 20 min, followed by centrifugation at 14,000 × g for 1 min. All amplified PCR products were visualised with the QIAxcel capillary electrophoresis system (Qiagen, Hilden, Germany).
Detection of virulence genes. A group of 15 virulence genes was used to determine the presence of six E. coli pathogroups, as presented in Table 1. A conventional PCR was performed for ipaH, aggR, aaiC, lt, bfp and EAST1. The primers used and amplification conditions for ipaH, aggR, aaiC and lt were those specified in EURL Methods 05, 07 and 08 (16). The bfp gene was detected according to Gunzburg et al. (19) and the EAST1 gene was amplified as described by Yamamoto and Echeverria (45). A multiplex PCR was performed to determine the presence of vtx1, vtx2, eae, saa and ehxA according to Paton and Paton (40) and the vtx1 and vtx2 subtypes were determined using EURL Method 06 (16). Detection of STI, STII, cnf1 and cnf2 was achieved using multiplex and conventional PCRs according to Pass et al. (39).
Pathogroups, virulence factors and virulence genes
Full name of virulence gene
verotoxin 1, 2
STEC autoagglutinating adhesin
aggregative adherence regulation factor
aggR-activated island C
heat-stable enterotoxin 1
bundle forming pili
invasion plasmid antigen H
heat-stable enterotoxin I, II
STI (stp, sth), STII
cytotoxic necrotizing factor 1, 2
VTEC – verotoxigenic E. coli; EHEC – enterohaemorraghic E. coli; STEC – Shiga toxin–producing E. coli; EAEC – enteroaggregative E. coli; EPEC – enteropathogenic E. coli; EIEC – enteroinvasive E. coli; ETEC – enterotoxigenic E. coli; ExPEC – extraintestinal pathogenic E. coli
Phylogenetic group determination. The strains were assigned to a phylogenetic group by using a multiplex PCR according to Clermont et al. (7).
Statistical analysis. Statistical analysis was performed using Stata 13.1. (StataCorp., College Station, TX, USA) and expressed in the binary variable 0/1 (yes/no). The connection between certain values was verified with the chi-squared and Fisher’s exact tests. Multivariant statistical analysis was conducted using a logistic regression model. Results with P ≤ 0.05 were considered statistically significant.
Distribution of potentially pathogenic E. coli strains. The occurrence of virulence genes within the strains is presented in Table 2. The presence of virulence genes was established in 36 (36%) of the 100 tested E. coli strains. The presence of EAST1, eae and cnf1/cnf2 virulence genes was detected in strains of various animal origins, with EAST1 present in 20 of the 100 (20%), eae in 9 (9%), cnf1 in 6 (6%) and cnf2 in 4 (4%) strains. Less varied by source, the STII, vtx and ehxA virulence genes were only detected in strains of game and pig origin. The STII gene was identified in 5 (5%) strains and vtx was detected in 2 (2%) strains and subtyped with the results vtx1c and vtx1d (game origin) and vtx2e (pig origin). The ehxA gene occurred in 2 of the 100 (2%) strains.
Occurrence of virulence genes and phylogenetic groups in different animal species
Number of positive strains, phylogenetic group and type of sample
Total gene-positive strains (%)
4 (2A, 1B1, 1F); 3 BM, 1 MSM
3 (1B1, 1D, 1A); FD, RD,WB
2 (2A); CS
4 (3A, 1C); CS
4 (3A, 1E); BM
1 (B1); RD
2 (2B1); MM
4 (3A, 1B1); DE, 2 FD, M
1 (B1); CS
1 (B1); BM
1 (B1); WB
1 (D); CS
1 (F); BM
1 (B2); DE
1 (B1); CS
1 (B1); MP
1 (B1); CS
1 (B2); FM
1 (E); RD
1 (C); CS
A, B1, B2, C, D, E, F – phylogenetic group; BM – broiler meat; MSM – mechanically separated meat; DE – fresh red deer meat; FD – fresh fallow deer meat; RD – fresh roe deer meat; WB – fresh wild boar meat; M – fresh moufflon meat ; CS – carcass swab; MM – minced meat; MP – meat preparation; FM – fresh meat
The animal origin and sample type in which virulence genes were detected is also presented in Table 2. Of the 27 poultry origin strains, the presence of virulence genes was established in 10 (9 isolated from broiler meat and 1 from mechanically separated meat), while in the other 17 strains (14 isolated from broiler meat, 1 from mechanically separated meat and 2 from meat preparations) none of the virulence genes were detected. Among the 14 strains isolated from game meat, 11 harboured virulence genes (2 strains being red deer isolates, 3 fallow deer, 3 roe deer, 2 wild boars and 1 moufflon) while the other 3 strains (isolated from red deer, fallow deer and roe deer) were found not to carry any of the target genes.
