Waterborne diseases are a major public health problem responsible for a high number of deaths worldwide, of which Escherichia coli is a major agent of contamination. This study investigates the occurrence of different diarrheagenic E. coli (DEC) pathotypes and its relationship with adherence patterns and biofilm formation. Between 2012 and 2014, a total of 1,780 drinking water samples were collected from different rural communities and urban water systems of north Paraná State. A total of 14% were positive for E. coli and 250 non-duplicate E. coli isolates were obtained. Between the E. coli isolates, 28 (11.2%) harbored DEC-associated genes, 10.7% being classified as Shiga toxin-producing E. coli (STEC), 64.3% enteroaggregative E. coli (EAEC) and 25% atypical enteropathogenic E. coli (aEPEC). The aggregative adherence (AA) was the predominant adherence pattern (84%), significantly associated with biofilm formation (p < 0.0001). On the other hand, the AA pattern and biofilm formation were not significantly associated to DEC pathotypes (p > 0.05). Therefore, we proposed that the AA pattern and biofilm formation in E. coli isolated from drinking water supplies could be associated with adherence and colonization of abiotic surfaces, such as pipes, leading to persistence and resistance to treatment or disinfection.

  • This study shows the relationship between DEC and non-DEC strains and adherence/biofilm formation.

  • The AA-positive strains were predominant and significantly associated to biofilm formation.

  • DEC and non-DEC strains present similar adherence patterns and biofilm formation values.

  • The AA pattern and biofilm formation could be responsible for the ability of these strains to persistence and resistance in the environment.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Waterborne diseases are an important public health problem worldwide and nearly 485,000 people die each year from diarrheal infections linked to contaminated drinking water (WHO 2019). In developing countries, such as Brazil, untreated water supplies are used as drinking water by millions of people who live in urban peripheries and rural communities (Bain et al. 2014b).

The Escherichia coli is an important indicator for detecting fecal contamination in drinking water supplies worldwide (Bain et al. 2014b). It is estimated that about 1.8 billion people use water sources contaminated by E. coli, being exposed to diarrheal diseases (Bain et al. 2014a; WHO 2019). Diarrheagenic E. coli (DEC) is an important etiologic agent responsible for intestinal infections, particularly in developing countries. This group is distributed into different pathotypes including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), Shiga toxin-producing E. coli (STEC) and enteroaggregative E. coli (EAEC) (Gomes et al. 2016).

EPEC and STEC are important enteric pathogens associated with diarrhea and foodborne outbreaks (Croxen et al. 2013). EPEC strains are further subdivided into two distinct groups, EPEC typical (tEPEC) and atypical (aEPEC), differentiated based on the presence of the bundle-forming pilus (BFP) found only in tEPEC (Hernandes et al. 2009). The island of pathogenicity called the locus of enterocyte effacement (LEE) contains genes associated with the formation of the attaching and effacing lesion, which include the eae gene responsible for encoding the outer membrane protein intimin (Clements et al. 2012; Gomes et al. 2016). STEC infections may result in severe complications such as hemorrhagic colitis and hemolytic-uremic syndrome (Croxen et al. 2013). STEC causes diseases in humans mainly due to the production of one or both types of potent cytotoxins called Shiga toxins (Stx1 and Stx2), which inhibit protein synthesis in the eukaryotic cell (Clements et al. 2012; Gomes et al. 2016).

EAEC has been considered a significant pathogen responsible for several cases of acute and chronic diarrhea worldwide (Hebbelstrup Jensen et al. 2014). Some molecular markers have been used for EAEC identification, including the plasmid genes aggR (aggregative adherence regulator) and aatA (anti-aggregation protein transporter), and chromosomal genes aaiA, aaiC and aaiG from a pathogenicity island denominated aai (aggR-activated island) (Lima et al. 2013; Andrade et al. 2014).

The adherence, colonization and biofilm formation are important characteristics associated to DEC pathotypes (Gomes et al. 2016). These features are closely associated with host pathogenesis, contributing to persistence of bacterial infection (Croxen et al. 2013). Distinct patterns of adherence to epithelial cells in vitro have been associated to DEC strains (Scaletsky et al. 1984, 1999; Nataro et al. 1987; Gomes et al. 2016). The localized adherence (LA) was initially linked with tEPEC strains, and this pattern is associated with the formation of microcolonies on the surface of cells (Hernandes et al. 2009). EAEC strains are identified by an aggregative adherence (AA) pattern characterized by a ‘stacked-brick-like’ arrangement on the surface of cells as well as abiotic surfaces (Nataro et al. 1987). In addition, some EAEC, STEC and non-DEC isolates express the AA and LA patterns or adhere with other adherence patterns such as localized adherence-like (LAL), diffuse adherence (DA), undefined patterns or non-adherent (NA) (Gomes et al. 2016).

