Escherichia coli is one of the most frequent causes of diarrhea in children in developing countries. However, characterization of truly diarrheagenic groups or strains can be a complex task because this species is one of the first colonizers of the human gut. Moreover, wild strains exhibit great genetic plasticity and heterogeneity 1 .

Diffusely adherent Escherichia coli have been considered a diarrheagenic group of E. coli (DEC). They are characterized by the diffuse adherence pattern on cultured epithelial cells HeLa or HEp-2 2 . Approximately 75% of DAEC harbor adhesins from the Afa/Dr family, responsible for this adherence phenotype 3 . Since Germani et al. 4 demonstrated that, among DAEC strains, only those that were positive to daaC probe - that recognize a conserved region from Afa/Dr adhesins operons - were found in higher frequency in diarrheic patients than asymptomatic controls, much attention has been given to DAEC strains possessing Afa/Dr adhesins.

The adhesins of Afa/Dr family have been implicated in DAEC pathogenesis. They include adhesins found in uropathogenic strains, like the Dr adhesin, in addition to AfaE-I, AfaE-II, AfaE-III, AfaE-V and F1845, which occur in diarrheagenic DAEC strains 5 . They recognize DAF (Decay Accelerating factor, CD55) and some of them also recognize CEACAMs (CEA-related molecules) as receptors 3 . The receptor is recruited around the bacteria after binding to the host cell 6 7 . The binding of strains expressing F1845 or Dr adhesin can promote the dismantling of the actin network in intestinal cells, causing elongation of microvilli 8 9 and redistribution of cytoskeleton-associated proteins in HeLa cells 10 .

However, in some studies, DAEC Afa/Dr⁺ strains are isolated from cases of diarrhea and controls in similar frequencies 11 12 , suggesting that additional factors may be necessary to trigger disease. In recent years, some of these potential virulence factors have been described. In addition, some studies have implicated DAEC strains as diarrheal agents only in children older than six months, depending on the study, and in adults. 4 13 14 15 16 17 18 19 .

Evidence of a type three secretion system (TTSS) in DAEC Afa/Dr⁺ isolated from cases of diarrhea in children has been demonstrated by Kyaw et al. 20 . The concomitant presence of Afa/Dr adhesins in these strains suggests that an adhesin-receptor-effector protein mechanism, similar to the one seen in EPEC (enteropathogenic E. coli), might occur in DAEC. After adhesion and intimate contact, EPEC strains use TTSS to inject effector proteins into the host cell, inducing lesions in the cytoskeleton.

Taddei et al. 21 reported the presence of the secreted autotransporter toxin (SAT) belonging to the family of serine protease autotransporters of Enterobacteriaceae (SPATE) in DAEC strains isolated from diarrhea. Guignot et al. 22 have demonstrated that SAT is able to cause lesions on tight junctions of epithelial cells, which in turn may lead to an increase in their permeability. They also found SAT more frequently in DAEC strains isolated from diarrheic children than from asymptomatic subjects, corroborating the role of SAT as a virulence factor.

DAEC strains have demonstrated pro-inflammatory effects, related to an increased secretion of interleukin-8 by epithelial cells. In T84 cells infected by wild-type strains, basolateral secretion of IL-8 promotes transmigration of polymorphonuclear leukocytes (PMNLs) across the epithelial monolayer 23 . The transmigrated PMNs increase apoptotic rates and reduce phagocytic activity 24 which can contribute to maintain the inflammatory response without eliminating the pathogen. Some studies 18 25 have found that the ability of increasing IL-8 secretion in epithelial cells by DAEC strains was associated with diarrhea in children.

One characteristic that has not been studied in DAEC is the ability to form biofilms. Although biofilm formation is a widespread phenomenon in bacteria, only recently has the importance of biofilms as a pathogenic factor been demonstrated for E. coli, such as in atypical EPEC strains 26 and in enteroaggregative E. coli (EAEC). The latter have biofilm formation as the only consensual virulence factor 27 .

