E. coli strains are highly affected by the propagation of resistance and virulence genes in urban rivers when compared to other Enterobacteriacea (Tortora, Funke, Case, 2008, Kittinger et al., 2016). However, there is still a poor understanding of the environmental factors that may alleviate the spread of antibiotic resistance. At present, it is not clear to what extent environmental antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) promote the acquisition and spread of antibiotic resistance among clinically relevant bacteria, or whether ARGs that are acquired by both clinically relevant bacteria and strictly environmental bacteria originate from the same reservoirs (Berendonk, et al., 2015).
In this present study, we documented the presence of resistant and MDR E. coli strains isolated from Joana river, located at Rio de Janeiro metropolitan area, Brazil. Heterogeneity among virulence pathotypes as well as host pathogen interaction with Caco-2 cells was verified among MDR river isolates. The presence of antimicrobial resistant E. coli in aquatic systems released from anthropogenic sources such as community, industries, veterinary and hospitals, is a public health concern in industrialized and developing countries due to relevance on environmental dissemination of antimicrobial resistance (Djordjevic, Stokes, Chowdhury, 2013; Berendonk, et al., 2015).
Although generally harmless, E. coli strains may express virulence potential properties that account for human localized and invasive infections in both communities and hospital enviroments (Hall-Stoodley, Costerson, Stoodley, 2004). The plasticity of the E. coli genome has hindered the identification of certain E. coli isolates as a pathotype, because some isolates combine the main virulence characteristics of different pathotypes and are thus considered potentially more virulent hybrid pathogenic strains. In this study, MDR E. coli river isolates presented the following distinct pathotypes: STEC, EHEC, EIEC/STEC.
MDR E. coli Ec31 isolated strain was classified as EHEC due to the ability to produces stx1 and stx2 Shiga toxin (Stx) cytotoxins associated with eaeA gene. MDR E. coli strains Ec21, Ec30 and Ec10 presented stx2 Shiga toxin (Stx) cytotoxins. The association of Stx cytotoxins, especially stx2, with severe diseases has been extensively studied by using endothelial cell lines and their ability to adhere is related to EHEC/STEC pathogenesis (Rivas et al., 2016). In addition, biofilm may act as bacterial protection against adverse environmental conditions, especially in aquatic envionment. A study conducted by Biscola and co-workers (2011) evaluated the capacity of biofilm formation in EHEC/STEC strains isolated from different reservoirs and demonstrated strongly ability to adhere on both glass and polystyrene surfaces. In fact, cell invasion and survival of EHEC/STEC strains in cultured human intestinal epithelial cells has been previously described (Cordeiro et al., 2013) and may be related to biofilm strongly adherence. It should be mentioned that this invasive characteristic has been identified in EHEC/STEC serotypes, responsible for human infections (Mateus-Guimarães et al., 2014; Cordeiro et al., 2013) and isolated from water representing an important vehicles of transmission (Lascowski et al., 2013).
Our studies also demonstrated the presence of MDR enteroinvasive E. coli Ec21 strain. EIEC is a causative agent of dysentery in humans, especially in developing countries, due to their ability to invade and penetrate cells by endocytosis, as shown in table 2. Despite the similarities invasion mechanism and symptoms of the disease, the infectious dose of EIEC appears to be a milder and self-limiting form when compared to Shigella, who leads to an exacerbation of proinflammatory response. EIEC was responsible for several outbreaks, but there are few reports on routes of transmission and distribution of this bacterium in nature, including water and cheese (Marier et al., 1973; Valentini et al., 1992) as well as the direct transmission through person-to-person contact. The isolation of EIEC in Brazil has ranged from 0.5 to 15%, depending on the population investigated (Moreno et al., 2010; Lozer et al., 2013). Toledo and Trabulsi (1990) investigated the presence of this microorganism in different areas of the city of São Paulo. This bacterium has been found related to children with diarrhea (15.9%). Studies performed outside the city of São Paulo showed a low prevalence of these bacteria, 0.5–2.5% (Oliveira et al., 1989).
Few studies have been conducted to investigate pathogenic E. coli strains in urban rivers, although pollution of surface waters with these pathogens has been implicated in an increased number of disease outbreaks and consequent deaths (Masters et al., 2011).
In attempt to investigate virulence potential of E. coli environmental isolates expressing MDR profiles and virulence genes, were investigated for ability of interaction with Caco-2 human intestinal epithelial cells. All MDR E. coli strains of STEC, EHEC and EIEC/STEC pathotypes were able to adhere to epithelial cells surfaces. MDR E. coli (Ec 10 and Ec30) isolates, classified as STEC pathotypes and presenting stx2 gene, expressed the higher ability of adherence, internalization and persistence within Caco-2 epithelial cells. Previous report documented that STEC annually was responsible to 2,801,000 cases of acute illness, 3890 HUS cases, 270 permanent end-stage renal disease and 270 deaths worldwide and cases of infections have been traced to person-to-person transmission (Duffy, Burgess, Bolton, 2014; Majowicz et al., 2014).
Presently, MDR E. coli strain (Ec21) of EIEC pathotypes and harboring ial gene, showed the ability of adherence, internalization and persistence within Caco-2 epithelial cells. EIEC infection occurs via fecal-oral route by the ingestion of contaminated food or water and invasion of colonic epithelium, causing abdominal cramps, bloody and mucous diarrhea. During the last decades, there are an increase number of EIEC cases in varied countries, including two large outbreaks in Europe (Thong et al., 2005; Bueris et al., 2007; Michelacci et al., 2020).
Moreover, one MDR E. coli (Ec31) river isolate was characterized as EHEC, presenting lower levels of adherence, internalization and persistence within Caco-2 epithelial cells. Virulence potential of this pathotypes is partially demonstrated by the ability of attaching intimately and effacing microvilli of epithelial intestinal cells that can directly induce renal and endothelial lesions due to expression of eaeA, Stx1 and Stx2 genes (Donnenberg, 1993; Maule, 2000; Gomes et al., 2016). Survival and persistence of EHEC in contact with surfaces and exposure to water environments among other conditions should be recognized as important risk factors in the spread of this pathogen, including rivers located at metropolitan areas. Data that deserves attention
In this study, one MDR E. coli (Ec31) river isolate was characterized as EHEC, presenting lower levels of adherence, internalization and persistence within Caco-2 epithelial cells, possibly related to previously described cytotoxicity abilities - whether apoptosis and/or necrosis (Donnenberg, 1993; Maule, 2000; Gomes et al., 2016; Abul-Milh et al., 2001). Data reinforce the fact that survival and persistence of EHEC in contact with surfaces and exposure to water environments among other conditions should be recognized as important risk factors in the spread of this pathogen, including rivers located at metropolitan areas. Information that deserves attention concerning the virulence potential and risk of contamination by EHEC pathotypes is the ability of causing acute infections with only ten bacterial cells indicating a high virulence level (Maule, 2000).