Thus, the investigation of virulence and phenotypic characteristics of E. coli strains in drinking water supplies have an epidemiological importance, especially regarding the monitoring of possible contamination sources. Therefore, the objective of this study was to investigate the prevalence of E. coli strains in rural communities and urban water systems of north Paraná State and to evaluate the genotypic and phenotypic properties of these strains, i.e., presence of DEC-associated genes, adherence patterns and biofilm formation.

Sample collection, laboratory testing and control strains

Between January 1, 2012 and December 31, 2014, a total of 1,780 drinking water samples used for human consumption from different rural communities and urban water systems located in 20 municipalities of north Paraná State were collected. All samples collected in rural communities were obtained from untreated water supplies such as deep boreholes, dug wells and spring water. Furthermore, all samples collected in urban water systems were obtained from treated water supplies.

Water samples (250 mL) were collected into sterile glass bottles, stored on ice and transported to the laboratory for analyses within 6 h. These samples were tested for the detection of E. coli using the Colilert system (IDEXX Laboratories, represented by SOVEREIGN, Brazil), according to the manufacturer's instructions. For analyses, the content of one pack was added into 100 mL of water sample and shaken until dissolved. The sample/reagent was mixed in a sterile bottle and incubated at 35±0.5 °C for 24 h. Yellow and fluorescent samples were considered positive for E. coli.

Samples positives for E. coli were sub-cultured in MacConkey agar (Difco, USA) at 37 °C for 18 h. Lactose-fermenting colonies were randomly selected from each MacConkey plate and identified biochemically using the kit EPM, MILi and Simmons’ Citrate Agar (PROBAC, Brazil), according to the manufacturer's protocol. The isolates identified as E. coli were stored in Brain–Heart Infusion (BHI) broth with 20% glycerol at −80 °C.

Different E. coli strains were used as controls in the various experiments described in this study. Their detailed descriptions and references are presented in Table S1 (Supplementary Material).

DNA extraction

E. coli isolates were grown in lysogeny broth (LB; Difco) for 18 h at 37 °C. 1.5 mL of the culture were added to microcentrifuge tubes and centrifuged at 1,200 × g for 10 min. The bacterial pellet was suspended in 200 μL of sterile ultrapure water, boiled for 10 min and centrifuged at 1,200 × g for 5 min. The supernatant was used as a template for polymerase chain reactions (PCRs).

Detection of DEC pathotypes by PCR

All E. coli isolates were subjected to PCR for detection of different markers for DEC pathotypes. Strains positive for stx or stx/eae genes were classified as STEC, whereas eae+/bfpA+ or eae+/bfpA– strains were classified as typical EPEC (tEPEC) or atypical EPEC (aEPEC), respectively. In addition, the aatA, aggR, aaiA and aaiC genes were used to identify EAEC strains; est and elt for identifying ETEC strains and ipaH for identifying EIEC. Primers, cycle conditions, sizes of amplified fragments and corresponding references are listed in Table S2 (Supplementary Material).

The PCRs were performed on Gene Amp PCR System 9700 thermal cycler (Applied Biosystems, USA), containing 25 μL with 2 μL of bacterial lysates, 200 μM of dNTPs, 1 × PCR buffer, 2 mM of MgCl2, 20 μM of each primer and 1 U of Taq DNA Polymerase (Invitrogen, USA). The amplified products were analyzed by 1.5% agarose gel electrophoresis, stained with SYBR Safe™ DNA Gel Stain solution (Invitrogen) and observed under ultraviolet light (Vilbert Loumart, France). A 100-bp DNA ladder (Invitrogen) was used as a molecular size maker.

Adherence assay

Bacterial adherence assay to HEp-2 (human laryngeal epithelial carcinoma ATCC CCL-23) cells was performed as described by Cravioto et al. (1979), with some modifications previously described (Puño-Sarmiento et al. 2014). The assay was performed allowing 6 h of bacterial–cell interaction at 37 °C in the presence of 1% d-mannose (Sigma-Aldrich, USA) and then evaluated using light microscopy. The adherence patterns were determined as previously described (Scaletsky et al. 1984, 1999; Nataro et al. 1987; Rodrigues et al. 1996).

