Diarrheal disease remains a relevant cause of morbidity and mortality among children, especially in nations marked by socioeconomic inequality, poor public health and inadequate sanitation service coverage  . Epidemiological studies have pointed to the persistence of large health disparities between Indigenous and non-Indigenous peoples, even in countries, which have developed their own culturally health indicator frameworks    . Despite differences in scale, Indigenous communities are also accompanied by poorer infrastructure, health care access and higher social inequity  . According to the First National Survey of Indigenous People’s Health and Nutrition in Brazil, the overall prevalence of diarrhea was 23.5% among children under five . Higher risk of diarrhea was observed among younger children and those who had less maternal care, lower household socioeconomic status, malnutrition, and occurrence of upper respiratory infection   .
Several studies have shown the relevance of Escherichia coli in the etiology of diarrheal disease worldwide. Intestinal E. coli pathotypes (or diarrheagenic E. coli, DEC) cause significant morbidity and mortality worldwide in children under 5 years of age, especially in the developing world   . DEC comprises important agents of endemic and epidemic diarrhea worldwide, associated with asymptomatic carriage or clinically symptomatic disease related to acute and persistent diarrhea    .
Diarrheagenic E. coli belong to at least six E. coli pathotypes that present a characteristic set of virulence factors responsible for different pathomechanism of infections. These pathotypes include: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), shiga toxin-producing E. coli (STEC), enteropathogenic E. coli (EPEC), and diffusely adherent E. coli (DAEC) pathotype. Besides the typical forms, which characterize each pathogenic group, antigenic and genetic variants can be found characterizing atypical lineages  .
Phylogroup characterization of E. coli also provides insight into virulence potential and is also useful for identifying human health risks . Commensal isolates mostly group into phylogroups A and B1, extraintestinal E. coli strains are derived from group B2 and to a lesser extent from D, whereas E. coli strains carrying diarrheagenic markers may be distributed across all phylogroups. Phylogroups differ according to metabolic properties, ecological niches, life-history characteristics and propensity to cause diseases .
Assessment of virulence and resistance profiles in clinical and environmental E. coli has revealed a high prevalence of antimicrobial resistance and the occurrence of the multidrug-resistant and possible extensively drug-resistant bacteria within phylogroups or among strains carrying diarrheagenic markers   . However, such properties among E. coli isolates from the Indigenous population is not documented and requires special attention given the vulnerability and risk of exposure of the population being studied  .
Diarrheagenic E. coli (DEC) is a group of microorganisms well known for its pathogenic role and associated severity. The current study describes the genotypic and phenotypic characteristics of E. coli isolates from the Indigenous population under five, belonging to the Guarani ethnic group in Brazil.
2.1. Indigenous Population
The indigenous population included in this study lives in communities in the State of Rio de Janeiro, and belongs to the Guarani ethnic group. It is distributed in five villages and two municipalities, Angra dos Reis (Sapukai village) and Paraty (Paraty-Mirim, Araponga, Rio Pequeno and Mamanguá villages) (Figure 1). The villages have an irregular water supply, besides precarious sewage treatment and waste collection. The Guarani are a semi-nomadic ethnic group, and the intense migration between villages belonging to the same ethnic group is a cultural characteristic that increases their vulnerability to infectious and parasitic diseases . The migration occurs between several Brazilian states, especially those located in the south and southeast regions, and South American countries (Argentina, Paraguay and Uruguay), which enables them to maintain relationships with relatives living in other villages. Organized health services are offered on a permanent basis, but medical care also occurs by spontaneous demand. Villages from the municipality of Paraty have a population of around 180 people, divided into 32 residences. The Sapukai village (Angra dos Reis municipality) is made up of 67 residences, housing around 360 indigenous people.
2.2. Bacterial Samples and Participants
A total of 314 E. coli isolates were included in the present study. Clinical samples were recovered during an active search aiming to outline the epidemiological profile of infectious diseases in the Guarani population living in the Araponga, Mamanguá, Rio Pequeno, Sapukai and Paraty-Mirim villages, in Rio de Janeiro state . E. coli isolates were obtained from 57 children younger than 5 years old, with (n = 10) and without (n = 47) diarrhea. Children who manifested an increased number of evacuations, with watery feces or with little consistency, showing the presence or not of mucous and/or blood, following observation and reporting by the person responsible at the time of collection of the material were considered to be a case. Diarrhea was considered to be a persistent diarrheic condition if it had a duration of 14 days or more. Children who did not manifest a diarrheic condition within the period that preceded the collection of material by at least 30 days were considered part of the control group. Stool samples were collected and placed in Clair-Blair transport medium and transported in iced boxes within 4 h to the laboratory. Samples were incubated in 3 ml of Tryptic Soy Broth (TSB, Difco) for 18 to 24 h at 37˚C. Following the incubation period, an aliquot of the bacterial growth was streaked onto Eosin Methylene Blue agar (EMB, Difco) and incubated at 37˚C for 18 to 24 h. Characteristics of the colonies were recorded. Up to ten 10 lactose positive and two lactose negative bacterial colonies were selected based on morphological and physiological characteristics suggestive of species. Putative E. coli colonies were tested for species-level
Figure 1. Location map of Araponga, Rio Pequeno, Mamanguá, Sapukai and Paraty-Mirim indigenous villages in the municipalities of Angra dos Reis and Paraty.
identification by traditional biochemical assays. Isolates identified as presumptive E. coli were assayed for the detection of the E. coli-specific uidA gene. E. coli isolates were kept frozen and screened for adherence phenotype, antimicrobial resistance, phylogrouping, genetic diversity and diarrheagenic potential. This study was approved by the Escola Nacional de Saúde Pública Sergio Arouca Ethics Committee on Human Research (CEP/ENSP), (reference number 0056.0.031.000-09), Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.
