Abstract
Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea among children, HIV-infected patients, and travelers to developing countries. We have searched for 18 genes-encoding virulence factors associated with aggregative adherence, dispersion, biofilm, toxins, serine protease autotransporters of Enterobacteriaceae (SPATEs) and siderophores, analyzed in 172 well-characterized EAEC strains (aggR+) isolated from stool samples of 97 children with diarrhea and 75 healthy controls from a passive surveillance diarrhea cohort study in Peru. Eighty-one different genetic profiles were identified, 37 were found only associated with diarrhea and 25 with control samples. The most frequent genetic profile was aggC+aatA+aap+shf+fyuA+, present in 19 strains, including diarrhea and controls. The profile set1A+set1B+pic+ was associated with diarrhea (P < 0.05). Of all genes evaluated, the most frequent were aatA (CVD 342) present in 159 strains (92.4%) and fyuA in 157 (91.3%). When EAEC strains were analyzed as a single pathogen (excluding co-infections), only pic was associated with diarrhea (P < 0.05) and with prolonged diarrhea (diarrhea ≥ 7 days) (P < 0.05). In summary, this is the first report on the prevalence of a large set of EAEC virulence genes and its association with diarrhea in Peruvian children. More studies are needed to elucidate the exact role of each virulence factor.
Keywords: EAEC, aggR gene, diarrhea, children, control, virulence genes
Eighteen genes-encoding virulence factors in EAEC strains were analyzed from stool samples of children with/without diarrhea. Only one gene (pic) was found more frequently in diarrhea than in control samples.
INTRODUCTION
Escherichia coli is a bacterium which normally lives in the intestinal tract of people and animals, many of them are usually harmless but some are pathogenic and cause various diseases such as diarrhea or extraintestinal infections (Clements et al. 2012). Diarrhea is a major cause of illness and death among children in developing countries, with children under 1 year of age being the most vulnerable age group (Liu et al. 2012). Among the bacterial agents causing diarrhea are several categories of diarrheagenic E. coli (DEC). Enteroaggregative E. coli (EAEC), a DEC pathotype, is one of the most common organisms isolated from acute and persistent diarrhea in infants and children worldwide (Huang and Dupont 2004; Weintraub 2007), as well as HIV-infected patients with persistent diarrhea (Huang et al. 2006), and adults with traveler's diarrhea (Huang et al. 2007). EAEC may contribute to malnutrition (Roche et al. 2010) and is associated with disease outbreaks in developed countries (Huang et al. 2006; Brzuszkiewicz et al. 2011).
Although not well established, EAEC pathogenesis is thought to relate to three major features: abundant adherence to intestinal mucosa (development of biofilm), elaboration of toxins (enterotoxins and cytotoxins) and induction of mucosal inflammation (Navarro-Garcia and Elias 2011). The pathogenesis is related to several putative virulence genes present on a plasmid associated with aggregative adherence (pAA) as ‘aggregative adherence fimbriae’ (AAFs): I, II, III and IV (Nataro et al. 1992; Czeczulin et al. 1997; Bernier, Gounon and Le Bouguénec 2002; Boisen et al. 2008); plasmid encoded toxin (Pet) (Eslava et al. 1998); enteroaggregative heat-stable toxin (EAST1) encoded by astA gene (Savarino et al. 1991); genes associated with dispersion: aap (dispersin) and aatA (transporter Aap protein) (Sheikh et al. 2002; Nishi et al. 2003); shf gene (Fujiyama et al. 2008), associated with biofilm formation; and aggR gene, characteristics of ‘typical EAEC’, the master regulator for some plasmid and chromosomal genes (Dudley et al. 2006). Additionally, some chromosomal genes appear to be important, including antigen 43 gene (agn43) (Roche, McFadden and Owen 2001), yersiniabactin siderophore gene: fyuA (Schubert et al. 1998). Some genes characteristic of Shigella have been identified, as those present in she pathogenicity island (PAI): pic gene, Shigella enterotoxin 1 (ShET1 or SET1) (Weintraub 2007) and sIgA gene (Boisen et al. 2009); and other genes that codify toxins such as sepA, sat (Boisen et al. 2009, 2012) and sen (Mendez-Arancibia et al. 2008) (additional information in Table S1, Supporting Information).