Of the 30 pig-origin strains, 7 (isolated from carcass swabs) harboured virulence genes, while these genes’ presence was not established in the other 23 strains (16 isolated from carcass swabs, 6 from minced meat and 1 from fresh meat). The 29 strains of cattle origin included 8 containing virulence genes (4 detected in carcass swabs, 2 in minced meat, 1 in a meat preparation and 1 in fresh meat) and 21 not carrying any of the tested virulence genes (11 yielded from carcass swabs, 4 from fresh meat, 4 from minced meat and 2 from meat preparations).
The distribution of virulence genes was uneven between strains of different origins and some harboured two such genes (Table 3), this state being frequently observed in game origin strains. Additionally, most of the virulence genes were detected in game origin strains (78.57%).
The frequency of virulent genes compared to the origin of the sample
Number of strains (%)
Virulence gene presence
Number of virulence genes in strains
Phylogenetic grouping of E. coli strains. The distribution of the strains within phylogenetic groups is presented in Table 4, which shows A (38%) and B1 (36%) as the most frequent groups, followed by D (9%), F (6%), B2 (4%), E (4%) and C (3%). The largest affiliation of strains isolated from poultry (11 out of 27; 40.74%) and pigs (13 of 30; 43.33%) was to phylogenetic group A, while for strains isolated from game (5 of 14; 35.71%) and cattle (13 of 29; 44.83%) it was to the B1 group. Group F was only present in strains of poultry origin, the B2 phylogenetic group was not confirmed in poultry or pig strains and C was not found in strains isolated from poultry and game.
Distribution of E. coli phylogenetic groups in different animal species
Total number of strains
Relationship between pathogroups and phylogenetic groups. In the 36 strains carrying virulence genes, A and B were the most prevalent phylogenetic groups (Fig. 1). The results revealed 14 out of 36 (38.89%) of the strains to be assigned to phylogenetic group A and 12 out of 36 (33.33%) to group B1, with the others (B2, C, D, E and F) represented equally (Table 5). Fourteen strains assigned to A group harboured EAST1, eae and STII virulence genes, while all of the analysed virulence genes except vtx were present in strains assigned to the B1 group. The results comprise two strains from the B2 group carrying the cnf1 and cnf2 genes, two strains from the F group with the EAST1, cnf1 and cnf2 genes, two strains from the E group carrying eae and vtx1, two strains from C group harbouring the vtx2 and EAST1 and two strains from group D with the EAST1, ehxA and eae genes. The distribution of the virulence genes within phylogenetic groups is displayed in Table 2.
The relationship between phylogenetic groups and virulence genes
Virulence gene confirmations
Statistical analysis. The distribution of the results revealed a relationship between the distribution of the virulence genes and the origin of the sample (P = 0.004) (Table 3). Compared to strain from poultry, pigs and cattle, most of the strains isolated from game harboured more than one virulence gene and the frequency of this difference was significant (P = 0.003) (Table 3). Statistical analysis also confirmed significance in the distribution of phylogenetic groups in host organisms, with P = 0.039 (Table 4). However, there was no significant correlation between the presence of virulence genes and phylogenetic group affiliation (P = 0.833) (Table 5).
This study analysed the occurrence and distribution of virulence genes and phylogenetic groups in E. coli strains isolated from food of different animal species and carcass swabs originating from Croatia. The results presented in this study confirmed the presence of various intestinal and extraintestinal E. coli strains. The presence of virulence genes in this bacteria in food of animal origin is a public health issue, and food contaminated with pathogenic E. coli could lead to illness. The research provides an insight into the diversity of E. coli strains isolated from food specimens as well as carcass swabs from different animal species originating from Croatia, for which the data are limited.
The most frequently occurring virulence genes were the ones associated with the EAEC, aEPEC and ExPEC pathogroups. These genes were detected in strains retrieved from samples of which the animal origins were varied, which could be explained by the genes’ broad distribution within different animal hosts and the environment (12, 24). In this study, EAST1 was the most prevalent gene, but its contribution to the virulence of EAEC remains unclear (6). However, previous research associated EAST1 toxin with severe cases of child diarrhoea in India (32), confirming its pathogenicity and indicating its significant association with EAEC causing diarrhoea. Furthermore, the zoonotic potential of the aEPEC strains confirmed in this investigation can also be assumed based on previous research. Moura et al. (34) described the relationship between aEPEC strains of human and animal origin, indicating that strains isolated from animals have the potential to cause diarrhoea in humans. As for ExPEC, previous studies indicate food of animal origin as a source of infection for humans (25). The presence of cnf1 and cnf2 detected in this study indicates a need for further research, since cnf1 is mainly associated with diarrhoea and extraintestinal infections in humans, and cnf2 with septicaemia or diarrhoea in cattle and sheep (26). Unlike that pathogroup, ETEC and VTEC were only present in strains of game and pig origin and harboured the vtx genes identified as vtxe2 (pig origin) and vtx1c and vtx1d (game origin). Based on the subtype, these strains cannot cause severe clinical illness in humans such as HUS or bloody diarrhoea. Furthermore, none of the VTEC strains harboured other vtx-related virulence genes analysed in this study (saa, ehxA or eae) and previous reports confirm the connection between vtx-eae and certain other gene combinations and the percentage of hospitalisations and HUS cases in humans (29). As a contrary indication however, vtx1c has often been isolated in patients with milder infections and, in combination with vtx1a, in patients with bloody diarrhoea (3). As for STII, its presence is almost exclusively linked to pigs (13) and further research is needed to establish possible role of the gene as a zoonotic pathogen. Furthermore, some genes (ipaH, bfp, aggR, aaiC and STI) were not confirmed in any of the strains, which was expected because of their adaptation to humans.