In a previous study performed in this laboratory 28 , it was found that EAEC biofilms could be enhanced by interaction with a Citrobacter freundii strain isolated concomitantly with EAEC from a diarrheic child. These mixed biofilms seem to be mediated by F pili. Aside from their role in conjugation, F pili have been considered important in establishing E. coli biofilms, in addition to other components like curli and cellulose 29 30 . In this work, we employed a C. freundii strain to assess the capacity of DAEC strains to form biofilms alone or with other bacteria, as well as to investigate the occurrence of synergistic effects as seen for EAEC strains. Furthermore, we analyzed characteristics potentially associated with virulence or related to biofilm formation, such as Afa/Dr adhesins, SAT toxin, TTSS, F pili, curli, cellulose and stimulus of IL-8 secretion by epithelial cells. The aim of this work is to study the overall profile of DAEC strains isolated from children and adults, both from cases of diarrhea and controls, thereby performing a systematic study of DAEC.

The human gut is colonized by a very complex and diversified microbiota. Bacteria in the gastrointestinal tract play multiple roles in human health, including metabolic features absent in humans 31 , modulation of gut morphology and physiology 32 and development of the immune system 33 34 35 . Colonization begins at birth, but maturation of the microbiota is a continuous process lasting for several years 36 37 38 .

One of the first facultative organisms to colonize the human gut is E. coli 39 40 . There is an ongoing debate on whether diffusely adherent E. coli (DAEC) represent normal inhabitants of the gut or diarrheagenic strains, because many epidemiological studies have shown inconsistent results 11 14 41 . As the controversy has been attributed, at least in part, to an age factor 13 14 15 16 17 18 we compared DAEC strains belonging to four different groups: children with diarrhea, asymptomatic children, adults with diarrhea and asymptomatic adults.

We have found remarkable differences between strains isolated from adults and from children regarding the characteristics analyzed in this work.

DAEC strains with undetermined afaE were first reported by Zhang et al. 42 that described new variants of Afa/Dr adhesins. In 20% (30/150) of afaB-C-positive strains in this study, the “E” gene was not identified, and the strains were referred to as “afa-X-positive” strains. In the adult group, afa-X was only found in strains isolated from cases of diarrhea. This result is similar to that found by Arikawa et al. 25 , who reported the presence of undetermined afaE in 26.3% (5/19) of DAEC strains isolated from cases of diarrhea (which they called “afaEX”). In contrast, in another work from Japan 43 the authors found afaEX strains isolated from healthy adults. It is unclear if afa-X and afaEX strains harbor the same or different Afa/Dr adhesins, since the afaE gene was not identified. It is likely that there are many yet undescribed variations of Afa/Dr adhesins. Korotkova et al. 44 showed that point mutations in Dr adhesin genes result in phenotypic variability with distinct binding properties. However, in a previous work performed in this laboratory 19 the analysis of surface proteins showed that all afa-X strains isolated from diarrheic adults had an identical electrophoretic profile, suggesting that all these strains harbor an identical member of Afa/Dr family. Further studies are required to identify Afa-X and clarify its role in the pathogenesis of diarrheas caused by DAEC in adults.

Strains from adults exhibit few types of adhesins in a characteristic pattern: AfaE-V associated with control and the putative Afa/Dr adhesin Afa-X with diarrhea. In contrast, several types of Afa/Dr adhesins are present in strains from children, with no one in particular associated with disease. Great diversity regarding the types of Afa/Dr adhesins was especially frequent among strains isolated from asymptomatic children, with 29.3% of strains harboring more than one Afa/Dr adhesin. The afaE1 and F1845 adhesins are always present in the associations. Both recognize DAF as a receptor, and F1845 also recognizes CEACAMs 2 . Since adhesins are involved in colonization, the presence of related adhesins able to recognize different receptors could provide an adaptive advantage to these bacteria and explain the apparent redundancy of Afa/Dr adhesins. Interestingly, DAF expression in erythrocytes is higher in adults than in children 45 , being especially low in children aged between 24 and 36 months 46 . If this differential expression were also found in enterocytes, it would help explain the advantage of strains from children in presenting adhesins able to bind to receptors other than DAF.

A factor frequently detected in strains isolated from children was the expression of curli. Curli is a bacterial structure involved in the adhesion to both fresh vegetables 47 48 49 and several proteins widespread in human cells or extracellular matrix, like MHC class I, TLR2, fibronectin and laminin 50 . Most DAEC strains from children that express curli at 37°C were also capable of expressing curli at 28°C (data not shown). Therefore, curli could facilitate further colonization by E. coli ingested through food sources mediating attachment once the bacteria are in the body.