2.3. Antimicrobial Susceptibility
Antimicrobial resistance was assessed using a standard disk diffusion method according to the guidelines published by the Clinical and Laboratory Standards Institute (CLSI) . Bacterial suspension was adjusted to a 0.5 McFarland standard and tested against the following antimicrobials: amikacin (AMI, 30 µg), ampicillin (AMP,10 µg), cephalotin (CFL, 30 µg), cefepime (CPM, 30 µg), ceftriaxone (CRO, 30 µg ), cefoxitin (CFO, 30 µg), ciprofloxacin (CIP, 5µg), gentamicin (GEN, 10 µg), nalidixic acid (NAL, 30 µg), nitrofurantoin (NIT, 300 µg), norfloxacin (NOR, 10 µg), trimethoprim-sulfamethoxazole (STU, 25 µg). Reference E. coli strains ATCC 25922 and ATCC 35218 were used as controls. The isolates were scored as susceptible, intermediate or resistant to a given antimicrobial according to the inhibition zone diameter around the disk. Intermediate and resistant E. coli isolates were subsequently grouped in the same resistant class and categorized as multidrug-resistant (MDR), extensively drug-resistant (XDR) or pandrug-resistant (PDR) .
2.4. Cell Adherence
E. coli isolates carrying diarrheagenic genetic markers were tested for adherence to epithelial cells as described . Adhesion assays were performed with HEp-2 cells cultivated in 24-well tissue culture plates (50% to 70% confluence) containing Dulbecco’s Modified Eagle Medium (DMEM, Gibco), supplemented with 50 µg/mL gentamicin, 2.5 µg/mL fungizone and 5% fetal calf serum (FCS) (Gibco). After two washes with phosphate buffered saline (PBS), 1.0 mL of fresh DMEM (supplemented with 1% D-mannose) was added to each well. E. coli strains were grown overnight in Luria Bertani (LB) broth under static conditions and an aliquot of the overnight culture (20 μL,) was added to the media contained on the microplates for 3 h/6h at 37˚C in 5% CO2. The infected monolayers were washed with sterile phosphate-buffered saline, fixed with 70% methanol, stained with 10% Giemsa stain, and examined for adherence patterns under light microscopy. Weakly adherent or non-adherent isolates were retested using the same procedure with an additional incubation period of 3 h (6 h assay). ATCC EAEC 042 (AA+), EPEC E2348/69 (LA+) and H1/1 (DA+) and non-adherent E. coli K12 (DH5-α) were used as controls.
2.5. Hybridization Assays for Diarrheagenic E. coli
All E. coli isolates were screened by colony DNA hybridization assays using specific radiolabelled DNA probes to identify the following DEC virulence sequences: st, lt, eae, bfpA, stx1, stx2, att and ipaC . E. coli isolates were classified as typical EPEC if they lacked stx DNA probe sequences, and carried the eae and bfpA genes, and as atypical EPEC if they carried eae only. E. coli isolates that carried st and/or lt genes were classified as ETEC, att gene as EAEC, ipaC gene as EIEC and stx1 and/or stx2 genes as STEC. E. coli HB101 (pBR 322) and E. coli K12 carrying recombinant plasmids were used as controls.
2.6. PCR-Multiplex for Diarrheagenic E. coli
The E. coli isolate was assigned to a particular pathotype as described . All strains were screened for the following target genes: escV, bfpB, stx1, stx2, elt, estIa, estIb, invE, astA, aggR, pic and uidA (E. coli marker). E. coli isolates were classified as tEPEC (escV positive, bfp positive, stx negative), aEPEC (escV positive, bfp negative, stx negative), STEC (escV positive/negative, bfp negative, stx positive), ETEC (elt positive and/or estIa positive, estIb positive), EIEC (invE positive) or EAEC (astA positive and/or aggR positive and/or pic positive). PCR-amplified fragments were separated on 2.0% (wt/vol) agarose gels, visualized under UV light and photographed using a digital image capture system (Silver UVIPro, Cambridge, UK). To estimate the size of the fragments, a 100 bp DNA ladder standard (Invitrogen) was used. Each PCR analysis included a negative DNA control for enterovirulence genes and a nontemplate control. Clinical E. coli isolates were used as positive controls for diarrheagenic genetic markers: 243IV (tEPEC, escV+ bfpB+), 36IV (ETEC, lt+), 245I (ETEC, st+), E30138 (STEC, stx2+), E40705 (STEC, stx1+), 103V (EAEC, aggR+) and 129III (EIEC, inv+). Isolates lacking one or more of the genes defining a given set were considered nonenteropathogenic.