The results demonstrate that most of the target genes were present in strains of game origin. The connection between the source of the strain and the presence of virulence genes was confirmed with P ≤ 0.05, suggesting game meat as a significant source of pathogenic E. coli. This could be the effect of many factors, including poor shot placement by hunters, delayed evisceration of the animal or inadequate carcass storage at inappropriate ambient temperatures (18). This study demonstrates the presence of VTEC and other E. coli pathogroups present in game meat, especially deer species, indicating the need for further research. These data are important, especially with regard to VTEC, because game is considered to be a reservoir and source of human pathogenic VTEC and EHEC strains. Examples are noted from studies from other European countries such as Spain, in which 40% of wildlife-isolated VTEC strains shared the same characteristics as the ones isolated from human patients from that geographic area (33). Research conducted in Germany also linked 32.8% (46/140) of VTEC strains isolated from wildlife meat with high-level virulence genes for humans (30). A report published by the EFSA also disclosed a high prevalence of VTEC in deer meat (26.7%), indicating it as one of the most contaminated foods of animal origin (15).
Other results mostly show broiler chicken meat contaminated with E. coli, which was probably because of poor sanitation standards of water used in the cutting and processing of chicken meat (28). All the positive pig-origin strains were isolated from carcass swabs. This is probably the result of cross-contamination during slaughter, which can occur with intestinal damage, faecal contamination, or contamination from the environment, equipment, or workers’ hands (43). Similarly, cattle carcass swabs were probably contaminated with E. coli during hide removal and evisceration (20). Other samples such as minced meat and meat preparations were contaminated during the slaughter process or subsequent handling (35). No differences investigated in the comparison of different samples from the same animal species were proved to be statistically significant, indicating the need for future research with a larger number of samples.
In the 100 E.coli strains analysed in this study, most were assigned to the A and B1 phylogenetic groups, including virulence gene-positive strains. These data are in agreement with the prevalence of the A and B1 groups in commensal strains isolated from different food and animal species (4, 9, 38) and the occurrence of enteric E. coli within these groups (8). The results of the study also indicate the phylogenetic group tendency toward a specific host (P = 0.039), with group A more frequently distributed in strains of pig origin and B1 more prevalent in game and cattle strains. The results confirm previous research suggesting that strains isolated from omnivores are mainly associated with group A, while B1 is more prevalent in strains isolated from herbivores (4). The analysed poultry strains had A as the dominant group, with group F appearing only in poultry, confirming the phylogenetic groups association with the species origin of the sample (9). Ultimately, the gathered information confirmed a connection between the source of the strain and phylogenetic group affiliation, verifying the groups’ adaptation to certain host species. This information could have a practical application as most virulence genes in survive phylogenetic groups best adapted to certain host species.
Escherichia coli are very diverse bacteria, and sometimes it is very difficult to place a strain in only one pathogroup. For example, EAST1, which was the most prevalent virulence gene in this study, is commonly associated with the EAEC pathogroup but is also present in others, such as EHEC, EPEC and ETEC (32, 37). Furthermore, strains carrying the EAST1 gene were present in all phylogenetic groups except for B2 and E, confirming its distribution over the entire phylogenetic tree (14) and in food of diverse origins. Similarly to EAST1, the eae gene is hosted by different animals (27) and has broad phylogenetic group distribution as a consequence. Unlike EAST1 and eae, the STII and vtx genes were identified in game- and pig-origin strains. Strains positive for STII were assigned to the A and B1 groups and those positive for vtx to the E and C groups, which is consistent with the outcomes described by Escobar-Páramo et al. (14). Previous reports emphasised the importance of assigning strains to certain phylogenetic groups, because they can be evaluated for their pathogenicity accordingly. For instance, Wang et al. (44) indicated an association between the strains isolated from cattle, humans with diarrhoea and the B1 group. This association could indicate a greater zoonotic potential of the eae-positive cattle strains isolated in this study. Along with enteric E. coli, the presence of ExPEC was also confirmed in strains isolated from samples of different animal origins. This pathogroup can be distributed in all phylogenetic groups, but is commonly associated with B2 and D (31), which harbour more virulence genes (25). Furthermore, previous studies confirm the connection between the B2 group and the occurrence of extraintestinal infections (8), which suggests that the ExPEC strains analysed in this study and identified as B2 (game and cattle) may possess greater zoonotic potential than those assigned to B1 (pig, cattle) and F (poultry) group.