Biofilm formation assay

Biofilm formation was assessed by the method described by Wakimoto et al. (2004). The cutoff (forming and non-forming biofilm) was established according to Stepanovic et al. (2000). Positive strains for biofilm formation were subdivided into three groups according to absorbance: weak (+), moderare (++) or strong (+++) following Stepanovic's criteria. This assay was performed in triplicate and E. coli HB101 (E. coli K-12) was used as a negative control.

Statistical analysis

Statistical analyses were performed using the R statistical software version 3.6.2. Results were analyzed by Fisher's Exact test, χ2 test or Mann–Whitney's U test. Statistical significance was established at p < 0.05.

During the study period, 1,780 drinking water samples were tested for E. coli presence using the Colilert system. The prevalence of E. coli-positive samples from untreated water was significantly higher than treated water supplies (p < 0.0001). Specific contamination rates in deep boreholes, dug wells, spring water (mines) and urban water systems are described in Table 1.

Table 1

Prevalence of E. coli in different drinking water supplies

SourceaNo. of samples (n = 1,780)No. (%) of samples positive for E. coli (n = 250)
DBH 222 84 (37.8) 
DGW 180 89 (49.4) 
SPW 136 67 (49.3) 
UWS 1,242 10 (0.8) 
SourceaNo. of samples (n = 1,780)No. (%) of samples positive for E. coli (n = 250)
DBH 222 84 (37.8) 
DGW 180 89 (49.4) 
SPW 136 67 (49.3) 
UWS 1,242 10 (0.8) 

aDBH, deep borehole; DGW, dug well; SPW, spring water; UWS, urban water system.

A total of 250 non-duplicate E. coli isolates were recovered and evaluated for the presence of DEC-associated genes. Of the 250 E. coli isolates analyzed, seven (2.8%) harbored genotypic characteristics of aEPEC (eae-positive and bfpA-negative) and three (1.2%) of STEC (stx-positive). Additionally, 18 E. coli isolates (7.2%) were positive to aaiC gene, being classified as EAEC. The specific genes used to identify ETEC and EIEC phatotypes were not detected. The general characteristics of DEC strains are presented in Table 2.

Table 2

Characteristics of DEC strains isolated from drinking water supplies

StrainOriginaDEC-associated genesPhenotypic assays
Pathotyped
Hep-2bBiofilm formationc
L232/12 SPW eae AA aEPEC 
L82/13 SPW eae AA ++ aEPEC 
L91/13 SPW eae AA aEPEC 
L109/13 DBH eae AA aEPEC 
L110/13 SPW eae AA +++ aEPEC 
L414/13 DGW eae AA aEPEC 
L536/14 SPW eae UND − aEPEC 
L95/13 DBH stx1 stx2 AA STEC 
L439/13 SPW stx1 AA STEC 
L414/13 SPW stx1 AA STEC 
L59/12 DGW aaiC AA +++ EAEC 
L110/12 DGW aaiC AA EAEC 
L206/12 DGW aaiC AA EAEC 
L265/12 DGW aaiC AA ++ EAEC 
L79/13 DGW aaiC AA +++ EAEC 
L160/13 DBH aaiC AA ++ EAEC 
L165/13 SPW aaiC AA ++ EAEC 
L200/13 DGW aaiC AA − EAEC 
L205/13 SPW aaiC AA ++ EAEC 
L227/13 SPW aaiC AA +++ EAEC 
L243/13 SPW aaiC AA +++ EAEC 
L305/13 DGW aaiC AA ++ EAEC 
L411/13 SPW aaiC AA EAEC 
L255/14 SPW aaiC AA ++ EAEC 
L554/14 SPW aaiC AA +++ EAEC 
L586/14 SPW aaiC AA +++ EAEC 
L617/14 SPW aaiC AA ++ EAEC 
L668/14 DGW aaiC AA ++ EAEC 
StrainOriginaDEC-associated genesPhenotypic assays
Pathotyped
Hep-2bBiofilm formationc
L232/12 SPW eae AA aEPEC 
L82/13 SPW eae AA ++ aEPEC 
L91/13 SPW eae AA aEPEC 
L109/13 DBH eae AA aEPEC 
L110/13 SPW eae AA +++ aEPEC 
L414/13 DGW eae AA aEPEC 
L536/14 SPW eae UND − aEPEC 
L95/13 DBH stx1 stx2 AA STEC 
L439/13 SPW stx1 AA STEC 
L414/13 SPW stx1 AA STEC 
L59/12 DGW aaiC AA +++ EAEC 
L110/12 DGW aaiC AA EAEC 
L206/12 DGW aaiC AA EAEC 
L265/12 DGW aaiC AA ++ EAEC 
L79/13 DGW aaiC AA +++ EAEC 
L160/13 DBH aaiC AA ++ EAEC 
L165/13 SPW aaiC AA ++ EAEC 
L200/13 DGW aaiC AA − EAEC 
L205/13 SPW aaiC AA ++ EAEC 
L227/13 SPW aaiC AA +++ EAEC 
L243/13 SPW aaiC AA +++ EAEC 
L305/13 DGW aaiC AA ++ EAEC 
L411/13 SPW aaiC AA EAEC 
L255/14 SPW aaiC AA ++ EAEC 
L554/14 SPW aaiC AA +++ EAEC 
L586/14 SPW aaiC AA +++ EAEC 
L617/14 SPW aaiC AA ++ EAEC 
L668/14 DGW aaiC AA ++ EAEC 