2.7. Random Amplification of Polymorphic DNA
RAPD-PCR was performed as previously described with the primers A04 (AATCGGGCTG), 1254 (CCGCAGCCAA), and M13 (GAGGGTGGCGCTTCT) . RAPD profiles were inspected visually and the genetic patterns were defined according to the presence or absence and intensity of polymorphic bands. A 1Kb DNA ladder was used as a molecular weight marker (Invitrogen, Rio de Janeiro, Brazil). Semiautomated analysis used the UVI Soft Image Acquisition and Analysis Software, program UVIPro Bandmap, version 11.9 (Uvitec, Cambridge). Cluster analysis was performed using the Unweighted Pair Group Method with Arithmetic averages (UPGMA) of the Image Analysis System. The percentages of similarity were estimated by Dice coefficient. The reproducibility of RAPD amplifications was assessed using the selected primers with different DNA samples isolated independently from the same strain and amplified at different times.
2.8. PCR-Triplex for E. coli Phylotyping
E. coli A, B1, B2 and D phylogroups were determined according to previously defined criteria in a triplex amplification of the chuA and yjaA genes and the DNA fragment (TSPE4.C2) . The characterization into phylotypes was based on the different combinations of these phylogroup genetic markers. Amplification products were visually inspected under UV light and photographed using a digital image capture system (silver UVIPro, Cambridge, UK). To estimate the size of the fragments, a 100 bp DNA ladder standard (Invitrogen) was used. Molecular assays included a nontemplate reaction and the clinical UPEC strain (L75A, chuA+ yjaA+ TSPE4.C2+) as the positive control for the phylogenetic markers.
Enterovirulence assays revealed that 136 (43%) of the 314 E. coli isolates were carriers of, at least one of the genetic sequences investigated, with the astA gene being the most frequent (26%, 83/314) followed by escV (8%, 24/314), aggR (5%, 17/314), pic (2%, 5/314), estIb and elt (1%, 3/314). All the isolates were carriers of the uidA gene. The following genetic profiles were detected: escV, aggR/astA, aggR/pic, estIb/elt/astA, astA, pic, aggR/pic. 15% (48/314) of the isolates were classified in the diarrheagenic categories: aEPEC (56%, 27/48), EAEC (35%, 17/48) and ETEC (8%, 4/48).
Distribution by indigenous village showed that 83% of the E. coli isolates from the Araponga village (n = 6) were carriers of the escV gene (aEPEC pathotype). Among the isolates from the Rio Pequeno village (n = 18), the genetic markers for enteropathogenicity were detected in 44% (8/18) of isolates, with the aggR gene found in 50% (4/8) (EAEC pathotype) and the astA gene in 25% (2/8). Among the isolates from the Mamanguá village (n = 22), genes for enterovirulence were detected in 45% (10/22), with only 30% (3/10) being carriers for the escV gene (aEPEC pathotype) and 50% (5/10) of isolates were carriers of the astA gene. Among the E. coli isolates from Sapukai (n = 135), the genetic markers for enterovirulence were detected in 44% of isolates (n = 56): astA (33/56), escV (aEPEC, 14/56), aggR (EAEC, 2/56), and pic (2/56). Among the isolates obtained from the Paraty Mirim village (n = 133), the genes for enterovirulence were detected in 43% of the samples (n = 57): astA (38/57), aggR (EAEC, 6/57), estlb/elt/astA (ETEC, 3/57), aggR/pic (EAEC, 3/57), aggR/astA (EAEC, 2/57), escV (aEPEC, 2/57). The hybridization assays detected the eae gene (aEPEC, 16/314) among the isolates from the Araponga and Sapukai villages, the att gene (EAEC, 13/314) in the Sapukai, Paraty Mirim and Rio Pequeno villages and the lt gene (ETEC, 1/314) in the isolates from the Sapukai village. From all the E. coli isolates identified as potential DEC strains (n = 48), 47 were identified by the amplification assays, and 30 by the DNA hybridization assays. Of these isolates, 29 showed results, which agreed with both the molecular tests. Results of phenotypic and genotypic characteristics of E. coli isolates carrying DEC markers are shown in Table 1. aEPEC (n = 27) was isolated from 10 children without diarrhea of up to six months of age, EAEC (n = 12) from 7 children, with and without diarrhea, with 6 to 60 months of age, and ETEC (n = 4) from two children with 36 months without diarrhea.
Table 1. Distribution of E. coli isolates carrying diarrheagenic genetic and phenotypic markers according to the indigenous villages.
DEC Diarrheagenic E. coli; ETEC enterotoxigenic E. coli; EAEC enteroaggregative E. coli; aEPEC atypical enteropathogenic E. coli; MDR multidrug-resistant; XDR extensively drug-resistant; PDR pandrug-resistant; RAPD random amplification of polymorphic DNA; AA aggregative adherence; U undefined adherence pattern.
