Loreny Gimenes Giugliano

Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells

BackgroundDiarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries. Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries. The ability of diarrhoeagenic strains of E. coli to adhere to and colonize the intestine is the first step towards developing the disease. EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence.Many epidemiological studies of diarrhoea have shown that breast-feeding protects infants from intestinal infections. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to the protection from diarrhoeal agents.ResultsThe effects of human milk and its protein components on the localized adherence of EPEC were investigated. Non-immunoglobulin components of human milk responsible for the inhibition of EPEC adhesion to HeLa cells were isolated by chromatographic fractionation of human whey proteins. Besides secretory immunoglobulin A, which has been previously reported to affect the adhesion of EPEC, free secretory component (fSC) and lactoferrin (Lf) were isolated. Even in concentrations lower than those usually found in whole milk, fSC and Lf were able to inhibit the adhesion of EPEC. α-lactalbumin was also isolated, but showed no activity on EPEC adhesion.ConclusionsThis study demonstrated that the immunoglobulin fraction, the free secretory component and lactoferrin of human milk inhibit EPEC adhesion to HeLa cells. These results indicate that fSC and Lf may be important non-specific defence factors against EPEC infections.

Virulence Markers of Enteroaggregative Escherichia coli Isolated from Children and Adults with Diarrhea in Brasília, Brazil

ABSTRACT Escherichia coli strains isolated from sporadic cases of acute diarrhea in children and adults and from children without diarrhea were investigated for the presence of the pAA plasmid. Strains harboring the pAA plasmid were isolated at similar frequencies from children with (19.6%) and without (10.8%) diarrhea and from adults with diarrhea (11.8%). The genotypic and phenotypic virulence markers of these strains were further analyzed. Most of the strains were positive for EAST1 (73%), and this toxin was detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.05). Likewise, pic sequences were detected significantly more frequently in strains from children with diarrhea than in strains from adults with diarrhea (P < 0.005) and controls (P < 0.025). Furthermore, the association of pAA positivity (pAA+) and pic positivity (pic+) was more frequently found for strains from children with diarrhea than for strains from controls, indicating that pAA+pic+ strains may represent a subset of pAA+ strains associated with disease in children. Most of the strains (82.5%) adhered to cells presenting the typical aggregative pattern. The frequency of occurrence of enteropathogenic E. coli (EPEC) serogroups in the strains from children with diarrhea was very high (56%), while none of the strains from adults with diarrhea belonged to EPEC serogroups. Extraintestinal virulence markers were very commonly found in strains from adults with diarrhea. The frequencies of occurrence of the adhesins AFA and SFA were significantly higher in strains from adults with diarrhea than in strains from children with diarrhea. More than one extraintestinal virulence marker was found in 58% of the strains from adults with diarrhea but in only 7.7% of the strains from children with diarrhea. Our results show that pAA+ strains isolated from children and adults with diarrhea present very different profiles when enteroaggregative E. coli virulence markers, serotypes, and extraintestinal virulence markers are considered.

Human Milk Glycoproteins Inhibit the Adherence of Salmonella typhimurium to HeLa Cells

The ability of human milk, as well as its protein fractions, to inhibit the adhesion and invasion of Salmonella typhimurium to HeLa cells was investigated. The results revealed that milk secretory immunoglobulin A (sIgA) inhibited neither the adherence nor the bacterial invasion; however, free secretory component and lactoferrin inhibited the bacterial adhesion and interacted with several bacterial proteins. Our data indicated that glycoproteins such as free secretory component and lactoferrin could act as protective compounds against infant enteric diseases, possibly binding to bacterial surface and blocking adhesion, the primordial step of S. typhimurium infection.

