[Show abstract][Hide abstract] ABSTRACT: Childhood diarrheal diseases remain highly endemic in developing areas of Brazil. The importance of Escherichia coli among children with diarrhea in these areas was unknown. This study determined the prevalence of different E. coli categories in symptomatic and asymptomatic children from low socioeconomic level rural communities in southeastern Brazil.
A total of 560 stool samples were collected from 141 children with diarrhea (< 10 years) and 419 apparently healthy controls who resided in 23 communities. E. coli isolates (n = 1943) were subjected to two multiplex PCRs developed for the detection of enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and Shiga toxin-producing E. coli (STEC). Strains were also examined for the presence of EPEC, EAEC, and DAEC by assays of adhesion to HEp-2 cells and by hybridization with specific DNA probes.
Diarrheagenic E. coli strains were isolated from 253 (45.2%) children, and were associated with diarrhea in children aged < 5 years (p < 0.001). EAEC (20.9%), DAEC (11.6%), EPEC (9.3%) were the most frequent pathotypes, followed by ETEC (2.7%), EIEC (0.5%), and STEC (0.2%). Depending of the assay, EPEC, EAEC, and DAEC (collectively termed enteroadherent E. coli) strains were isolated in 45% to 56% of diarrhea cases, a significantly higher incidence than in controls (P < 0.05). Individually, only DAEC showed significant association with diarrhea (p < 0.05), particularly in children aged 2--5 years.
This study indicates that enteroadherent E. coli is an important cause of diarrhea in children living in low socioeconomic level communities in southeastern Brazil. Our results reveal that the PCR1 assay is an excellent tool for the identification of EAEC and DAEC.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Helicobacter pylori infection is usually acquired in childhood and persists into adulthood if untreated. The bacterium induces a chronic inflammatory response, which is associated with epigenetic alterations in oncogenes, tumor-suppressor genes, cell-cycle regulators, and cell-adhesion molecules. AIM: The aim of this study was to analyze the effect of H. pylori infection on the methylation status of Thrombospondin-1 (THBS1), Hypermethylated in cancer 1 (HIC1) and Gata binding protein-4 (GATA-4) in gastric biopsy samples from children and adults infected or uninfected with the bacterium and in samples obtained from gastric cancer patients. METHODS: The methylation pattern was analyzed with methylation-specific PCR. RESULTS: Our results showed that H. pylori infection was associated with methylation of the promoter regions of the THBS1 and GATA-4 genes in pediatric and adult samples (p < 0.01). HIC1 showed the lowest level of methylation, which was not an early event during gastric carcinogenesis. CONCLUSIONS: The results from this study indicate that methylation of THBS1 and GATA-4 occurs in the early stages of chronic gastritis and gastric cancer in association with H. pylori infection; however, in gastric cancer samples, other mechanisms cooperate with the down-regulation of these genes. Methylation of HIC1 may not be the principal mechanism implicated in its down-regulation in gastric cancer samples.
Digestive Diseases and Sciences 06/2013; 58(10). · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A real-time multiplex PCR assay was designed to amplify the virulence genes eae, pEAF, aatA, daaC, elt, est, ipaH, stx1 and stx2, for detecting all diarrheagenic E. coli pathotypes. This assay proved to be more sensitive and rapid than conventional multiplex PCR in DEC isolates from children with diarrhea.
Journal of clinical microbiology 01/2013; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To analyze small intestinal bacterial overgrowth in school-aged children and the relationship between hydrogen and methane production in breath tests.
This transversal study included 85 children residing in a slum and 43 children from a private school, all aged between 6 and 10 years, in Osasco, Brazil. For characterization of the groups, data regarding the socioeconomic status and basic housing sanitary conditions were collected. Anthropometric data was obtained in children from both groups. All children completed the hydrogen (H(2)) and methane (CH(4)) breath test in order to assess small intestinal bacterial overgrowth (SIBO). SIBO was diagnosed when there was an increase in H(2) ≥ 20 ppm or CH(4) ≥ 10 ppm with regard to the fasting value until 60 min after lactulose ingestion.
Children from the slum group had worse living conditions and lower nutritional indices than children from the private school. SIBO was found in 30.9% (26/84) of the children from the slum group and in 2.4% (1/41) from the private school group (P = 0.0007). Greater hydrogen production in the small intestine was observed in children from the slum group when compared to children from the private school (P = 0.007). A higher concentration of hydrogen in the small intestine (P < 0.001) and in the colon (P < 0.001) was observed among the children from the slum group with SIBO when compared to children from the slum group without SIBO. Methane production was observed in 63.1% (53/84) of the children from the slum group and in 19.5% (8/41) of the children from the private school group (P < 0.0001). Methane production was observed in 38/58 (65.5%) of the children without SIBO and in 15/26 (57.7%) of the children with SIBO from the slum. Colonic production of hydrogen was lower in methane-producing children (P = 0.017).