By contrast, curli expression was frequent in strains isolated from diarrheic adults but rare in strains from asymptomatic adults, suggesting a potential involvement with diarrheal disease in adults. Several studies have associated curli to virulence of E. coli. Besides being a colonization factor 50 , curli leads to the stimulation of inflammatory response in its host 50 51 , which is mediated by TLR1/TLR2 52 . Curli was associated to higher rates of invasion of epithelial cells 53 and increased virulence in mice 54 .

Curli shares many characteristics with human amyloids 55 . Amyloid deposits induce chronic inflammation, which in turn results in tissue injuries associated with neurodegenerative diseases, with Alzheimer’s disease being the most notorious example. Some lines of evidence suggest that old cells (at least neurons) can be more susceptible to beta-amyloids 56 57 58 . Analogously, adults could be more susceptible to bacterial amyloids than children, helping to explain why curli might be associated to diarrhea in adults, but not in children. Furthermore, the immune system in children is not fully developed 33 , leading us to speculate that while curli expressing E. coli strains might be carried by asymptomatic children, healthy adults’ immune systems could exclude those potentially virulent strains.

In EPEC strains, the TTSS is part of the the LEE pathogenicity island 3 . Pathogenicity islands (PAIs) are large portions of microbial genomes that are present in pathogens but not in non pathogenic strains of the same species 59 . In this study, genes that are part of the TTSS apparatus were found in strains isolated from asymptomatic children. Despite considering that they were detected too frequently to be found incidentally, we do not know whether these strains possess a functional TTSS. Blanc-Potard et al. 60 found that Afa/Dr DAEC strains C1845 and IH11128 harbor part of a PAI described for an uropathogenic E. coli strain. Analogously, some DAEC strains from children could harbor part of a LEE, including part of a TTSS, but not necessarily the complete functional apparatus. Interestingly, TTSS genes were found in strains from children, but not in strains from adults. Many strains from children also belong to some classical EPEC serogroup - again not found in strains from adults - leading us to wonder whether the strains from children may be more closely related to EPEC in evolutionary terms. Although TTSS has been associated to virulence in a broad range of Gram-negative bacteria 61 , we have found it in control strains. Even though much emphasis has been given to the role of TTSS in pathogenesis, its presence was recorded in non-pathogenic bacteria such as Pseudomonas fluorescens 62 and Sodalis glossinidius 63 .

By the late fifties, the development of seroagglutination assays enabled the establishment of the classical groups of EPEC. These serogroup-marked strains were frequently associated with sporadic cases of infantile diarrhea as well as outbreaks 64 . In virtue of the current molecular characterization adopted for typing E. coli strains, nowadays it is known that some of the so-called classical EPEC serogroups are shared with other diarrheagenic categories 65 66 67 . The World Health Organization recognized that EPEC comprises strains of 12 O serogroups known as the classical EPEC serogroups: O26, O55, O86, O111, O114, O119, O125,O126, O127, O128, O142 and O158 68 In this work, we found 30.5% of DAEC isolated from children belonging to serogroups O86, O127, O142 or O158. Serogroup O86 was very frequent, corresponding alone to 20% of DAEC strains isolated from children. This serogroup seems to be widely distributed among different E. coli pathotypes, since it has been found in EAEC 65 , DAEC 67 and STEC strains 69 . Interestingly, we have not found DAEC strains from adults belonging to EPEC serogroups, reinforcing the differences between DAEC strains isolated from children and adults.

Arikawa et al. 25 found that some DAEC strains are able to stimulate IL-8 secretion by epithelial cells and suggested that strains possessing this ability could be implicated in the establishment of diarrhea. The importance of IL-8 stimulus in the pathogenesis of DAEC strains was reinforced by the study of Meraz et al. 18 . In a more recent work Arikawa et al. 70 found that high levels of IL-8 secretion by epithelial cells were associated with stimulation by flagella via the TLR5 receptor.

We found some DAEC strains stimulating IL-8 secretion by HeLa cells. Meanwhile, association with the motility of strains, and consequently to flagella, was not found, perhaps because almost all DAEC strains in this work were mobile. Interestingly, we found more strains able to stimulate IL-8 secretion cells among strains isolated from asymptomatic children. However, most of DAEC strains stimulated only low levels of IL-8 secretion, which could simultaneously explain the lack of association with diarrhea and the presence of the flagella.