The pathophysiology of EAEC infection is not completely understood. It is not clear why some children infected with EAEC develop severe or persistent illness, while others do not. It is not known if the duration and severity of illness are due to pathogen specific factors, host susceptibility or both. Previous studies have evaluated the association of some specific EAEC genes with virulence (Zamboni et al. 2004; Mendez-Arancibia et al. 2008; Boisen et al. 2012; Lima et al. 2013); however, it is important to take into account that EAEC is composed by multiple lineages which may carry on different virulence factors and which may present geographical variations (Chattaway et al. 2014). Regarding host factors, it has been proposed that the association of diarrhea with the aggregative adherence pattern (AA) genotype and the polymorphism at the –251 promoter position of IL-8 is due to the increased levels of this interleukin in feces (Jiang et al. 2003). However, there is a gap in the literature relating relevance of these factors to the development of watery acute or prolonged diarrhea, since previous studies have only evaluated a small collection of strains with limited clinical data. The aims of this study were to evaluate the genes associated with the pathogenesis of EAEC in a collection of strains isolated from Peruvian children; to compare the genetic profile of strains isolated from diarrhea and healthy controls; and to determine their association with acute or prolonged diarrhea.
MATERIALS AND METHODS
Bacterial strains
EAEC strains were isolated from a passive surveillance cohort study in children younger than 24 months of age, conducted between September 2006 and July 2008, in four peri-urban communities in Lima, Peru (Ochoa et al. 2009). These strains were identified as EAEC by the presence of the aggR gene, a major EAEC virulence regulator encoded within the pAA (Dudley et al. 2006), using a multiplex real-time PCR for detection of DEC (Guion et al. 2008; Barletta et al. 2009). One hundred seventy-two EAEC strains were analyzed: 75 EAEC strains from healthy ‘controls’ collected in the absence of diarrhea or other gastrointestinal symptoms 7 days before and after the stool sample collection and 97 EAEC strains from patients with diarrhea (3 or more loose stools in 24 h, or 1 loose stool with blood in 24 h). Since co-infection with other pathogens is common in developing countries, we have analyzed the clinical characteristics of EAEC episodes only as single-pathogen infection, excluding co-infections (with virus, other bacteria and parasites). Diarrheal episodes were divided into two groups based on duration: acute (<7 days) and prolonged (≥7 days) (Moore et al. 2010). The study was approved by the Institutional Ethics Committee of Universidad Peruna Cayetano Heredia.
Identification of virulence genes
We have searched for 18 genes-encoding virulence factors associated with aggregative adherence, dispersion, biofilm, toxins, serine protease autotransporters of Enterobacteriaceae (SPATEs) and siderophores. We have used primers previously described in the literature (Table S1, Supporting Information), and have adapted the previously described PCR conditions in order to evaluate some of the genes in a more cost-effective manner. Five duplex-PCR were designed following these conditions: for set1A/set1B, denaturation: 94°C: 30 s, annealing: 55°C: 10 s and extension: 72°C: 15 s; and for sen/astA, sat/sepA, pet/sigA and aap/aatA, denaturation: 94°C: 45 s, annealing: 56°C: 45 s, 58°C: 45 s, 58°C: 45 s and 55°C: 30 s and extension: 72°C: 45 s, respectively.
The PCR was performed in the Thermal cycler ABI 2720 (Applied Biosystems, CA, USA) in a volume of 25 μL reaction mixture containing 0.63 μL of 10 mM of dNTPs mix, 2.5 μL of 10× buffer, 1.5 μL of 25 mM MgCl2, 0.5 U of Taq polymerase GeneCraft, Cologne, Germany and 2 μL of DNA template. The PCR products were detected by electrophoresis on 2% agarose gel and stained with ethidium bromide (0.5 mg L−1).