In conclusion, the study revealed the presence of intestinal and extraintestinal pathogenic E. coli in food of various animal origin as well as carcass swabs, indicating a potential public health risk. This study confirmed the wide distribution of certain pathogroups (EAEC and aEPEC) within different animal hosts and the importance of game meat as a source of potentially pathogenic E. coli, indicating the need for further research. Additionally, this research provides information on the presence of E. coli phylogenetic groups in domestic and wild animals in Croatia, which has not been exhaustively analysed. The study also confirms a connection between the source of a strain and phylogenetic group affiliation, verifying the groups’ adaptation to certain host species. However, further research is needed to assess the epidemiological association between strains obtained from this study and the strains isolated from the environment, animals, other food and humans suffering from E. coli infections.
Baldy-Chudzik K., Mackiewicz P., Stosik M.: Phylogenetic background, virulence gene profiles, and genomic diversity in commensal Escherichia coli isolated from ten mammal species living in one zoo. Vet Microbiol 2008, 131, 173–184, doi: 10.1016/j.vetmic.2008.02.019.Baldy-ChudzikK.MackiewiczP.StosikM.Phylogenetic background, virulence gene profiles, and genomic diversity in commensal Escherichia coli isolated from ten mammal species living in one zoo200813117318410.1016/j.vetmic.2008.02.01918423907DOI öffnenSearch in Google Scholar
Begum Y.A., Talukder K.A., Nair G.B., Svennerholm A.M., Sack R.B., Qadri F.: Enterotoxigenic Escherichia coli isolated from surface water in urban and rural areas of Bangladesh. J Clin Microbiol 2005, 43, 3582–3583, doi: 10.1128/JCM.43.7.3582-3583.2005.BegumY.A.TalukderK.A.NairG.B.SvennerholmA.M.SackR.B.QadriF.Enterotoxigenic Escherichia coli isolated from surface water in urban and rural areas of Bangladesh2005433582358310.1128/JCM.43.7.3582-3583.2005116909416000515DOI öffnenSearch in Google Scholar
Buvens G., De Gheldre Y., Dediste A., De Moreau A.-I., Mascart G., Simon A., Allemeersch D., Scheutz F., Lauwers S., Piérard D.: Incidence and Virulence Determinants of Verocytotoxin-Producing Escherichia coli Infections in the Brussels-Capital Region, Belgium, in 2008–2010. J Clin Microbiol 2012, 50, 1336–1345, doi: 10.1128/JCM.05317-11.BuvensG.De GheldreY.DedisteA.De MoreauA.-I.MascartG.SimonA.AllemeerschD.ScheutzF.LauwersS.PiérardD.Incidence and Virulence Determinants of Verocytotoxin-Producing Escherichia coli Infections in the Brussels-Capital Region, Belgium, in 2008–20102012501336–134510.1128/JCM.05317-11331857022238434DOI öffnenSearch in Google Scholar
Carlos C., Pires M.M., Stoppe N.C., Hachich E.M., Sato M.I.Z., Gomes T.A.T., Amaral L.A., Ottoboni L.M.M.: Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination. BMC Microbiol 2010, 10, 161, doi: 10.1186/1471-2180-10-161.CarlosC.PiresM.M.StoppeN.C.HachichE.M.SatoM.I.Z.GomesT.A.T.AmaralL.A.OttoboniL.M.M.Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination20101016110.1186/1471-2180-10-161288995320515490DOI öffnenSearch in Google Scholar
Chapman T.A., Wu X.-Y., Barchia I., Bettelheim K.A., Driesen S., Trott D., Wilson M., Chin J.J.-C.: Comparison of virulence gene profiles of Escherichia coli strains isolated from healthy and diarrheic swine. Appl Environ Microbiol 2006, 72, 4782–4795, doi: 10.1128/AEM.02885-05.ChapmanT.A.WuX.-Y.BarchiaI.BettelheimK.A.DriesenS.TrottD.WilsonM.ChinJ.J.-C.Comparison of virulence gene profiles of Escherichia coli strains isolated from healthy and diarrheic swine2006724782479510.1128/AEM.02885-05148937516820472DOI öffnenSearch in Google Scholar
Clements A., Young J.C., Constantinou N., Frankel G.: Infection strategies of enteric pathogenic Escherichia coli. Gut Microbes 2012, 3, 71–87, doi: 10.4161/gmic.19182.ClementsA.YoungJ.C.ConstantinouN.FrankelG.Infection strategies of enteric pathogenic Escherichia coli20123718710.4161/gmic.19182337095122555463DOI öffnenSearch in Google Scholar