Most of the E. coli isolates were identified as phylogroup A (56%), followed by B1 (26%), B2 and D (9%, each). In the phylogroup A the following genetic variants were detected: A1 −,−,− (40%, 19/48) and A2 −,+,− (16%, 8/48), in the phylogroup B2 the genotypes B21 +,+,− (2%, 1/48) and B22 +,+,+ (7%, 3/48) and in the phylogroups B1 and D the phylotypes −,+,+ 26% (13/48) and +,-,- 9% (4/48), respectively. ETEC were classified in the phylogroup A, EAEC in A, B1, B2 and D and the pathotype aEPEC in the phylogroups A, B1, and B2. Considering the distributions of the phylotypes according to indigenous village, the following occurrences were detected: Araponga village with 60% (3/5) of the DEC belonging to phylotype A1 and 40% (2/5) to group B1; the Rio Pequeno village, 50% (2/4) belonged to the phylotype A1 and 50% (2/4) to B1; the Mamanguá village, phylotypes A1 (33%, 1/3) and A2 (67%, 2/3); Sapukai village, the phylotypes A1 (60%, 12/20), B1 (30% (3/20), A2 (5%, 1/20) and B21 (5%, 1/20) and in the Paraty Mirim village, phylotypes A1 (6%, 1/16), A2 (31%, 5/16), B1 (19%, 3/16), B22 (19%, 3/16) and D (25%, 4/16).
Adherence tests revealed that 100% (17/17) of the EAEC exhibited the typical aggregative pattern (AA) in the 3 and 6 hour assays. ETEC exhibited the AA phenotype (AA) in the 3 hour assay (75%, 3/4) and one isolate (25%, 1/4) was adherent, however, without exhibiting a defined pattern. aEPEC exhibited the AA pattern (11%, 3/27) in 3-hour assays and the other isolates showed a discrete and moderate adhesion. E. coli isolates which were only carriers of the pic gene showed the typical aggregative pattern, while the isolates, which were only carriers of the ast gene exhibited the phenotypes of localized, aggregative and undefined adherence patterns in 6-hour assays.
Antimicrobial susceptibility tests revealed that 88% (276/314) of the E. coli isolates were resistant to, at least one of the 11 antimicrobials tested characterizing 72 distinct profiles (I to LXXII). Eight single resistance profiles (I to VIII) were observed for AMI, AMP, CFL, COM, GEN, NIT, NOR and SUT. The percentage of resistance for each antimicrobial was 69% (216/314) AMP, 63% (197/314) CFL, 54% (171/314) SUT, 18% (58/314) GEN, 12% (39/314) AMI, 9% (28/314) CRO, 6% (19/314) NAL, 6% (20/314) CFO, 6% (19/314) NIT, 4% (11/314) CIP, 3% (14/314) NOR and 2% (5/314) CPM. The intermediary resistance phenotypes were found in 65% of the isolates (203/314). The multiresistance classified 26% of the isolates as MDR (80/314) and 11% as possible XDR/PDR (36/314). The distribution of resistant isolates by indigenous village showed that 91% (20/22) from the Mamanguá village, 88% (121/135) from Sapukai, 87% (116/133) from Paraty Mirim and 83% in the Rio Pequeno (15/18) and Araponga (5/6) villages were resistant. In the group of E. coli isolates that carried diarrheagenic genetic markers (n = 48), 24 profiles with resistance to up to 9 antimicrobials were detected, with the profile XXIV shared by the ETEC, aEPEC and EAEC pathotypes and, types III, XII and XXXVIII by the EAEC and aEPEC pathotypes. The MDR phenotype was observed in 23 isolates from the Paraty Mirim, Sapukai, and Araponga villages, belonging to the EAEC, aEPEC and ETEC pathotypes. Two EAEC isolates obtained from diarrheal children exhibited the MDR phenotype. Possible XDR/PDR isolates were observed among the EAEC and aEPEC isolated from Paraty Mirim, Rio Pequeno, Sapukai and Araponga villages.
RAPD typing based on 1254, A04 and M13 primers revealed a genetically diverse bacterial population. The amplification reactions generated polymorphic, stable, and reproducible profiles, composed of 2 to 6 bands (primer 1254), 2 to 8 bands (primer A04) and 5 to 10 bands (primer M13), ranging from 500 - 2000 bp, 500 - 3000 bp, 500 - 2000 bp, respectively. RAPD clustering showed a bacterial population arranged into separate branches or organized in small clonal groups, exhibiting Dice similarity indices ranging from 10% to 100% (1254), 2% to 100% (A04) and 5% to 100% (M13). The genetic relationship of the E. coli isolates belonging to the same pathotype was in agreement in terms of the primers used and higher indices of similarity were observed among the isolates from the same child and indigenous village.