Children who live in inadequate environmental conditions are at risk of bacterial overgrowth and methane production. Hydrogen is a substrate for methane production in the colon.
World Journal of Gastroenterology 11/2012; 18(41):5932-9. · 2.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Helicobacter pylori ClariRes assay is a novel commercially available real-time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens.
The aim of this study was to validate the novel biprobe real-time assay in stool specimens from 217 dyspeptic children.
DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing.
The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance.
This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin-resistant strains is suspected.
[Show abstract][Hide abstract] ABSTRACT: We isolated 45 Helicobacter pylori strains from 217 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of cagA, vacA s1m1, iceA2, and babA2 genes.
Journal of clinical microbiology 11/2010; 48(11):4266-8. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We recently observed an association of resistance with a certain enteropathogenic Escherichia coli (EPEC) serotypes and identified a conjugative plasmid, similar to plasmid pED208, that was conserved among archival O111:H2/NM and O119:H2 strains of diverse geographical origin. In this study, we sought to determine the prevalence and distribution of this plasmid among a collection of EPEC isolates from Brazil, as well as to study the susceptibilities of these isolates to antimicrobial agents.
Resistance was more commonly seen in typical EPEC than atypical strains. The most prevalent resistances were to ampicillin, tetracycline, streptomycin and the sulfonamides. Markers for the EPEC conjugative multiresistance plasmid, were detected in 21 (30%) of typical but only 4 (5%) of atypical strains (p = 0.001, Chi-squared test). This plasmid, previously reported from only O111 and O119 strains was found in O55 and O127 strains and was associated with the presence of class 1 integrons.
Our data suggest a limited but expanding host range for the EPEC resistance plasmid.
[Show abstract][Hide abstract] ABSTRACT: Although atypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in childhood diarrhea in developing countries, not much is known about their adherence properties. The phenotypic and genotypic characterization of 29 aEPEC strains expressing the localized adherence-like pattern points toward the involvement of E. coli common pilus (ECP), intimins, and other known E. coli adhesins in this pattern.
Journal of clinical microbiology 10/2009; 48(1):302-6. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the presence of diarrheagenic Escherichia coli and antibiotic resistance in asymptomatic school-age children living in an area with defective environmental sanitation, comparing with children registered at a private school, both in the city of Osasco, Brazil.
Seventy-nine school-age children between 5 and 10 years living in a slum and 35 children who attended a private school of the same city were included in the study.
DEC was found in 58% of the children living in the slum and in 17% of the control group (P=0.001). Resistance to at least one antimicrobial drug was found in 65% of DEC strains; resistant to two or more antimicrobial drugs was found in 46% of strains.
The high carriage status among the slum children point towards the widespread environment contamination in low socio-economic housing conditions, in conformance with the pediatric population at higher risk for developing DEC diarrhea.
The Journal of infection 09/2009; 59(4):247-51. · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea.
Journal of clinical microbiology 09/2009; 47(11):3756-9. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the number of lactobacillus and bifidobacterium colonies in the feces of schoolchildren from two different socioeconomic levels.
We analyzed fecal samples of children aged 6 to 10 years without gastrointestinal symptoms or recent use of antimicrobials. The first group included 86 children living in a favela in the city of Osasco, state of São Paulo, southeastern Brazil. The second group included 36 children attending a private school in the same city. Body mass index (BMI) was used to assess nutritional status according to the reference values of the National Center for Health Statistics (NCHS). Specific anaerobic culture media were used for isolation of colonies for 48 and 72 hours at 37 degrees C. The number of colonies was determined using the plate-counting method.
The mean lactobacillus (1.125 x 10(9) colony-forming units, CFU/g) and bifidobacterium (1.675 x 10(9) CFU/g) counts in the private school group were higher (p < 0.001) than those in the favela group: 0.250 x 10(9) and 0.350 x 10(9) CFU/g, respectively. In the favela group, children with BMI z score < -1.0 standard deviation (SD) (n = 28) showed lower mean (p < 0.05) lactobacillus (0.100 x 10(9) CFU/g) and bifidobacterium (0.095 x 10(9) CFU/g) counts than the children with BMI >or= -1.0 SD (n = 57): 0.350 x 10(9) and 0.420 x 10(9) CFU/g, respectively.
The microbiota of schoolchildren living in unfavorable environmental conditions shows lower numbers of fecal lactobacillus and bifidobacterium colonies, especially in children with lower BMI values.