Clermont O., Christenson J.K., Denamur E., Gordon D.M.: The Clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep 2013, 5, 58–65, doi: 10.1111/17582229.12019.ClermontO.ChristensonJ.K.DenamurE.GordonD.M.The Clermont Escherichia coli phylo-typing method revisited: Improvement of specificity and detection of new phylo-groups20135586510.1111/17582229.12019DOI öffnenSearch in Google Scholar
Clermont O., Olier M., Hoede C., Diancourt L., Brisse S., Keroudean M., Glodt J., Picard B., Oswald E., Denamur E.: Animal and human pathogenic Escherichia coli strains share common genetic backgrounds. Infect Genet Evol 2011, 11, 654–662, doi: 10.1016/j.meegid.2011.02.005.ClermontO.OlierM.HoedeC.DiancourtL.BrisseS.KeroudeanM.GlodtJ.PicardB.OswaldE.DenamurE.Animal and human pathogenic Escherichia coli strains share common genetic backgrounds20111165466210.1016/j.meegid.2011.02.00521324381DOI öffnenSearch in Google Scholar
Coura F.M., Diniz S.D.A., Silva M.X., Mussi J.M.S., Barbosa S.M., Lage A.P., Heinemann M.B.: Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples. Sci World J 2015, 2015, 258424, doi: 10.1155/2015/258424.CouraF.M.DinizS.D.A.SilvaM.X.MussiJ.M.S.BarbosaS.M.LageA.P.HeinemannM.B.Phylogenetic Group Determination of Escherichia coli Isolated from Animals Samples2015201525842410.1155/2015/258424457246026421310DOI öffnenSearch in Google Scholar
Croxen M.A., Law R.J., Scholz R., Keeney K.M., Wlodarska M., Finlay B.B.: Recent Advances in Understanding Enteric Pathogenic Escherichia coli. Clin Microbiol Rev 2013, 26, 822–880, doi: 10.1128/CMR.00022-13.CroxenM.A.LawR.J.ScholzR.KeeneyK.M.WlodarskaM.FinlayB.B.Recent Advances in Understanding Enteric Pathogenic Escherichia coli20132682288010.1128/CMR.00022-13381123324092857DOI öffnenSearch in Google Scholar
Donnenberg M.S., Whittam T.S.: Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli. J Clin Invest 2001, 107, 539–548, doi: 10.1172/JCI12404.DonnenbergM.S.WhittamT.S.Pathogenesis and evolution of virulence in enteropathogenic and enterohemorrhagic Escherichia coli200110753954810.1172/JCI1240419943111238553DOI öffnenSearch in Google Scholar
Dubreuil J.D.: EAST1 toxin: An enigmatic molecule associated with sporadic episodes of diarrhea in humans and animals. J Microbiol 2019, 57, 541–549, doi: 10.1007/s12275-019-8651-4.DubreuilJ.D.EAST1 toxin: An enigmatic molecule associated with sporadic episodes of diarrhea in humans and animals20195754154910.1007/s12275-019-8651-431016564DOI öffnenSearch in Google Scholar
Dubreuil J.D., Isaacson R.E., Schifferli D.M.: Animal Enterotoxigenic Escherichia coli. EcoSal Plus 2016, 7, doi: 10.1128/ecosalplus.ESP-0006-2016.DubreuilJ.D.IsaacsonR.E.SchifferliD.M.Animal Enterotoxigenic Escherichia coli2016710.1128/ecosalplus.ESP-0006-2016512370327735786DOI öffnenSearch in Google Scholar
Escobar-Páramo P., Clermont O., Blanc-Potard A.-B., Bui H., Le Bouguénec C., Denamur E.: A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli. Mol Biol Evol 2004, 21, 1085–1094, doi: 10.1093/molbev/msh118.Escobar-PáramoP.ClermontO.Blanc-PotardA.-B.BuiH.Le BouguénecC.DenamurE.A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli2004211085109410.1093/molbev/msh11815014151DOI öffnenSearch in Google Scholar
European Food Safety Authority: The European Union One Health 2020 Zoonoses Report. EFSA J 2021, 19, 6971, doi: 10.2903/j.efsa.2021.6971.European Food Safety AuthorityThe European Union One Health 2020 Zoonoses Report202119697110.2903/j.efsa.2021.6971DOI öffnenSearch in Google Scholar
European Union Reference Laboratory for Escherichia coli: Laboratory methods. https://www.iss.it/web/iss-en/vtec-laboratory-methods.Laboratory methodshttps://www.iss.it/web/iss-en/vtec-laboratory-methodsSearch in Google Scholar
Fratamico P.M., Smith J.: Escherichia coli. In: Foodborne Infections and Intoxications, edited by H.P. Riemann, D.O. Cliver, Academic Press, Amsterdam, 2005, pp. 205–258.FratamicoP.M.SmithJ.Escherichia coliInedited byHRiemann.P.CliverD.O.Academic PressAmsterdam2005pp205258Search in Google Scholar
Gill C.O.: Microbiological conditions of meats from large game animals and birds. Meat Sci 2007, 77, 149–160, doi: 10.1016/j.meatsci.2007.03.007.GillC.O.Microbiological conditions of meats from large game animals and birds20077714916010.1016/j.meatsci.2007.03.00722061585DOI öffnenSearch in Google Scholar