Studies carried out around the world report that the pathogenic groups of Escherichia coli associated with human diseases and in animals are markedly diversified, being able to cause intestinal or extra-intestinal infections according to their repertoire of virulence traits   . E. coli as an etiological agent of intestinal infections can be associated with manifestations with broad clinical spectrum, ranging from asymptomatic and subclinical forms to clinical conditions of great severity potentially leading to death. Epidemiological surveys of indigenous populations conducted in countries on the American continent, have shown that diarrhea contributes significantly to rates of morbi-mortality and hospitalization of indigenous populations     . In Brazil, these populations generally live in villages with inadequate sewage treatment and in conditions of poverty  . Studies on the etiology of diarrhea in indigenous populations are very limited and there are no reports regarding the occurrence of diarrheic E. coli in indigenous children of the Guarani ethnicity. Our findings revealed the circulation of the diarrheagenic pathotypes among indigenous children younger than 5 years of age, with and without diarrhea.
aEPEC was the most prevalent pathotype recovered from children without diarrhea residing in Sapukai, Mamaguá, Araponga and Paraty Mirim villages. aEPEC has not yet been described in indigenous populations, however this pathotype is gaining greater significance in non-indigenous populations associated with symptomatic and asymptomatic intestinal disorders   . Recent studies have shown that infections by aEPEC seem to surpass those caused by tEPEC, both in developed and developing countries, mainly associated with human persistent diarrhea    . In Brazil, aEPEC has been implicated as the cause of diarrhea in different urban areas   . aEPEC has also been isolated from animal sources, including dogs, rabbits, monkeys and sheep, which has led to suppose the role of these animals as environmental reservoirs of these enteropathogens  . The precarious sanitation infrastructure where Guarani communities reside and the close coexistence with domestic and forest animals, are unfavorable environmental conditions which may have contributed to the DEC spread and contamination of the indigenous children    . As well as via direct contact, transmission of the microorganism can also take place in an indirect manner through ingesting contaminated water and food, representing an additional risk of exposure for the health of the indigenous community. Our data from the adherence assays (3 and 6 hours) showed that aEPEC isolates were adherent but did not express a defined pattern and 11% (3/27) exhibited the aggregative phenotype (AA). These findings are in agreement with previous observations, which underline that adherence is also a variable characteristic of the pathotype, even among epidemiologically related bacterial isolates  . Phylogrouping categorized the pathotype mainly in groups A and B1. In general, A and B1 strains appear to be found within guts of a range of vertebrates and also are more prevalent in freshwater samples than other strains . So, we assume that the aEPEC isolates are microorganisms possibly derived from non-human sources or other environmental compartments    . These findings reinforce the role of the environment as a reservoir and source of contamination of these pathogens. aEPEC isolates were also categorized in B1, B2, and D phylogroups, which shows the concept of the diverse structure of the category and its different potential risks to human health as well as their high adaptability to environments or diverse niches . Results from the antimicrobial resistance showed that 81% of aEPEC isolates (22/27) were resistant to at least one of the 11 antibiotics tested and that the MDR and possible XDR/PDR phenotypes were observed for 37% and 11% of aEPEC isolates, respectively. Our findings are in accordance with previous studies carried out in Brazil and in other geographic regions, that report the emergence of resistance among aEPEC clinical isolates from non-indigenous children    . This phenomenon alerts us to the inappropriate use of antimicrobials with humans and animals, which ends up favoring the selection of resistant bacteria in the environment. The impact of the spread of resistance at high levels can assume even greater proportions, especially if we consider that their genetic determinants can move quickly between different bacterial species and genera    .
EAEC was the second most frequent pathotype isolated from children both with and without diarrhea, resident in Sapukai, Paraty Mirim and Rio Pequeno villages. The diarrheic children associated with EAEC isolates manifested acute diarrhea, liquid feces with an absence of fever, and vomiting or abdominal pain. Epidemiological studies have associated EAEC infection with acute cases but especially with persistent diarrhea, particularly in developing countries      . The pathogenic properties of EAEC require more detailed study, especially seeking to investigate its clinical significance through long term studies, given that the pathotype is also isolated from asymptomatic adult and child carriers  . EAEC is a pathotype of recognized variability, exhibiting elevated genetic and antigenic diversity. Though a great diversity of adhesins, toxins and proteins are possibly involved in the pathogenesis of EAEC, the prevalence of these associated factors or genes is highly variable and is not found in all the isolates, which has hampered diagnostic accuracy for this pathotype    . The virulence properties of the pathotype that are associated with symptoms of diarrhea include the production of biofilm and of diverse enterotoxins, such as thermo-stable enteroaggregative toxin (EAST-1), the Pet cytotoxin and the anti-aggregative protein known as dispersin      . EAST-1 is codified by the astA gene, located in a transposon, and has high homology with the amino-acid sequence of the thermo-stable entertoxin (ST) of ETEC. In our study, the astA gene was the most frequent genetic marker in EAEC isolates, obtained from acute diarrhea, suggesting the possible role of EAST-1 in the manifestation of the indigenous clinical status. As well as toxins, EAEC strains may exhibit diverse adhesins such as the Pic protein, associated or not, with other virulence factors of the pathotype . In our study, the pic gene was only detected among EAEC obtained from asymptomatic children. In these E. coli isolates, no other EAEC virulence associated markers were observed suggesting that colonization can require additional factors for the efficiency of the infection. Despite the genotypic differences detected in EAEC, all the isolates originating both from asymptomatic and diarrheal indigenous children were classified as typical (tEAEC) due to presence of the aggR gene, which codes for the transcriptional activator AggR   . Typical EAEC has closely associated with the expression of the typical aggregative pattern, corroborating with the adherence