Statistical analysis
The allelic frequencies obtained from each group, the allelic distributions and association/combination of the virulence genes in each population were compared using the GenALEx 6.501 web tool (http://genepop.curtin.edu.au/). The classification and regression tree (CART) software (Salford Predictive Modeler v8.0; Salford Systems) inputing all genes of interest as binary (present/absent) independent predictive variables was used. Diarrhea/control status was the binary dependent outcome variable. Comparisons between groups were made using chi square analysis; P-values of <0.05 were considered significant.
RESULTS
Using the GenAlex program on 18 genes, we were able to identify 81 different genetic profiles among 172 EAEC strains; 37 and 25 profiles were only associated with diarrhea and control samples, respectively (Table 1). The most common profile among diarrheal samples was aggC+aatA+aap+shf+fyuA+ present in 11 strains (11.3%), followed by agg3C+aatA+aap+astA+set1A+set1B+pic+sat+ag43+fyuA+ in five strains (5.2%). Among control samples, the most common profile was agg3C+aatA+aap+set1A+set1B+pic+sat+ag43+fyuA+ present in nine strains (12.0%) and aggC+aatA+aap+shf+fyuA+ in eight strains (10.7%). Eight strains were negative for all searched virulence genes, three (3.1%) and five (6.7%) strains among diarrhea and control samples, respectively.
Table 1.
Genetic profile of EAEC strains isolated from children with diarrhea and asymptomatic controls.
| Plasmidic genes | Chromosomal genes | |||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Profile | aggC | aafC | agg3C | agg4C | aatA | aap | astA | sen | pet | sepA | shf | set1A | set1B | pic | sigA | sat | ag43 | fyuA | Diarrhea 97 | Control 75 |
| 1 | + | – | - | – | + | + | – | – | – | – | + | – | – | – | – | – | – | + | 11 | 8 |
| 2 | – | – | + | – | + | + | – | – | – | – | – | + | + | + | – | + | + | + | 4 | 9 |
| 3 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | 3 | 5 |
| 4 | – | + | + | – | + | + | + | – | + | – | + | + | + | + | – | – | + | + | 4 | 3 |
| 5 | – | – | + | – | + | + | – | – | – | – | – | – | – | – | – | – | – | + | 2 | 3 |
| 6 | – | – | + | – | + | + | + | – | – | – | – | + | + | + | – | + | + | + | 5 | 0 |
| 7 | + | – | – | – | + | + | – | – | – | – | – | – | – | – | – | – | – | + | 3 | 2 |
| 8 | + | – | – | – | + | + | – | – | – | – | – | + | + | + | – | + | + | + | 3 | 2 |
| 9 | – | – | – | – | + | – | + | – | – | + | – | + | + | + | – | – | + | + | 2 | 2 |
| 10 | – | – | – | + | + | + | – | – | – | + | + | + | + | + | – | + | + | + | 3 | 1 |
| 11 | – | – | + | – | + | + | – | – | – | – | – | – | – | – | – | – | + | + | 2 | 2 |
| 12 | – | – | + | – | + | + | – | – | – | – | – | + | + | + | – | – | + | + | 2 | 2 |
| 13 | + | – | – | – | + | + | – | – | – | – | + | + | + | + | – | – | – | + | 4 | 0 |
| 14 | – | – | – | + | + | + | – | – | – | + | + | + | + | + | – | – | + | + | 2 | 1 |