Jornal de pediatria 05/2009; 85(4):307-14. · 1.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In a prospective study between February 2003 and June 2004, stool specimens of children less than 2 years of age with diarrhoea (n=218) and without diarrhoea (n=86), living in Vitória, Espírito Santo, Brazil, were examined for the presence of diarrhoeagenic Escherichia coli. E. coli isolates were tested by colony blot hybridization with specific DNA probes designed to detect EPEC, ETEC, EIEC, EAEC, DAEC and EHEC/STEC. Diarrhoeagenic E. coli strains were detected as the sole pathogen in stools of 92 (30.3 %) children, including 72 (33.0 %) with diarrhoea and 20 (23.2 %) without diarrhoea. DAEC was the most frequent pathotype and was found significantly more often from patients (18.3 %) than from controls (8.1 %) (P<0.05), particularly among children more than 1 year of age (P=0.01). Atypical EPEC and EAEC isolates were isolated from both patients (5.5 % and 4.6 %, respectively) and controls (6.9 % and 6.9 %, respectively). ETEC was more frequently isolated from patients (3.2 %) than controls (1.2 %). Typical EPEC (0.9 %) and EIEC (0.4 %) isolates were detected only in children with diarrhoea. In conclusion, our data suggest that DAEC should be considered potential pathogens in the region of Brazil studied.
Journal of Medical Microbiology 04/2008; 57(Pt 3):359-63. · 2.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 1-year prospective study was carried out in two large urban centers of São Paulo State, Brazil, to determine the prevalences and roles of the different Escherichia coli pathotypes in children less than 5 years of age with diarrhea presenting to the emergency rooms of public hospitals or visiting private pediatricians' offices. Of the pathotypes sought, typical enteroaggregative and atypical enteropathogenic types of E. coli were isolated for 8.9% and 5.4% of 774 diarrhea cases, respectively, and were found to be dominant and significantly associated with diarrhea.
Journal of Clinical Microbiology 11/2007; 45(10):3396-9. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Atypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in infant diarrhoea in developing countries. Not much is known about the adherence properties of aEPEC; however, it has been shown that these strains can adhere to tissue-cultured cells. A chromosomal region designated the locus for diffuse adherence (LDA) confers aEPEC strain 22 the ability to adhere to culture cells. LDA is an afimbrial adhesin that contains a major subunit, LdaG, whose expression is induced on MacConkey agar at 37 degrees C. We hypothesized that the bile salts found in this culture media induce the expression of LdaG. Strain 22 and the LdaG mutant were grown in Luria-Bertani (LB) media in the presence or absence of bile salts and heat-extracted surface-expressed proteins were separated by SDS-PAGE to determine whether expression of the 25 kDa LdaG protein was induced. Western blot analysis with anti-LdaG confirmed that bile salts enhance LdaG expression at 37 degrees C. Adhesion assays on HeLa cells revealed that adhesion in a diffuse pattern of strain 22 increased in the presence of bile salts. We also confirmed that expression of the localized adherence pattern observed in the ldaG mutant required the presence of a large cryptic plasmid found in strain 22 and that this phenotype was not induced by bile salts. At the transcriptional level, the ldaG-lacZ promoter fusion displayed maximum beta-galactosidase activity when the parent strain was grown in LB supplemented with bile salts. Fluorescence Activated Cell Sorting analysis, immunogold labelling electron microscopy and immunofluorescence using anti-LdaG sera confirmed that LDA is a bile salts-inducible surface-expressed afimbrial adhesin. Finally, LdaG expression was induced in presence of individual bile salts but not by other detergents. We concluded that bile salts increase expression of LDA, conferring a diffuse adherence pattern and having an impact on the adhesion properties of this aEPEC strain.
[Show abstract][Hide abstract] ABSTRACT: A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.
[Show abstract][Hide abstract] ABSTRACT: The O26 serogroup of enteropathogenic Escherichia coli (EPEC) is one of the serogroups most frequently implicated in infant diarrhea and is also common among enterohemorrhagic E. coli (EHEC) strains. The most common O26 strains belong to EPEC/EHEC serotype O26:H11 and are generally Shiga toxin (Stx) positive. Stx-negative E. coli strains that are negative for the EPEC EAF plasmid and bundle-forming pilus (Bfp) are classified as atypical EPEC. Here, we report a novel adhesin present in an stx-negative bfpA-negative atypical EPEC O26:H11 strain isolated from an infant with diarrhea. A cloned 15-kb genomic region from this strain, designated the locus for diffuse adherence (lda), confers diffuse adherence on HEp-2 cells when expressed in E. coli K-12. Sequence analysis of lda revealed a G+C content of 46.8% and 15 open reading frames sharing homology with the E. coli K88 fae and CS31A clp fimbrial operons. The lda region is part of a putative 26-kb genomic island inserted into the proP gene of the E. coli chromosome. Hybridization studies have demonstrated the prevalence of the minor structural subunit gene, ldaH, across E. coli serogroups O5, O26, O111, and O145. A second plasmid-encoded factor that contributed to the Hep-2 adherence of this strain was also identified but was not characterized. Null mutations that abolish adherence to HEp-2 cells can be restored by plasmid complementation. Antiserum raised against the major structural subunit, LdaG, recognizes a 25-kDa protein from crude heat-extracted protein preparations and inhibits the adherence of the E. coli DH5alpha lda(+) clone to HEp-2 cells. Electron microscopy revealed a nonfimbrial structure surrounding the bacterial cell.