In vitro adhesion and invasion inhibition of Shigella dysenteriae, Shigella flexneri and Shigella sonnei clinical strains by human milk proteins

BackgroundShigella is the etiological agent of shigellosis, a disease responsible for more than 500,000 deaths of children per year, in developing countries. These pathogens colonize the intestinal colon, invade, spreading to the other enterocytes. Breastfeeding plays a very important role in protecting infants from intestinal infections. Amongst milk compounds, glycosylated proteins prevent the adhesion of many enteropathogens in vitro. The aim of this work was to determine the effect of human milk proteins on the colonization potential of Shigella dysenteriae, S. flexneri and S. sonnei. To fulfill this purpose, pooled milk samples from five donors, were fractionated by gel filtration and affinity chromatography. Using tissue culture, the milk fractions obtained were tested in Shigella adhesion and invasion assays.ResultsOur revealed showed that both adhesion and invasion of Shigella species were inhibited by low concentration of secretory immunoglobulin A, lactoferrin and free secretory component. This work also showed that, these proteins bind to superficial and whole-cell Shigella proteins.ConclusionsOur findings suggest that human milk may act inhibiting adhesion and, consequently, invasion of Shigella, thereafter preventing shigellosis in infants.

Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili.

BackgroundEnteroaggregative Escherichia coli (EAEC) are enteropathogenic strains identified by the aggregative adhesion (AA) pattern that share the capability to form biofilms. Citrobacter freundii is classically considered as an indigenous intestinal species that is sporadically associated with diarrhea.ResultsDuring an epidemiologic study focusing on infantile diarrhea, aggregative C. freundii (EACF) and EAEC strains were concomitantly recovered from a severe case of mucous diarrhea. Thereby, the occurrence of synergic events involving these strains was investigated. Coinfection of HeLa cells with EACF and EAEC strains showed an 8-fold increase in the overall bacterial adhesion compared with single infections (P < 0.001). The synergic effect was mediated by physical interactions among the bacteria and primed in the absence of chemical signaling and without the participation of host cells. Thus, significant increases (2.7-fold on average) in bacterial adhesion were also observed during the formation of mixed biofilms on abiotic surfaces. Bacterial settling assays showed that EAEC strains harboring F-pili genes (traA) were capable of forming bacterial aggregates only in the presence of EACF. Scanning electronic microscopy analyses revealed that bacterial aggregates as well as enhanced biofilms formed by EACF and traA-positive EAEC were mediated by non-bundle forming, flexible pili. Moreover, mixed biofilms formed by EACF and traA-positive EAEC strains were significantly reduced using nonlethal concentration of zinc, a specific inhibitor of F pili. In addition, EAEC strains isolated from diarrheic children frequently produced single biofilms sensitive to zinc.ConclusionsPutative F pili expressed by EAEC strains boosted mixed biofilm formation when in the presence of aggregative C. freundii.

Enteroaggregative Escherichia coli Virulence Markers: Positive Association with Distinct Clinical Characteristics and Segregation into 3 Enteropathogenic E. coli Serogroups

BACKGROUND Enteroaggregative Escherichia coli (EAEC) are a heterogeneous category of emerging pathogens associated with acute and persistent diarrhea. METHODS We performed a case-control study to evaluate the association of EAEC strains and their virulence markers with infantile diarrhea in Brasilia, Brazil. RESULTS Of the EAEC markers that were tested, only EAEC heat-stable toxin (EAST1), protein involved in colonization (Pic), and the CVD432 sequence showed positive correlation with diarrhea; however, these virulence traits presented associations with distinct clinical characteristics. Although CVD432-positive strains were associated with persistent diarrhea, EAST1-positive strains were statistically correlated with acute diarrhea only in children >6 months of age. Typical EAEC strains were found with equal frequency in case patients and control subjects. O antigens of enteropathogenic E. coli (EPEC) were also sought, and the data showed that the EAEC markers are frequently segregated into 3 EPEC serogroups: O126, O128, and O158. CONCLUSIONS The presence of EPEC O antigens along with EAEC markers influenced the positive association of E. coli strains with diarrhea. Concerning the detection of the AA phenotype, the CVD432 sequence presented a low positive predictive value, regardless of its high specificity as a marker.