aSPW, spring water; DBH, deep borehole; DGW, dug well.

bAdherence patterns to HEp-2 cells: AA, aggregative adherence; UND, undefined pattern.

cThe strains were classified into four groups according to absorbance: no biofilm production (−), weak biofilm production (+), moderate biofilm production (++), and strong biofilm production (+++), following the Stepanovic's criteria (Stepanovic et al. 2000).

daEPEC, atypical enteropathogenic E. coli; STEC, Shiga toxin-producing E. coli; EAEC, enteroaggregative E. coli.

In adherence assay, 240 E. coli isolates (96%) adhered to HEp-2 cells after 6 h of incubation and 10 (4%) were NA. The AA and DA patterns (Figure 1) were observed in 210 (84%) and four (1.6%) strains, respectively. The remaining strains (26/250; 10.4%) showed a distinct adherence pattern from those previously described, being classified as undefined pattern (UND). Strains presenting LA or LAL patterns were not found. The prevalence of different adherence patterns in DEC and non-DEC strains is presented in Table 3. There was no significant association between adherence patterns and DEC pathotypes (p > 0.05).
Table 3

Prevalence of adherence patterns in E. coli strains

Patterns of adherenceaNo. (%) of isolates
p-Valueb
Non-DEC (n = 222)aEPEC (n = 7)STEC (n = 3)EAEC (n = 18)
AA 183 (82.4) 6 (85.7) 3 (100) 18 (100) 0.197 
DA 4 (1.8) 1.000 
NA 10 (4.5) 1.000 
UND 25 (11.3) 1 (14.3) 0.498 
Patterns of adherenceaNo. (%) of isolates
p-Valueb
Non-DEC (n = 222)aEPEC (n = 7)STEC (n = 3)EAEC (n = 18)
AA 183 (82.4) 6 (85.7) 3 (100) 18 (100) 0.197 
DA 4 (1.8) 1.000 
NA 10 (4.5) 1.000 
UND 25 (11.3) 1 (14.3) 0.498 

aAA, aggregative adherence; DA, diffuse adherence; NA, non-adherent; UND, undefined pattern.

bDetermined by Fisher's Exact test.

Figure 1

Adherence patterns exhibited by E. coli strains. The adherence assays were performed with HEp-2 cells, allowing 6 h of bacterial–cell interaction at 37 °C in the presence of 1% d-mannose. (a) Aggregative adherence (AA) pattern (strain L265/12), (b) diffuse adherence (DA) pattern (strain L23/14) and (c) non-adherent (NA) (strain L166/13). Bars = 50 μm.

Figure 1

Adherence patterns exhibited by E. coli strains. The adherence assays were performed with HEp-2 cells, allowing 6 h of bacterial–cell interaction at 37 °C in the presence of 1% d-mannose. (a) Aggregative adherence (AA) pattern (strain L265/12), (b) diffuse adherence (DA) pattern (strain L23/14) and (c) non-adherent (NA) (strain L166/13). Bars = 50 μm.

Close modal
E. coli isolates were also phenotypically examined by quantitative biofilm assay presenting optical density (OD570 nm) values in the range of 0.05–1.2. First, we compared the biofilm formation between AA-positive and AA-negative E. coli isolates. The AA-positive strains showed significantly stronger biofilm than non-AA strains (p < 0.0001; Figure 2(a)), demonstrating the relationship between AA pattern and biofilm formation. In the AA-positive isolates, there was no significant difference in biofilm formation between DEC and non-DEC strains (p = 0.1; Figure 2(b)). Therefore, biofilm formation was only associated to AA pattern and not associated to DEC pathotypes. In addition, among DEC isolates, ten (35.7%) were classified as weak biofilm producers, nine (32.1%) as moderate biofilm producers, seven (25%) as strong biofilm producers and only two (7.1%) did not produce biofilm Figure S1 (Supplementary Material).
Figure 2

Biofilm formation of E. coli strains isolated from drinking water supplies. (a) Comparison of biofilm formation between AA-positive and AA-negative strains. (b) Comparison of biofilm formation between AA-positive strains classified as non-DEC or DEC. Box plots show median and the 10th, 25th, 75th and 90th percentiles, whereas p-values were determined by Mann–Whitney U test.