Infection and Immunity 09/2005; 73(8):4753-65. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One hundred twelve diffusely adherent Escherichia coli strains isolated from children in a case control study were evaluated for virulence-associated characteristics, serotyping, antibiotic resistance, and plasmid profiles. Half of the strains hybridized with the probes for icuA (aerobactin) and fimH (type 1 pili); daaE (F1845 fimbriae), afa (afimbrial Dr adhesin), agg-3A (aggregative adhesion fimbria type III fimbriae), pap (P fimbriae), astA (EAST1 toxin), and shET1 (Shigella enterotoxin 1) sequences were present in <20% of the strains. The shET1 gene was noted most frequently in strains isolated from patients. A minority (7%) of the strains produced hemolysin or colicin or showed cytotoxic effects on Vero cells. Forty-five different serotypes were found. The majority (70%) of the strains presented multiple antibiotic resistance. Antibiotic resistance and diffuse adherence were located on the same conjugative plasmids. These results suggest that the transfer of these potential virulence markers could be important in the epidemiology of diffusely adherent E. coli.
Journal of Clinical Microbiology 04/2005; 43(4):1968-72. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previously, we have identified a large gene (lifA, for lymphocyte inhibitory factor A) in enteropathogenic Escherichia coli (EPEC) encoding a protein termed lymphostatin that suppresses cytokine expression in vitro. This protein also functions as an adhesion factor for enterohemorrhagic E. coli (EHEC) and Shiga toxin-producing E. coli and is alternatively known as efa1 (EHEC factor for adherence 1). The lifA/efa1 gene is also present in Citrobacter rodentium, an enteric pathogen that causes a disease termed transmissible murine colonic hyperplasia (TMCH), which induces colitis and massive crypt cell proliferation, in mice. To determine if lifA/efa1 is required for C. rodentium-induced colonic pathology in vivo, three in-frame mutations were generated, disrupting the glycosyltransferase (GlM12) and protease (PrMC31) motifs and a domain in between that does not encode any known activity (EID3). In contrast to infection with wild-type C. rodentium, that with any of the lifA/efa1 mutant strains did not induce weight loss or TMCH. Enteric infection with motif mutants GlM12 and PrM31 resulted in significantly reduced colonization counts during the entire 20-day course of infection. In contrast, EID3 was indistinguishable from the wild type during the initial colonic colonization, but cleared rapidly after day 8 of the infection. The colonic epithelium of all infected mice displayed increased epithelial regeneration. However, significantly increased regeneration was observed by day 20 only in mice infected with the wild-type in comparison to those infected with lifA/efa1 mutant EID3. In summary, lifA/efa1 is a critical gene outside the locus for enterocyte effacement that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration.
Infection and Immunity 04/2005; 73(3):1441-51. · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have previously shown that enteroaggregative Escherichia coli (EAEC) is an important pathogen among Brazilian infants. Most EAEC strains harbor a plasmid (pAA) from which a DNA fragment has been used as a probe (EAEC probe). To better understand the characteristics of EAEC in Brazil, 109 strains carrying and lacking the EAEC probe sequence were tested for the presence of pAA plasmid-borne and chromosomal factors. Common virulence factors of probe-positive and probe-negative isolates included the presence of the Pet, EAST-1, Shf, Irp2, ShET1/Pic, and Hly virulence markers. The presence of AggR or one other virulence factor (AAF/I, AAF/II, AAF/III, or Aap) was predominantly identified only in probe-positive strains. In EAEC probe-positive strains, the virulence marker Aap was found significantly more frequently (P = 0.023) in isolates from children with diarrhea (22%) than in isolates from controls (3%). EAST-1 and Shf were the markers most frequently detected (61%) in EAEC probe-negative strains and were found to be significantly associated with diarrhea (P = 0.003 and P = 0.020, respectively). Furthermore, our data suggest that AggR can be used as an important genetic marker for EAEC probe-positive strains.
Journal of Clinical Microbiology 04/2004; 42(3):1058-63. · 4.23 Impact Factor