Detection of calicivirus from fecal samples from children with acute gastroenteritis in the West Central region of Brazil

The objective of this study was to describe the circulation of caliciviruses in the West Central region of Brazil and its correlation with childrens gender and age, as well as with the year and months of the sample collection. Reverse transcriptase-polymerase chain reaction was performed to detect the human calicivirus genome in 1006 fecal samples that were collected in Goiânia (n = 696) and Brasília (n = 310). Viral RNA was detected in 8.6% of the samples. No significant difference in viral prevalence was found regarding gender, age or year of the sample. However, it was observed that in Goiânia, there is a higher incidence of caliciviruses from September to March. The analysis employing three primer pairs demonstrated that the Ni/E3 or JV12/13 primer pairs, which detect norovirus (NoV), detected 41 positive samples while the 289/290 primer pair, which detects NoV or sapovirus, detected the remaining 46 samples. Calicivirus circulates in the West Central region of Brazil and for better detection of this virus it is important to use more than one primer pair. Also, we conclude that the seasonality presented by this virus is related to higher humidity in the period.

Diffusely adherent Escherichia coli strains isolated from children and adults constitute two different populations

BackgroundDiffusely adherent Escherichia coli (DAEC) have been considered a diarrheagenic category of E. coli for which several potential virulence factors have been described in the last few years. Despite this, epidemiological studies involving DAEC have shown inconsistent results. In this work, two different collections of DAEC possessing Afa/Dr genes, from children and adults, were studied regarding characteristics potentially associated to virulence.ResultsDAEC strains were recovered in similar frequencies from diarrheic and asymptomatic children, and more frequently from adults with diarrhea (P < 0.01) than from asymptomatic adults. Association with diarrhea (P < 0.05) was found for SAT-positive strains recovered from children and for curli-positive strains recovered from adults. Mixed biofilms involving DAEC and a Citrobacter freundii strain have shown an improved ability to form biofilms in relation to the monocultures. Control strains have shown a greater diversity of Afa/Dr adhesins and higher frequencies of cellulose, TTSS, biofilm formation and induction of IL-8 secretion than strains from cases of diarrhea in children.ConclusionsDAEC strains possessing Afa/Dr genes isolated from children and adults represent two different bacterial populations. DAEC strains carrying genes associated to virulence can be found as part of the normal microbiota present in asymptomatic children.

Astrovirus infection in children living in the Central West region of Brazil

This study presents data regarding the circulation of astrovirus in Goiânia-GO and Brasília-DF. These viruses were detected in fecal samples from hospitalized children up to five years old with and without acute gastroenteritis. A total of 1244 fecal samples were collected in two periods, 1994 to 1996 (Brasília) and 1998 to 2002 (Goiânia and Brasília), and were analyzed for viral RNA using the reverse transcription-polymerase chain reaction (RT-PCR). Positivity rates of 4.3 and 0.5% for astrovirus were observed in children with acute gastroenteritis and those without gastroenteritis, respectively. Among children with gastroenteritis no statistically significant difference was seen with regards to viral positivity rates in relation to gender and age. However, a higher incidence rate was observed for children from Brasília aged 36 months or more. Overall, astroviruses occurred predominantly from September to March in the two cities, suggesting a seasonal pattern for these viruses which coincides with the highest relative air humidity period. The results of this study highlight the importance of astrovirus as an etiologic agent of acute gastroenteritis in children of the Central West region of Brazil.

The circulation of human astrovirus genotypes in the Central West Region of Brazil

Out of 1,588 faecal samples of children taken from three locations of the Central West Region of Brazil, 57 were positive for astroviruses (HAstVs) using reverse transcription-polymerase chain reaction (RT-PCR). They were genotyped by nested RT-PCR and/or genomic sequencing. HAstV-1 (42.8%), HAstV-2 (23.2%), HAstV-3 (3.6%), HAstV-4 (14.3%) and HAstVs -5, -6, -7 and -8 (1.8% each) were detected. In Goiânia and Campo Grande, HAstV-1 was the most frequently detected genotype while in Brasília (DF) it was HAstV-2. Shifts in the circulation of astrovirus genotypes were observed in DF and Campo Grande. All samples collected by rectal swabs were viral negative. The astrovirus genotypes were detected in all age groups and there was no correlation between genotype and age group.