Gunzburg S.T., Tornieporth N.G., Riley L.W.: Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene. J Clin Microbiol 1995, 33, 1375–1377, doi: 10.1128/jcm.33.5.1375-1377.1995.GunzburgS.T.TornieporthN.G.RileyL.W.Identification of enteropathogenic Escherichia coli by PCR-based detection of the bundle-forming pilus gene1995331375–137710.1128/jcm.33.5.1375-1377.19952281707615759DOI öffnenSearch in Google Scholar
Gutema F.D., Abdi R.D., Agga G.E., Firew S., Rasschaert G., Mattheus W., Crombe F., Duchateau L., Gabriël S., De Zutter L.: Assessment of beef carcass contamination with Salmonella and E. coli O 157 in slaughterhouses in Bishoftu, Ethiopia. Int J Food Contam 2021, 8, doi: 10.1186/s40550-021-00082-1.GutemaF.D.AbdiR.D.AggaG.E.FirewS.RasschaertG.MattheusW.CrombeF.DuchateauL.GabriëlS.De ZutterL.Assessment of beef carcass contamination with Salmonella and E. coli O 157 in slaughterhouses in Bishoftu, Ethiopia2021810.1186/s40550-021-00082-1DOI öffnenSearch in Google Scholar
Gyles C.L.: Shiga toxin-producing Escherichia coli: an overview. J Anim Sci 2007, 85, E45–E62, doi: 10.2527/jas.2006-508.GylesC.L.Shiga toxin-producing Escherichia coli: an overview200785E45E6210.2527/jas.2006-50817085726DOI öffnenSearch in Google Scholar
International Organization for Standardization (ISO): ISO 166492:2001: Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 2: Colony-count technique at 44 degrees using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide. ISO, Geneva, 2001.International Organization for Standardization (ISO)Geneva2001Search in Google Scholar
International Organization for Standardization (ISO): ISO/TS 13136:2012: Microbiology of food and animal feeding stuffs – Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens – Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups. ISO, Geneva, 2001.International Organization for Standardization (ISO)ISOGeneva2001Search in Google Scholar
Ishii S., Meyer K.P., Sadowsky M.J.: Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources. Appl Environ Microbiol 2007, 73, 5703–5710, doi: 10.1128/AEM.00275-07.IshiiS.MeyerK.P.SadowskyM.J.Relationship between phylogenetic groups, genotypic clusters, and virulence gene profiles of Escherichia coli strains from diverse human and animal sources2007735703571010.1128/AEM.00275-07207492617644637DOI öffnenSearch in Google Scholar
Jakobsen L., Spangholm D.J., Pedersen K., Jensen L.B., Emborg H.-D., Agersø Y., Aarestrup F.M., Hammerum A.M., Frimodt-Møller N.: Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients. Int J Food Microbiol 2010, 142, 264–272, doi: 10.1016/j.ijfoodmicro.2010.06.025.JakobsenL.SpangholmD.J.PedersenK.JensenL.B.EmborgH.-D.AgersøY.AarestrupF.M.HammerumA.M.Frimodt-MøllerN.Broiler chickens, broiler chicken meat, pigs and pork as sources of ExPEC related virulence genes and resistance in Escherichia coli isolates from community-dwelling humans and UTI patients201014226427210.1016/j.ijfoodmicro.2010.06.02520656368DOI öffnenSearch in Google Scholar
Kadhum H.J., Finlay D., Rowe M.T., Wilson I.G., Ball H.J.: Occurrence and characteristics of cytotoxic necrotizing factors, cytolethal distending toxins and other virulence factors in Escherichia coli from human blood and faecal samples. Epidemiol Infect 2008, 136, 752–760, doi: 10.1017/S0950268807009247.KadhumH.J.FinlayD.RoweM.T.WilsonI.G.BallH.J.Occurrence and characteristics of cytotoxic necrotizing factors, cytolethal distending toxins and other virulence factors in Escherichia coli from human blood and faecal samples200813675276010.1017/S0950268807009247287087117678557DOI öffnenSearch in Google Scholar
Krause G., Zimmermann S., Beutin L.: Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types. Vet Microbiol 2005, 106, 87–95, doi: 10.1016/j.vetmic.2004.11.012.KrauseG.ZimmermannS.BeutinL.Investigation of domestic animals and pets as a reservoir for intimin- (eae) gene positive Escherichia coli types2005106879510.1016/j.vetmic.2004.11.01215737477DOI öffnenSearch in Google Scholar