phenotype observed during the 3 and 6-hour assays  . Variations in this adherence pattern have been observed and seem to involve adhesins of a diverse nature. Molecular methods for diagnosis purposes based on hybridization tests and amplification assays are being widely used as alternative tools to the gold-standard test with epithelial cells in culture  . In the present study, molecular assays were used as the EAEC diagnostic method, and subsequently, the isolates were tested on HEp-2 cells for the observation of the expression of the adhesion phenotypes. Our results showed the importance of molecular diagnosis for tEAEC, given that all the EAEC isolates exhibited the typical aggregative adherence (AA) pattern. However, considering that not all the EAEC strains were found to harbor the pAA plasmid, the diagnosis of the pathotype in the present study could have been underestimated in the indigenous population. Results from phylogrouping and genome typing agree with previous reports that describe the elevated genetic diversity of this pathotype observed both in clinical and environmental isolates  . EAEC typing revealed that EAEC isolates belong to the main four phylogroups, suggesting distinct pathogenic potentials for these isolates    . The absence of a correlation with phylogenetic markers or specific virulence patterns with a diarrhea condition is also observed in other studies with non-indigenous populations   . These results underline the need to investigate pathogenic specific characteristics, especially when considering the potential aggressiveness of the diverse virulence factors already described and the existence of cases of significant severity associated with the pathotype. 94% of EAEC isolates (16/17) were resistant to at least one of the 11 antimicrobials tested and the multidrug-resistance was also a phenomenon observed for EAEC, characterizing the MDR and possible XDR/PDR phenotypes for 65% and 12% of the isolates, respectively. Resistance to antimicrobials is also reported worldwide in clinical EAEC strains among the isolates from hospitals and communities      . However the occurrence of MDR and possible XDR/PDR phenotypes among EAEC is not reported and reinforces the need for environmental monitoring aiming to minimize the exposure of human populations and the limitation of treatment options.
ETEC is one of the classic E. coli pathotypes mainly in non-indigenous children younger than five years of age and in travelers coming from endemic regions  . Studies with indigenous populations report that ETEC is a diarrheagenic pathotype common in children who live in rural communities in Guatemala, associated both with acute and persistent diarrhea as well as among control children . In our study, ETEC presented low prevalence and was only isolated from children without diarrhea, suggesting that this pathotype does not represent a relevant etiological agent for diarrhea in the indigenous Guarani population. The virulence genotype detected, codifying for both classes of enterotoxin, has been detected in ETEC isolates from non-indigenous children without diarrhea, which is in accordance with our findings   . ETEC clinical isolates frequently exhibit diverse adhesins capable of mediating the bacterial adherence with the surface of intestinal cells, contributing in this way to the full expression of the enteroinfection  . Given that in our study, these adhesins were not investigated, it was not possible to define a more complete ETEC virulence profile, and it was consequently difficult to establish an association with the clinical status. In addition to enterotoxins, the astA gene is recognized as an accessory gene for pathogenic E. coli populations, including diarrheagenic pathotypes such as ETEC and extra-intestinal lineages     . However, in our study the detection of this gene among bacterial isolates obtained from asymptomatic individuals may be explained as a consequence of a continuous process of genetic exchange, between pathogenic and non-pathogenic E. coli populations and/or other bacterial genes which share the same ecological niche   . Phylogrouping assays classified the ETEC isolates as belonging to group A. These results are in accordance with previous findings, which revealed that phylogroup A is associated with clinical isolates of ETEC obtained from asymptomatic individuals, commensals of environmental origin or even of low virulence potential     . Our results from adherence assays revealed that the ETEC isolates were adherent, exhibiting the aggregative phenotype, typically observed in EAEC, and undefined patterns. ETEC isolates of environmental and asymptomatic origin commonly exhibit elevated antigenic heterogeneity, influencing in this manner, the expression of its specific virulence factors . Recent studies that correlate the dynamics of microbial populations and evolution of bacterial resistance have revealed that increased resistance is associated in most cases, either directly or indirectly, with decreased virulence  . In our study, the MDR phenotypes and multidrug-resistance to up to four antimicrobials were observed among ETEC isolates originated from asymptomatic children, which reinforces this observation. RAPD typing confirms the high variability among ETEC isolates thus characterizing this bacterial population as being epidemiologically unrelated  .
Studies on the molecular epidemiology of diarrheagenic E. coli in indigenous populations is very limited worldwide and non-existent in Brazil. Our results revealed that the aEPEC pathotype was predominant in the indigenous Guarani children, followed by EAEC and ETEC. This data draws our attention considering the association of these pathotypes with malnutrition and infant development. The precarious sanitation conditions and the life style of the population studied, which routinely maintains a close relationship with diverse environmental reservoirs, create conditions of greater vulnerability for the establishment of antimicrobial resistance and bacterial virulence, thus facilitating the rapid dissemination of these properties.
This is the first report regarding the circulation and characterization of diarrheagenic E. coli among indigenous Guarani children residing in villages located in the south of the state of Rio de Janeiro. The variability of genotypes and resistance to a wide range of antibiotics reflects the inherent genomic plasticity of E. coli, which involves a dynamic genetic exchange between commensal and pathogenic microorganisms. This epidemiological context could lead to serious problems in public health through the emergence of highly virulent new lineages and to the spread of resistance in the environment. These findings alert us to the need for strategic actions in the prevention and health surveillance of this indigenous population, especially considering that the child population is exposed to adverse environmental conditions and inadequate coverage of the health services, which makes it even more vulnerable to infections caused by these microorganisms and serious complications.