| 15 | – | – | – | – | + | – | + | – | – | + | – | + | + | + | – | – | + | – | 2 | 0 |
| 16 | – | – | – | – | + | + | – | – | – | – | – | – | – | – | – | + | + | + | 1 | 1 |
| 17 | – | – | – | – | + | + | – | – | + | + | + | + | + | + | – | + | + | + | 2 | 0 |
| 18 | – | – | + | – | + | + | – | – | – | – | – | – | – | – | – | + | + | + | 0 | 2 |
| 19 | – | – | + | – | + | + | + | – | – | – | – | – | – | – | – | + | + | + | 0 | 2 |
| 20 | – | – | + | – | + | + | – | – | – | + | – | + | – | – | – | + | + | + | 1 | 1 |
| 21 | – | – | + | – | + | + | – | – | – | – | + | + | + | + | – | + | + | + | 2 | 0 |
| 22 | – | – | + | – | + | + | + | – | – | + | – | + | + | + | – | + | + | + | 1 | 1 |
| 23 | – | – | + | – | + | + | + | – | – | – | + | + | + | + | + | – | – | + | 0 | 2 |
| 24 | + | – | – | – | + | + | – | – | – | – | – | – | – | – | – | – | + | + | 1 | 1 |
| 25 | + | – | – | – | + | + | + | – | – | – | + | – | – | – | – | + | + | + | 2 | 0 |
| 26 | + | – | – | – | + | + | + | – | – | – | + | + | – | – | – | + | + | + | 1 | 1 |
| 27 | + | – | – | – | + | + | – | – | – | + | – | + | + | + | – | – | + | + | 2 | 0 |
| 28 | + | – | – | – | + | + | + | – | – | – | – | + | + | + | – | – | + | + | 1 | 1 |
| 29 | + | – | – | – | + | + | + | – | – | – | + | + | + | + | – | + | + | + | 1 | 1 |
| 30 | – | – | – | – | – | – | – | – | – | – | – | – | – | – | + | – | – | – | 1 | 0 |
| 31 | – | – | – | – | – | – | + | – | – | – | + | – | – | – | – | – | – | – | 1 | 0 |
| 32 | – | – | – | – | – | + | + | – | – | + | – | + | + | + | – | – | – | – | 1 | 0 |
| 33 | – | – | – | – | + | – | + | – | – | + | – | + | + | + | – | + | + | + | 1 | 0 |
| 34 | – | – | – | – | + | + | – | – | – | – | + | – | – | – | – | – | – | + | 0 | 1 |
| 35 | – | – | – | – | + | + | – | – | – | – | – | – | – | – | – | – | + | + | 1 | 0 |
| 36 | – | – | – | – | + | + | + | – | – | + | + | – | – | – | – | – | – | + | 1 | 0 |
| 37 | – | – | – | – | + | + | – | – | – | – | – | – | + | – | – | – | + | + | 1 | 0 |
| 38 | – | – | – | – | + | + | – | – | – | – | – | + | – | – | – | – | + | + | 0 | 1 |
| 39 | – | – | – | – | + | + | – | – | – | – | – | + | + | + | – | – | + | + | 1 | 0 |
| 40 | – | – | – | – | + | + | – | – | – | + | + | + | + | + | – | + | – | + | 1 | 0 |
| 41 | – | – | – | – | + | + | – | – | + | – | + | + | + | + | – | + | + | + | 1 | 0 |
| 42 | – | – | – | – | + | + | – | – | + | + | + | + | + | + | – | + | – | + | 1 | 0 |
| 43 | – | – | – | – | + | + | – | + | + | + | + | + | + | + | – | + | + | + | 0 | 1 |
| 44 | – | – | – | + | + | – | – | – | – | + | + | + | + | + | – | – | – | + | 0 | 1 |
| 45 | – | – | – | + | + | + | – | – | – | – | + | – | – | – | + | – | – | + | 0 | 1 |
| 46 | – | – | – | + | + | + | – | – | – | – | + | – | – | – | + | – | + | + | 1 | 0 |
| 47 | – | – | – | + | + | + | + | – | – | – | + | – | – | – | + | – | + | + | 1 | 0 |
| 48 | – | – | – | + | + | + | – | – | – | – | + | + | + | + | – | + | + | + | 0 | 1 |