Liur I.J., Veerman M.: Level of Escherichia coli contamination of broiler chicken meat in Ambon City Market. IOP Conf Ser Earth Env 2021, 883, 012036, doi: 10.1088/1755-1315/883/1/012036.LiurI.J.VeermanM.Level of Escherichia coli contamination of broiler chicken meat in Ambon City Market202188301203610.1088/1755-1315/883/1/012036DOI öffnenSearch in Google Scholar
Messens W., Bolton D., Frankel G., Liebana E., McLauchlin J., Morabito S., Oswald E., Threlfall E.J.: Defining pathogenic verocytotoxin-producing Escherichia coli (VTEC) from cases of human infection in the European Union, 2007–2010. Epidemiol Infect 2015, 143, 1652–1661, doi: 10.1017/S095026881400137X.MessensW.BoltonD.FrankelG.LiebanaE.McLauchlinJ.MorabitoS.OswaldE.ThrelfallE.J.Defining pathogenic verocytotoxin-producing Escherichia coli (VTEC) from cases of human infection in the European Union, 2007–201020151431652166110.1017/S095026881400137X25921781DOI öffnenSearch in Google Scholar
Miko A., Pries K., Haby S., Steege K., Albrecht N., Krause G., Beutin L.: Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans. Appl Environ Microbiol 2009, 75, 6462–6470, doi: 10.1128/AEM.00904-09.MikoA.PriesK.HabyS.SteegeK.AlbrechtN.KrauseG.BeutinL.Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans2009756462647010.1128/AEM.00904-09276514619700552DOI öffnenSearch in Google Scholar
Mitchell N.M., Johnson J.R., Johnston B., Curtiss R. 3rd, Mellata M.: Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs. Appl Environ Microbiol 2015, 81, 1177–1187, doi: 10.1128/AEM.03524-14.MitchellN.M.JohnsonJ.R.JohnstonB.CurtissR. 3rdMellataM.Zoonotic potential of Escherichia coli isolates from retail chicken meat products and eggs2015811177–118710.1128/AEM.03524-14429250625480753DOI öffnenSearch in Google Scholar
Modgil V., Mahindroo J., Narayan C., Kalia M., Yousuf M.D., Shahi V., Koundal M., Chaudhary P., Jain R., Singh Sandha K., Tanwar S., Gupta P., Thakur K., Singh D., Gautam N., Kakkar M., Bharti B., Mohan B., Taneja N.: Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative Ecoli in India, PLoS Negl Trop Dis 2020, 14, e0008769, doi: 10.1371/journal.pntd.0008769.ModgilV.MahindrooJ.NarayanC.KaliaM.YousufM.D.ShahiV.KoundalM.ChaudharyP.JainR.SinghSandha K.TanwarS.GuptaP.ThakurK.SinghD.GautamN.KakkarM.BhartiB.MohanB.TanejaN.Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E202014e000876910.1371/journal.pntd.0008769767354733206643DOI öffnenSearch in Google Scholar
Mora A., López C., Dhabi G., López-Beceiro A.M., Fidalgo L.E., Díaz E.A., Martínez-Carrasco C., Mamani R., Herrera A., Blanco J.E., Blanco M., Blanco J.: Seropathotypes, Phylogroups, Stx Subtypes, and Intimin Types of Wildlife-Carried, Shiga Toxin-Producing Escherichia coli Strains with the Same Characteristics as Human-Pathogenic Isolates. Appl Environ Microbiol 2012, 78, 2578–2585, doi: 10.1128/aem.07520-11.MoraA.LópezC.DhabiG.López-BeceiroA.M.FidalgoL.E.DíazE.A.Martínez-CarrascoC.MamaniR.HerreraA.BlancoJ.E.BlancoM.BlancoJ.Seropathotypes, Phylogroups, Stx Subtypes, and Intimin Types of Wildlife-Carried, Shiga Toxin-Producing Escherichia coli Strains with the Same Characteristics as Human-Pathogenic Isolates2012782578258510.1128/aem.07520-11DOI öffnenSearch in Google Scholar
Moura R.A., Sircili M.P., Leomil L., Matté M.H., Trabulsi L.R., Elias W.P., Irino K., Pestana de Castro A.F.: Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans. Appl Environ Microbiol 2009, 75, 7399–7408, doi: 10.1128/AEM.00636-09.MouraR.A.SirciliM.P.LeomilL.MattéM.H.TrabulsiL.R.EliasW.P.IrinoK.Pestanade Castro A.F.Clonal relationship among atypical enteropathogenic Escherichia coli strains isolated from different animal species and humans2009757399740810.1128/AEM.00636-09278640719801470DOI öffnenSearch in Google Scholar
Mrdovic B., Nastasijevic I., Brankovic Lazic I., Jovanovic J., Nikolic A., Petrovic Z., Raseta M.: Examination of meat preparations in order to control process hygiene in retail, IOP Conf Ser Earth Env 2019, 333, 012083, doi: 10.1088/17551315/333/1/012083.MrdovicB.NastasijevicI.BrankovicLazic I.JovanovicJ.NikolicA.PetrovicZ.RasetaM.Examination of meat preparations in order to control process hygiene in retail201933301208310.1088/17551315/333/1/012083DOI öffnenSearch in Google Scholar
