Brazilian Indigenous Children as Carriers of Diarrheagenic <i>Escherichia coli</i> Pathotypes

Introduction: Diarrheagenic Escherichia coli (DEC) is a relevant cause of diarrhea, particularly among infants and young children in developing countries. Methodology: We compared the frequency, antimicrobial resistance, adherence, enterovirulence and genetic diversity of DEC isolates from Guarani indigenous population under five living in distinct villages in Brazil. Results: Of the 314 E. coli isolates from 57 children, with and without diarrhea, 15% (48/314) were classified in DEC categories: aEPEC (56%, 27/48), EAEC (35%, 17/48) and ETEC (8%, 4/48). ETEC belonged to plylogroup A, EAEC to groups A, B1, B2 and D, and aEPEC to phylogroups A, B1, and B2. EAEC exhibited the aggregative adherence phenotype while ETEC and aEPEC the aggregative and undefined patterns. Multidrug-resistance was detected in aEPEC, ETEC and EAEC while extensive drug-resistance was found in EAEC and aEPEC. RAPD typing revealed a genetically diverse bacterial population. Conclusion: This is the first report regarding aspects of DEC in an indigenous Brazilian population, showing that Guarani children are DEC carriers and that antimicrobial resistance at high levels is widely disseminated among these enteropathogens.

dies 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 [3] [4] [5] [6]. Despite differences in scale, Indigenous communities are also accompanied by poorer infrastructure, health care access and higher social inequity [3] [7]. 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 [8]. 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 [4] [7] [8].
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 [9] [10] [11]. DEC comprises important agents of endemic and epidemic diarrhea worldwide, associated with asymptomatic carriage or clinically symptomatic disease related to acute and persistent diarrhea [9] [11] [12] [13]. 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 [12] [13].
Phylogroup characterization of E. coli also provides insight into virulence potential and is also useful for identifying human health risks [13]. 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 [13].
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 [14] [15] [16]. 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 [5] [8].
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 pop-

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 [17].

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) [18]. Bacterial suspension was adjusted to a 0.

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 [21]. 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.

Random Amplification of Polymorphic DNA
RAPD-PCR was performed as previously described with the primers A04 (AATCGGGCTG), 1254 (CCGCAGCCAA), and M13 (GAGGGTGGCGCTTCT) [23]. RAPD profiles were inspected visually and the genetic patterns were defined according to the presence or absence and intensity of polymorphic bands.
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 as-tA gene. Among the E. coli isolates from Sapukai (n = 135), the genetic markers for enterovirulence were detected in 44% of isolates (n = 56): astA ( 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. 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.

Discussion
Studies carried out around the world report that the pathogenic groups of  [29]. 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 [12] [27]. 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 [24]. So, we assume that the aEPEC isolates are microorganisms possibly derived from non-human sources or other environmental compartments [9] [14] [15] [24]. 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 [29]. 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 studies, given that the pathotype is also isolated from asymptomatic adult and child carriers [12] [13]. 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 [9] [12] [13] [31]. 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 [9]  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 [9] [12]. 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 [5]. 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 [9] [11] [12]. 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 [9] [12]. 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 [9] [15] [32] [34] [35]. 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 [28] [30] [36]. 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 [9] [12] [14] [15] [37]. 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 [36]. 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 [30] [37]. 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 [15] [37].

Conclusion
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.