| 49 | – | – | – | + | + | + | + | – | – | + | + | + | + | + | – | + | + | + | 0 | 1 |
| 50 | – | – | + | – | – | – | – | – | – | – | – | – | – | – | – | + | + | – | 0 | 1 |
| 51 | – | – | + | – | + | – | + | – | – | + | – | + | + | + | – | – | + | + | 1 | 0 |
| 52 | – | – | + | – | + | + | – | – | – | – | + | – | – | – | – | – | – | + | 0 | 1 |
| 53 | – | – | + | – | + | + | – | – | – | – | + | – | – | – | – | – | + | + | 1 | 0 |
| 54 | – | – | + | – | + | + | – | – | – | – | + | – | – | – | – | + | + | + | 1 | 0 |
| 55 | – | – | + | – | + | + | – | – | – | – | – | – | – | + | – | + | + | + | 1 | 0 |
| 56 | – | – | + | – | + | + | – | – | – | – | – | – | + | – | – | + | + | + | 0 | 1 |
| 57 | – | – | + | – | + | + | – | – | – | – | – | + | – | – | – | – | – | + | 0 | 1 |
| 58 | – | – | + | – | + | + | – | – | – | – | + | – | – | – | – | + | + | 0 | 1 | |
| 59 | – | – | + | – | + | + | – | – | – | – | – | + | + | + | – | – | – | + | 1 | 0 |
| 60 | – | – | + | – | + | + | + | – | – | – | + | + | + | + | – | + | – | + | 1 | 0 |
| 61 | – | – | + | – | + | + | + | – | – | + | – | + | + | – | – | + | + | + | 0 | 1 |
| 62 | – | – | + | – | + | + | + | – | – | – | + | + | + | + | + | – | + | + | 0 | 1 |
| 63 | – | – | + | – | + | + | + | – | + | – | + | + | + | + | – | + | + | + | 1 | 0 |
| 64 | – | + | + | – | + | + | + | – | + | – | + | + | + | – | – | – | + | + | 1 | 0 |
| 65 | + | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | – | + | 0 | 1 |
| 66 | + | – | – | – | + | – | – | – | – | – | – | – | – | – | – | – | – | + | 0 | 1 |
| 67 | + | – | – | – | + | – | – | – | – | – | – | + | + | + | – | – | – | + | 1 | 0 |
| 68 | + | – | – | – | + | + | – | – | – | – | – | – | – | – | – | – | + | – | 0 | 1 |
| 69 | + | – | – | – | + | + | – | – | – | – | – | – | – | –– | – | + | + | + | 0 | 1 |
| 70 | + | – | – | – | + | + | + | – | – | – | + | – | – | – | – | + | – | + | 1 | 0 |
| 71 | + | – | – | – | + | + | – | – | – | – | + | + | – | – | – | – | – | + | 0 | 1 |
| 72 | + | – | – | – | + | + | – | – | – | – | – | + | – | – | – | – | + | + | 0 | 1 |
| 73 | + | – | – | – | + | + | – | – | – | – | + | + | + | + | – | + | – | + | 0 | 1 |
| 74 | + | – | – | – | + | + | – | – | – | + | – | + | + | + | – | + | – | + | 1 | 0 |
| 75 | + | – | – | – | + | + | + | – | – | – | – | + | + | + | – | + | + | + | 1 | 0 |
| 76 | + | – | – | – | + | + | + | – | – | + | – | + | + | + | – | – | + | + | 1 | 0 |
| 77 | + | – | + | – | + | + | – | – | – | – | – | – | – | – | – | – | – | + | 1 | 0 |
| 78 | + | – | + | – | + | + | + | – | – | – | + | – | – | – | – | + | – | + | 1 | 0 |
| 79 | + | – | + | – | + | + | – | – | – | – | – | + | + | + | – | – | + | + | 1 | 0 |
| 80 | + | – | + | – | + | + | – | – | – | – | – | + | + | – | – | + | + | + | 0 | 1 |
| 81 | + | – | + | – | + | + | – | – | – | – | + | + | + | + | – | + | + | + | 1 | 0 |
Combination of set1A/set1B/pic genes are shaded in gray.