Opeyemi U.L, Parreira V., Goodridge L.: The Biology and the Evolutionary Dynamics of Diarrheagenic Escherichia coli Pathotypes. In: Escherichia coli, edited by M. Starčič Erjavec, IntechOpen, London, 2022, pp. 1–37, doi: 10.5772/intechopen.101567OpeyemiU.LParreiraV.GoodridgeL.The Biology and the Evolutionary Dynamics of Diarrheagenic Escherichia coli PathotypesInedited byStarčič ErjavecM.IntechOpenLondon2022pp13710.5772/intechopen.101567DOI öffnenSearch in Google Scholar
Pakbin B., Allahyari S., Amani Z., Brück W.M., Mahmoudi R., Peymani A.: Prevalence, Phylogroups and Antimicrobial Susceptibility of Escherichia coli Isolates from Food Products. Antibiotics 2021, 10, 1291, doi: 10.3390/antibiotics10111291.PakbinB.AllahyariS.AmaniZ.BrückW.M.MahmoudiR.PeymaniA.Prevalence, Phylogroups and Antimicrobial Susceptibility of Escherichia coli Isolates from Food Products202110129110.3390/antibiotics10111291861517434827229DOI öffnenSearch in Google Scholar
Pass M.A., Odedra R., Batt R.M.: Multiplex PCRs for identification of Escherichia coli virulence genes. J Clin Microbiol 2000, 38, 2001–2004, doi: 10.1128/jcm.38.5.2001-2004.2000.PassM.A.OdedraR.BattR.M.Multiplex PCRs for identification of Escherichia coli virulence genes2000382001200410.1128/jcm.38.5.2001-2004.2000DOI öffnenSearch in Google Scholar
Paton A.W., Paton J.C.: Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1stx2eaeehxA, and saa. J Clin Microbiol 2002, 40, 271–274, doi: 10.1128/JCM.40.1.271-274.2002.PatonA.W.PatonJ.C.Direct detection and characterization of Shiga toxigenic Escherichia coli by multiplex PCR for stx1 stx2 eae ehxA, and saa20024027127410.1128/JCM.40.1.271-274.200212013611773130DOI öffnenSearch in Google Scholar
Pitout J.D.D.: Extraintestinal pathogenic Escherichia coli: A combination of virulence with antibiotic resistance. Front Microbiol 2012, 3, 9, doi: 10.3389/fmicb.2012.00009.PitoutJ.D.D.Extraintestinal pathogenic Escherichia coli: A combination of virulence with antibiotic resistance20123910.3389/fmicb.2012.00009326154922294983DOI öffnenSearch in Google Scholar
Qadri F., Svennerholm A.-M., Faruque A.S.G., Sack R.B.: Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 2005, 18, 465–483, doi: 10.1128/CMR.18.3.465-483.2005.QadriF.SvennerholmA.-M.FaruqueA.S.G.SackR.B.Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention20051846548310.1128/CMR.18.3.465-483.2005119596716020685DOI öffnenSearch in Google Scholar
Wang L., Liu N., Gao Y., Liu J., Huang X., Zhang Q., Li Y., Zhao J., Wang J., Zhao G.: Surveillance and Reduction Control of Escherichia coli and Diarrheagenic E. coli During the Pig Slaughtering Process in China. Front Vet Sci 2021, 8, 735076, doi: 10.3389/fvets.2021.735076.WangL.LiuN.GaoY.LiuJ.HuangX.ZhangQ.LiY.ZhaoJ.WangJ.ZhaoG.Surveillance and Reduction Control of Escherichia coli and Diarrheagenic E2021873507610.3389/fvets.2021.735076852388834676255DOI öffnenSearch in Google Scholar
Wang L., Wakushima M., Aota T., Yoshida Y., Kita T., Maehara T., Ogasawara J., Choi C., Kamata Y., Hara-Kudo Y., Nishikawa Y.: Specific Properties of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients and Comparison to Strains from Foods and Fecal Specimens from Cattle, Swine, and Healthy Carriers in Osaka City, Japan. Appl Environ Microbiol 2013, 79, 1232–1240, doi: 10.1128/AEM.03380-12.WangL.WakushimaM.AotaT.YoshidaY.KitaT.MaeharaT.OgasawaraJ.ChoiC.KamataY.Hara-KudoY.NishikawaY.Specific Properties of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients and Comparison to Strains from Foods and Fecal Specimens from Cattle, Swine, and Healthy Carriers in Osaka City, Japan2013791232124010.1128/AEM.03380-12356861623220963DOI öffnenSearch in Google Scholar
Yamamoto T., Echeverria P.: Detection of the enteroaggregative Escherichia coli heat stable enterotoxin 1 gene sequences in enterotoxigenic E. coli strains pathogenic for humans. Infect Immun 1996, 64, 1441–1445, doi: 10.1128/iai.64.4.1441-1445.1996.YamamotoT.EcheverriaP.Detection of the enteroaggregative Escherichia coli heat stable enterotoxin 1 gene sequences in enterotoxigenic E1996641441144510.1128/iai.64.4.1441-1445.19961739408606115DOI öffnenSearch in Google Scholar