The authors would like to thank Rose Mary Pimentel Bezerra for technical assistance in the bacteriological procedures.
 Keusch, G.T., Walker, C.F., Das, J.K., Horton, S. and Habte, D. (2016) Diarrheal Diseases. In: Disease Control Priorities, Chapter 9, 3rd Edition, 163-185.
 Global Burden of Disease Pediatrics Collaboration (2016) Global and National Burden of Diseases and Injuries among Children and Adolescents between 1990 and 2013. Findings from the Global Burden of Disease 2013 Study. JAMA Pediatrics, 170, 267-287.
 Halpenny, C.M., Koski, K.G., Valdés, V.E. and Scott, M.E. (2012) Prediction of Child Health by Household Density and Asset-Based Indices in Impoverished Indigenous Villages in Rural Panama. The American Journal of Tropical Medicine and Hygiene, 86, 280-291.
 Escobar, A.L., Coimbra Jr., C.E.A., Welch, J.R., Horta, B.L., Santos, R.V., James, R. and Welch, J.R. (2015) Diarrhea and Health Inequity among Indigenous Children in Brazil: Results from the First National Survey of Indigenous People’s Health and Nutrition. BMC Public Health, 15, 1-11.
 Torres, O.R., Gonzalez, W., Lemus, O., Pratdesaba, R.A., Matute, J.A. and Wiklund, G. (2015) Toxins and Virulence Factors of Enterotoxigenic Escherichia coli Associated with Strains Isolated from Indigenous Children and International Visitors to a Rural Community in Guatemala. Epidemiology and Infection, 143, 1662-271.
 Echagüe, G., Sosa, L., Díaz, V., Ruiz, I., Rivas, L., Granado, D., Funes, P., Zenteno, J., Pistilli, N. and Ramírez, M. (2015) Enteroparasitosis en niños bajo 5 años de edad, indígenas y no indígenas, de comunidades rurales del Paraguay. Revista Chilena de Infectología, 32, 649-657.
 Souza, L.G., Santos, R.V. and Coimbra Jr., C.E.A. (2010) Age Structure, Natality and Mortality of the Xavante Indigenous People of Mato Grosso State, Brazilian Amazon. Ciência & Saúde Coletiva, 15, 1465-1473.
 Coimbra Jr., C.E.A. (2014) Health and Indigenous Peoples in Brazil: Reflections Based on the First National Survey of Indigenous People’s Health and Nutrition. Cadernos de Saúde Pública, 30, 855-859.
 Zheng, S., Yu, F., Chen, X., Cui, D., Cheng, Y. and Xie, G. (2016) Enteropathogens in Children Less than 5 Years of Age with Acute Diarrhea: A 5-Year Surveillance Study in the Southeast Coast of China. BMC Infectious Diseases, 16, 434.
 Onanuga, A., Igbeneghu, O. and Lamikanra, A. (2014) A Study of the Prevalence of Diarrhoeagenic Escherichia coli in Children from Gwagwalada, Federal Capital Territory, Nigeria. The Pan African Medical Journal, 17, 146.
 Gomes, T.A.T., Elias, W.P., Scaletsky, I.C.A., Guth, B.E.C., Rodrigues, J.F., Piazza, R.M.F., Ferreira, L.C.S. and Martinez, M.B. (2016) Diarrheagenic Escherichia coli. Brazilian Journal of Microbiology, 47, 3-30.
 Croxen, M.A., Law, R.J., Scholz, R., Keeney, K.M., Wlodarska, M. and Finlay, B.B. (2013) Recent Advances in Understanding Enteric Pathogenic Escherichia coli. Clinical Microbiology Reviews, 26, 822-880.
 Mosquito, S., Pons, M.J., Riveros, M., Ruiz, J. and Ochoa, T.J. (2015) Diarrheagenic Escherichia coli Phylogroups Are Associated with Antibiotic Resistance and Duration of Diarrheal Episode. The Scientific World Journal, 2015, Article ID: 610403.
 Rebello, R.C.L. and Regua-Mangia, A.H. (2014) Potential Enterovirulence and Antimicrobial Resistance in Escherichia coli Isolates from Aquatic Environments in Rio de Janeiro, Brazil. Science of the Total Environment, 490, 19-27.
 Randrianirina, F., Ratsima, E.H., Ramparany, L., Randremanana, R., Rakotonirina, H.C. and Andriamanantena, T. (2014) Antimicrobial Resistance of Bacterial Enteropathogens Isolated from Stools in Madagascar. BMC Infectious Diseases, 14, 1-7.
 Nery, J.A.C., Pimentel, M.I.F., Lyra, M.R., Lyra, M.R., Sohsten Bde, L., Marinho, D.P. and Périssé, A.R. (2012) Detection of Clusters of Leprosy Cases among Guarani Indians in the Southern Region of the State of Rio de Janeiro, Brazil. Revista da Sociedade Brasileira de Medicina Tropical, 45, 538-539.