We analyzed the combination of the chromosomal genes set/pic, since both are in the same genetic locus but encoded in different strands, and found that the profile set1A+set1B+pic+ (highlighted on Table 1) was present in 88 strains (51.2%), including 57 from diarrheal (58.8%) and 31 from controls (41.3%) samples, this difference was significant (P < 0.05);
A specific virulence gene profile associated with the status of diarrhea or control was not found. When we analyzed the presence of the virulence profile, including the aggR, aap, aatA, astA genes with or without the set1A genes in the diarrhea group, it was more frequent in prolonged diarrhea episodes (50%) than acute episodes (24%) (P < 0.05) (data not show); however, when we analyzed this combination of genes on EAEC as a single-pathogen infection (excluding co-infections) (Table 2), this significant difference was lost.
Table 2.
Prevalence of individual virulence genes among EAEC strains isolated as single-pathogen infection (excluding co-infections) among acute and prolonged diarrhea episodes and asymptomatic controls.
| Function | Genes | Acute diarrhea n = 34 | Prolonged diarrhea n = 24 | All diarrhea episodes n = 58 | Control n = 63 |
|---|---|---|---|---|---|
| N (%) | N (%) | N (%) | N (%) | ||
| AAFs I–IV | aggC | 12 (35.3) | 6 (25.0) | 18 (31.0) | 21 (33.3) |
| aafC | 3 (8.8) | 1 (4.2) | 4 (6.9) | 2 (3.2) | |
| agg3C | 13 (38.2) | 13 (54.2) | 26 (44.8) | 28 (44.4) | |
| agg4C | 3 (8.8) | 1 (4.2) | 4 (6.9) | 5 (7.9) | |
| Associated with dispersion | aatA | 31 (91.2) | 23 (95.8) | 54 (93.1) | 57 (90.5) |
| aap | 31 (91.2) | 21 (87.5) | 52 (89.6) | 54 (85.7) | |
| Toxins | set1A | 19 (55.9) | 20 (83.3) | 39 (67.2) | 37 (58.7) |
| set1B | 18 (52.9) | 20 (83.3) | 38 (65.5) | 31 (49.2) | |
| astA | 12 (35.3) | 8 (33.3) | 20 (34.5) | 13 (20.6) | |
| sen | 0 (0) | 0 (0) | 0 (0) | 1 (1.6) | |
| SPATEs | pic | 17 (50.0) | 20 (83.3)a | 37 (63.8)b | 28 (44.4) |
| sat | 14 (41.2) | 11 (45.8) | 25 (43.1) | 25 (39.7) | |
| sepA | 7 (20.6) | 7 (29.2) | 14 (24.1) | 10 (15.9) | |
| sigA | 1 (2.9) | 1 (4.2) | 2 (3.4) | 3 (4.8) | |
| pet | 4 (11.8) | 3 (12.5) | 7 (12.1) | 3 (4.8) | |
| Associated with biofilm | shf | 19 (55.9) | 9 (37.5) | 28 (48.3) | 24 (38.1) |
| ag43 | 20 (58.8) | 16 (66.7) | 36 (62.1) | 40 (63.5) | |
| Siderophore | fyuA | 31 (91.2) | 23 (95.8) | 54 (93.1) | 57 (90.5) |
P < 0.05 for the comparison between prolonged and acute diarrhea.
P < 0.05 for the comparison between diarrhea and control samples.