Developing microbiota in children is not formed by random bacterial groups, but instead consisting of bacterial consortia that interact among themselves 71 . Thus, the chance of a given E. coli strain establishing itself will be determined, in large part, by the partners previously found in the gut environment and by the relationships among them. A C. freundii strain (Cf 205) that was shown to be capable of increasing biofilm formation of EAEC strains isolated from cases of diarrhea was selected from a previous study 28 . Since many DAEC strains were not able to form biofilms alone, or only form weak biofilms, we decided to investigate the effect of Cf 205 in DAEC mixed biofilm assays. Consortia DAEC-C. freundii showed not only increased biofilm formation, but also higher adhesion to cultured cells, suggesting that bacterial combinations can be decisive for colonization. A great increase in biofilm formation was observed especially when strains isolated from asymptomatic children were employed in mixed biofilm assays, perhaps because these strains possess greater diversity of adhesins that could help interactions with C. freundii. Those strains also showed greater production of cellulose, which is an important component of biofilms, and cellulose could facilitate adherence of bacterial consortia both to abiotic surfaces and cell surfaces.

Other bacterial components possibly involved in formation of mixed biofilms are F pili. It has been demonstrated that the presence of natural conjugative plasmids promotes biofilm formation 29 and that F pili are used in the initial stages of E. coli biofilm formation 30 . We believe that F pili are involved in mixed biofilms since most of them were inhibited by zinc in a concentration that does not affect bacterial growth. Furthermore, Pereira et al. 28 demonstrated that cell-to-cell interactions involved in EAEC-Cf 205 biofilms were mediated by putative F pili, leading us to hypothesize that F pili also mediate DAEC – Cf 205 biofilms.

The effect of a toxin and the resulting association to diarrhea depend on its effective concentration at the site of infection, which in turn depends on the density of producing bacterial cells. Although SAT is highly prevalent in strains from adults, association with diarrhea was found only in strains from children – precisely the strains which showed greater diversity of adhesins, greater expression of cellulose and biofilm formation, in other words, that possess greatest capacity of colonization.

Recent works in the field of microbial ecology that take advantage of non-cultivating methods are elucidating the gut colonization process. Here, we have found that DAEC strains possessing Afa/Dr genes may reflect some principles that apply to the microbiota in general. First, as microbiota composition is different in children and adults, we found that DAEC from children and from adults represent two different populations, with distinct profiles regarding the characteristics studied in this work. Second, as microbiota seems to be more diversified in control subjects than in diarrhea patients 72 , DAEC strains isolated from asymptomatic controls present greater diversity of genes related to virulence. Quiroga et al. 73 demonstrated that strains of E. coli belonging to four different diarrheagenic categories – including DAEC and EPEC – can be found colonizing infants in the first months of life. Here, we refined the analysis of DAEC strains and found that potentially diarrheagenic strains can be found as part of gut microbiota in children. We also demonstrated that many DAEC strains possessing Afa/Dr genes belong to serogroups associated with EPEC, reflecting perhaps an evolutionary relationship.

DAEC strains as etiological agents of diarrhea are still a matter of controversy. We found that DAEC strains possessing Afa/Dr genes from children and adults possibly possess distinct virulent mechanisms. DAEC strains from children apparently have greater ability of colonizing the gastrointestinal tract, which may contribute to the effective action of a toxin, such as SAT. We also demonstrated for the first time, to the authors’ knowledge, that curli can play a role in pathogenesis of DAEC strains isolated from adults. Further studies are warranted to conclusively demonstrate this involvement.

DAEC strains possessing Afa/Dr genes isolated from children and adults have shown very distinct profiles regarding the distribution of the characteristics analyzed in this work. Strains from children are more diverse than strains from adults in relation to the studied characteristics. Most characteristics were more frequent in strains from asymptomatic children. In contrast, virulence factors were less frequent in strains from adults, which seem to form a more homogeneous group. Characteristics potentially associated to virulence are distinct in DAEC strains from adults and children. The results confirm the importance of SAT in diarrhea caused by DAEC in children and suggest that its action may be enhanced as a result of their efficiency in colonization. Moreover, curli is a potential virulence factor for DAEC strains that cause diarrhea in adults. Together, these results indicate that DAEC strains possessing Afa/Dr genes isolated from children and adults represent two different bacterial populations. Furthermore, the results also indicate that DAEC strains with virulence markers can be found as part of the normal microbiota present in healthy children.

The authors declare that they have no competing interests.

RMA conceived the study and designed the experiments. RMA, ALP and LGG analyzed the data, wrote the manuscript and were responsible for concepts, vision and direction for the study. All authors read and approved the final manuscript.