 Magiorakos, A.-P., Srinivasan, A., Carey, R.B., Carmeli, Y., Falagas, M.E. and Giske, C.G. (2012) Multidrug-Resistant, Extensively Drug-Resistant and Pandrug-Resistant Bacteria: An International Expert Proposal for Interim Standard Definitions for Acquired Resistance. Clinical Microbiology and Infection, 18, 268-281.
 Rosa, A.C.P., Mariano, A.T., Pereira, M.A.S., Tibana, A., Gomes, T.A.T. and Andrade, J.R. (1998) Enteropathogenicity Markers in Escherichia coli Isolated from Infants with Acute Diarrhoea and Healthy Controls in Rio de Janeiro, Brazil. Journal of Medical Microbiology, 47, 781-790.
 Muller, D., Greune, L., Heusipp, G., Karch, H., Fruth, A., Tschäpe, H. and Schmidt, M.A. (2007) Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR. Applied and Environmental Microbiology, 73, 3380-3390.
 Rebello, R.C.L., Gomes, K.M., Duarte, R.S., Rachid, C.T.C.C., Rosado, A.S. and Regua-Mangia, A.H. (2013) Diversity of Mercury Resistant Escherichia coli Strains Isolated from Aquatic Systems in Rio de Janeiro, Brazil. International Journal of Biodiversity, 2013, Article ID: 265356.
 Clermont, O., Bonacorsi, S. and Bingen, E. (2000) Rapid and Simple Determination of the Escherichia coli Phylogenetic Group. Applied and Environmental Microbiology, 66, 4555-4558.
 Tobias, J., Kassem, E., Rubinstein, U., Bialik, A., Vutukuru, S.R. and Navaro, A. (2015) Involvement of Main Diarrheagenic Escherichia coli, with Emphasis on Enteroaggregative E. coli, in Severe Non-Epidemic Pediatric Diarrhea in a High-Income Country. BMC Infectious Diseases, 15, 1-7.
 Pitondo-Silva, A., Nakazato, G., Falcão, J.P., Irino, K., Martinez, R. and Darini, A.L. (2015) Phenotypic and Genetic Features of Enteropathogenic Escherichia coli Isolates from Diarrheal Children in the Ribeirão Preto Metropolitan Area, São Paulo State, Brazil. Apmis, 123, 128-135.
 Braga, R.M., Dourado, M.N. and Araújo, W.L. (2016) Microbial Interactions: Ecology in a Molecular Perspective. Brazilian Journal of Microbiology, 47, 86-98.
 Karesh, W.B., Dobson, A., Lloyd-Smith, J.O., Lubroth, J. and Dixon, M.A. (2012) Ecology of Zoonoses: Natural and Unnatural Histories. The Lancet, 380, 1936-1945.
 Schubert, S., Darlu, P., Clermont, O., Wieser, A., Magistro, G. and Hoffmann, C. (2009) Role of Intraspecies Recombination in the Spread of Pathogenicity Islands within the Escherichia coli Species. PLoS Pathogens, 5, 1-10.
 Ali, M.M.M., Ahmed, S.F., Klena, J.D., Mohamed, Z.K., Moussa, T.A.A. and Ghenghesh, K.S. (2014) Enteroaggregative Escherichia coli in Diarrheic Children in Egypt: Molecular Characterization and Antimicrobial Susceptibility. The Journal of Infection in Developing Countries, 8, 589-596.
 Seo, D.J., Choi, S., Jeon, S.B., Jeon, S., Park, H., Lee, B.-H., Kim, G.-B., Yang, S.-J., Nishikawa, Y. and Choi, C. (2017) Comparative Sequence Analysis of Enteroaggregative Escherichia coli Heat-Stable Enterotoxin 1 Identified in Korean and Japanese Escherichia coli Strains. International Journal of Food Microbiology, 243, 1-8.
 Harrington, S.M., Sheikh, J., Henderson, I.R., Ruiz-Perez, F., Cohen, P.S. and Nataro, J.P. (2009) The Pic Protease of Enteroaggregative Escherichia coli Promotes Intestinal Colonization and Growth in the Presence of Mucin. Infection and Immunity, 77, 2465-2473.
 Regua-Mangia, A.H., Gomes, T.A.T., Vieira, M.A.M., Irino, K. and Teixeira, L.M. (2009) Molecular Typing and Virulence of Enteroaggregative Escherichia coli Strains Isolated from Children with and without Diarrhoea in Rio de Janeiro City, Brazil. Journal of Medical Microbiology, 58, 414-422.
 Patzi-Vargas, S., Zaidi, M.B., Perez-Martinez, I., León-Cen, M., Michel-Ayala, A., Chaussabel, D. and Estrada-Garcia, T. (2015) Diarrheagenic Escherichia coli Carrying Supplementary Virulence Genes Are an Important Cause of Moderate to Severe Diarrhoeal Diseae in Mexico. PLOS Neglected Tropical Diseases, 9, 1-18.
 Hoseinzadeh, T., Ghanbarpour, R. and Rokhbakhsh-Zamin, F. (2016) Phylogenetic Background of Enterotoxigenic and Enteroinvasive Escherichia coli from Patients with Diarrhea in Sirjan, Iran. Iranian Journal of Microbiology, 8, 187-192.