The CART model analysis (Figure 1) showed that the presence of the pic gene was associated with diarrhea independently of the presence/absence of other genes. In absence of the pic gene, only the astA gene (EAST1) was associated with diarrhea. The most common genes, identified from the analysis of EAEC as a single-pathogen infection, were aatA, formerly known as CVD432 probe, and fyuA, both of them present in 111 strains (92%), followed by aap in 149 strains (88%) (Table 2). Among the AAFs genes, the most frequent was agg3C, present in 54 strains (45%); in the case of the toxins, set1 in 76 strains (63%); and among the SPATEs, pic in 65 strains (54%). The overall distribution of each individual gene was similar between diarrhea and control samples, except for pic, which was found more frequently in diarrhea than control samples (64% versus 44%, P < 0.05) and also was associated with prolonged diarrhea (≥7 days) (83% versus 50%, P < 0.05; Table 2).
Figure 1.
CART model in EAEC strains from diarrhea and control samples. For the analysis, the software Salford Predictive Modeler 8.0 considering the 18 virulence genes analyzed was used. Each node is related with the presence (1) or absence (0) of each gene (red boxes are terminal nodes). Inside each box the distribution of the gene among diarrhea (blue bars) and control samples (red bars) is shown.
DISCUSSION
EAEC strains are microorganisms whose pathogenic mechanisms are not fully understood. They have been characterized as a heterogeneous group due to the great variability of virulence factors (Zamboni et al. 2004; Mendez-Arancibia et al. 2008; Boisen et al. 2012; Lima et al. 2013). In the present study, after the analysis of 18 different virulence genes, we found 81 different genetic profiles without significant differences among diarrhea and control samples. In accordance with other reports (Mendez-Arancibia et al. 2008; Boisen et al. 2012), in this study, the most common genes detected were aatA, formerly known as CVD432 probe (92%), followed by fyuA (92%) and aap (88%). Both aatA and aap are important EAEC virulence genes that are used alone or in combination with aggR as a method of EAEC detection as an alternative to the AA detection on tissue culture cells (Cerna, Nataro and Estrada-Garcia 2003). However, Escherichia coli carrying aatA, aggR or aap, or in fact any other recognized EAEC virulence factor may be recovered from feces of healthy people (Jenkins et al. 2007; Monteiro et al. 2009), showing that development of diarrhea may not be attributable in a single manner to the presence of a specific virulence gene. Thus bacterial virulence might be related to specific virulence factors combinations. The fyuA gene within the high pathogenicity island (HPI) produces a protein involved in iron transport, however, previous studies on EAEC isolates (Hu et al. 2005) showed that it is necessary to establish the presence of the integrated functional core region of HPI to determine its full functionality. The fyuA gene has been previously described in Yersinia spp (Schubert et al. 1998), as well as in other diarrheagenic E. coli (DEC) and extraintestinal E. coli (Koczura and Kaznowski 2003; Mendez-Arancibia et al. 2008). The presence of the fyuA gene, which encodes a siderophore receptor involved in ferric caption, has also been associated with the formation of biofilm in uropathogenic E. coli especially in environments with ferric limitations (Hancock, Ferrières and Klemm 2008). Similarly, Agn43, a SPATE, has also been related with biofilm formation (Ulett et al. 2007). However, multiple alleles of agn43 can be found in a single strain, as has been observed in the EAEC strain 042 (Chaudhuri et al. 2010). Thus, no absolute correlation exists between the clinical disease and the presence or absence of a particular allele of agn43 (Chaudhuri et al. 2010).
In relation to the AA genotype, the most frequent genes were agg3C (45%) and aggC (32%). However, there were no significant differences when comparing diarrhea versus control strains. Previous studies have detected agg3C and aggC as the most common fimbriae (genes) (Kahali et al. 2004; Jenkins et al. 2006).