Figure 2

Biofilm formation of E. coli strains isolated from drinking water supplies. (a) Comparison of biofilm formation between AA-positive and AA-negative strains. (b) Comparison of biofilm formation between AA-positive strains classified as non-DEC or DEC. Box plots show median and the 10th, 25th, 75th and 90th percentiles, whereas p-values were determined by Mann–Whitney U test.

Close modal

Drinking water supplies have been used for human consumption and food preparation worldwide (WHO 2019). In this study, E. coli was detected in 14% of drinking water samples, being higher in untreated samples (44.6%). The absence of treatment for drinking water in poorer regions (urban peripheries and rural communities) may lead to a higher fecal contamination and, consequently, of E. coli (Bain et al. 2014b). Overall, the E. coli in drinking water supplies represent a potential threat for human health and their occurrence may be an indicator of diarrheal risk (Bain et al. 2014a).

In Brazil, we previously documented the presence of DEC strains in drinking water supplies (Lascowski et al. 2013; da Silva et al. 2019) reinforcing the role of the water as an important source for these strains. Although phenotypic assays have been widely used for the characterization of DEC strains (Gomes et al. 2016), to our knowledge, there are no studies comparing phenotypical characteristics such as adherence patterns and biofilm formation between DEC and non-DEC strains isolated from drinking water.

The presence of bacterial strains presenting the AA pattern and biofilm formation is an important factor that contributes to the persistence of bacterial infection, making the microorganism more resistant to treatment or disinfection (Hebbelstrup Jensen et al. 2014). As previously described (Wakimoto et al. 2004), we confirmed the relationship between the AA pattern and biofilm formation in E. coli strains. On the other hand, this pattern was not associated to DEC pathotypes, where several AA-positive strains (87.1%) were negative to DEC-associated genes. Although AA pattern has been originally described as the gold standard for EAEC identification (Nataro et al. 1987), this phenotype has been detected in STEC, aEPEC and non-DEC strains (Gomes et al. 2016). Also, the AA pattern has been considered prevalent in E. coli strains isolated from drinking water supplies and other environmental sources (Lascowski et al. 2013; Puño-Sarmiento et al. 2014; Schuroff et al. 2014; Gazal et al. 2015; da Silva et al. 2019). At present, it has been considered flawed to classify only AA-positive strains into the EAEC category (Boisen et al. 2020). Thus, it has been proposed that a sensitive and specific diagnosis of EAEC could include the detection of AA pattern and different genetic markers presenting in plasmid (aatA/aggR) and/or chromosomal genes (aai) (Lima et al. 2013; Andrade et al. 2014). Therefore, only the AA pattern was not enough to classify our strains into EAEC pathotype.

The capability of drinking water strains to adhere and form biofilm on abiotic surfaces is extremely effective, enabling the colonization of piping and water reservoirs (Wingender & Flemming 2011; Abberton et al. 2016). In this study, the high prevalence of AA-positive and biofilm-forming strains reinforces that these phenotypes could be associated with adherence and colonization of abiotic surfaces, leading to persistence and resistance to treatment or disinfection and allowing the bacterial survival in drinking water systems. Additionally, future questions about the virulence factors responsible for AA pattern and/or biofilm formation and its role in environmental colonization and pathogenicity in the host need to be better understood.

In summary, our results emphasize that drinking water supplies harbored virulence factors associated with DEC (i.e., EAEC, aEPEC and STEC), reinforcing the role of water as an important source of these pathotypes. In addition, the high prevalence of AA pattern and biofilm formation between DEC and non-DEC strains reinforce that these phenotypes could be associated to colonization in abiotic surfaces and persistence in the environment. Finally, new studies are required to better understand virulence factors responsible for AA/biofilm formation in environmental E. coli strains.

The authors thank the Virology Laboratory (UEL) for supplying the HEp-2 cell cultures and Barbara B. Fernandes for English support.

P.A.S. performed the isolation and experiments with E. coli strains; P.A.S. and F.B.A. conceptualized, prepared, and wrote the original draft; J.S.P. reviewed and edited the article. All authors have read and agreed the article to be published.

This study was supported by a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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