SPATEs proteins, distributed both in plasmid and chromosomal genes, are present in several pathotypes of E. coli and Shigella spp. (Ruiz et al. 2002; Boisen et al. 2009). Our study showed that Pic was more frequently found in diarrheal samples than in controls (64% versus 44%). Other studies have found that SepA is associated with episodes of diarrhea but not Pic (Boisen et al. 2012). In relation to other types of toxins, the most frequently found were set1A (57%) and set1B (53%), while astA and sen were detected with a low frequency. Our results differ from what has been presented by other authors (Mendez-Arancibia et al. 2008; Aslani et al. 2011), in which the presence of astA gene (EAST1) was equal or higher than the presence of set1A gene. EAST1 encoded by the astA gene is structurally similar to the heat-stable toxin A (STa) of enterotoxigenic E. coli, which is known to cause diarrhea (Navarro-Garcia and Elias 2011); however, this gene can also be found in commensal E. coli (Kaper, Nataro and Mobley 2004).
The analysis of the combinations of genes according to pathogenic profile (aggregation, dispersion, serine transportation) showed that the combination SET1/Pic was more frequent in diarrhea samples compared with controls, but She PAI, previously distributed and identified in Shigella, has also been found in EAEC (Weintraub 2007) presenting two SPATEs (Pic, sigA) and the toxin Set1 (ShET1). Set1 is composed of two subunits (Set1A and Set1B), being functional only when both subunits are present (Navarro-Garcia and Elias 2011). Set1 is found in the complementary strand and is antisense to Pic. Analyzing the combination Pic/Set1, a significant association with diarrhea was found, while SigA was only detected in a few strains (4%). The presence of only set1 or pic genes in some strains could be related to a different phenomenon, including the presence of allelic variants that were unable to be detected by the primers used. This possibility is specially reinforced by the spatial position of the used pic gene primers, which amplify all set1a but the first 2 bp, and by the fact that an analysis of the pic, set1a and set1b genes present in GenBank has showed the presence of allelic variants of all three genes. Similar dissociated results have been previously described in Shigella spp strains, including isolates from the same geographic area (Yang et al. 2005; Lluque et al. 2015).
It has been previously reported (Cennimo et al. 2009) that the concomitant presence of the aggR, aap, aatA, astA genes with or without set1A gene (or the presence of this later gene alone) is associated with diarrhea cases. In our study, there was no difference when comparing diarrhea (34%) versus control samples (35%); however, when we analyzed prolonged-persistent diarrhea (50%) versus acute diarrhea (24%), there was a significant difference. As indicated, when the analysis was done considering only the EAEC single infections, this significance disappeared. This could be the result of the small sample size analyzed. Strains isolated from controls may reflect a low bacterial load, as has been described in enteropathogenic E. coli infections (Barletta et al. 2011). The same reason might explain the presence of the same genetic profile in control and diarrhea samples. Subsequent studies focusing on the quantification of the bacterial load of EAEC from stool samples should be performed to elucidate the exact role of each virulence factor and their combinations. Virulence association studies will require the study of multiple markers, probably some yet unknown factors, with specific gene combinations, in select host sub-populations or in select bacterial lineages.
In summary, this is the first report on the prevalence of a large set of virulence genes of EAEC and its association within Peruvian children. We found that the pic/set locus is associated with diarrhea; however, when analyzed EAEC as single-pathogen infection (excluding co-infections with virus, other bacteria and parasites), only pic gene is associated with diarrhea and prolonged diarrhea (diarrhea ≥ 7 days). In this study the loci sought are mostly, but not entirely plasmid-borne. This could be a limitation; however, even studies that included more chromosomal loci did not find significant associations.
Supplementary Material
SUPPLEMENTARY DATA
FUNDING
This work was supported by the Agencia Espan˜ ola de Cooperaci ´on Internacional para el Desarrollo (AECID) [grants nos D/019499/08, D/024648/09, D/030509/10 and A1/035720/11]. JR has a fellowship from the program I3 of the Instituto de Salud Carlos III (ISCII, Spain) [grant no. CES11/012].
Conflict of interest. None declared.
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