Enterotoxigenic Escherichia coli in children with acute diarrhoea and controls in Teresina/PI, Brazil: distribution of enterotoxin and colonization factor genes.

AIMS: To investigate the distribution of the genes that encode enterotoxins and the colonization factors (CF) types as well as the antibiotic susceptibility profile of enterotoxigenic Escherichia coli (ETEC) isolated from children from the Brazilian Northeast. METHODS AND RESULTS: We conducted a 3·5-year prospective study that involved 250 children with and 150 without diarrhoea, aged 1-60 months, from low-income families in Teresina/Brazilian Northeast. All samples were assayed for E. coli, enterotoxin and CF genes and antimicrobial susceptibility by microbiological methods and PCR. ETEC strains were isolated from 9·2% children with and 4·0% without diarrhoea. Infection was more common in children aged 6-24 months in rainy months. elt⁺ /CFA/IV⁺ and elt⁺ /CS14⁺ were the most frequent genotypes. Susceptibility to nalidixic acid, ciprofloxacin and gentamicin and resistance to ampicillin, cephalothin and sulfamethoxazole-trimethoprim were common. CONCLUSIONS: elt ⁺isolates and ETEC strains harbouring genes encoding CFA/IV and CS/14 were the most common ETEC found in Brazilian Northeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data, the first generated for north-eastern Brazilian children, may be important for the development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveller's diarrhoea in the area studied.

Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Physiological alterations of a Fusobacterium nucleatum strain exposed to oxidative stress.

AIM: The purpose of this study was to investigate the effect of oxidative stress on physiological and genetic characteristics of Fusobacterium nucleatum and its interference on this microbial identification methods. METHODS AND RESULTS: Fus. nucleatum ssp. nucleatum ATCC 25586 (wt-strain) and an oxidative-stress-adapted strain derived from the wt-strain (aero-strain) were employed in the study. Cell-free crude protein extracts were obtained from both strains and differentially expressed proteins were identified by two-dimensional electrophoresis. Bacterium identification was performed by conventional biochemical tests, automated Rapid ID 32A system and specific PCR analysis. Genetic diversity between wt- and aero-strain was assessed by arbitrarily-primed (AP)-PCR. There were significant changes in the protein profile of aero-strain. The identification of the wt-strain was confirmed by all methods employed. Similar results were obtained for aero-strain when conventional biochemical tests and PCR were used. However, aero-strain was identified as Fusobacterium varium when submitted to Rapid ID 32A system. According to AP-PCR analysis, no significant genetic alteration was detected in aero-strain. CONCLUSIONS: The adaptive response of Fus. nucleatum to oxidative stress is associated with changes on its biology, which may lead to misidentification of the organism, according to the conventional identification methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxidative stress may act as a cause of adaptive response in Fus. nucleatum with consequences to its biology, such as alterations on biochemical and physiological profile.

iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults.

Data concerning the geographic distribution of iceA alleles are scarce, and information on the association of the gene with the disease is rare and still controversial. Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common. We investigated, by PCR, the frequency of iceA alleles and cagA status in Helicobacter pylori strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma. iceA was identified in bacterium samples obtained from all patients. Eleven (7.7%) of them were infected with multiple strains. Among the patients with nonmixed infection, iceA2 allele was detected in 118 (90.1%). iceA2 allele was associated with ulcer (P = 0.02) and with carcinoma (P = 0.001). iceA2 amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder. iceA2 strains were more frequent in patients older than 7 years (P = 0.001). The gene was also more frequent in strains obtained from males (P = 0.02). cagA was more common in strains obtained from carcinoma (P = 0.0008) and ulcer patients (P < 0.006). cagA-positive strains were more frequent in children older than 7 years (P < 0.003). No association between cagA status and sex was found (P = 0.28). In conclusion, we think iceA should not be used as a reliable marker for predicting the clinical outcome of H. pylori infection.

'Flexispira rappini' strains represent at least 10 Helicobacter taxa.

'Flexispira rappini' is a provisional name given to gram-negative, microaerophilic, motile, spindle-shaped micro-organisms with spiral periplasmic fibres and bipolar tufts of sheathed flagella. Several investigators, including Kirkbride, Romero, and Archer isolated strains possessing this morphology. Previously, the phylogenetic position of three 'Flexispira rappini' strains was determined by 165 rRNA sequencing, which indicated that flexispira were members of the genus Helicobacter. As more organisms with 'F. rappini' morphology were isolated, it became apparent that there were multiple Helicobacter taxa with this distinctive morphology. The purpose of this study was to examine a collection of 36 'F. rappini' strains from diverse habitats by using 165 rRNA sequence analysis. The strains fell into 10 taxa, each possibly representing a novel Helicobacter species. Two of these flexispira taxa were previously named, by us, Helicobacter bilis and Helicobacter trogontum. Currently, none of the flexispira taxa contains enough phenotypically and genotypically characterized strains to be formally named 'Helicobacter rappinii'.

Factors associated with Helicobacter pylori infection by a cagA-positive strain in children.

Although infection with a cagA-positive Helicobacter pylori strain is considered a risk factor for the development of duodenal peptic ulcer in adults, this association has not been demonstrated in children. The presence of cagA was investigated by polymerase chain reaction in H. pylori strains isolated from 27 children with duodenal ulcer and 53 without duodenal ulcer. All patients (100%) with duodenal ulcer and 33 (62.3%) without ulcer were colonized by a cagA-positive strain (P=.00007). A cagA-positive status was also associated with a more marked macroscopic gastritis, with a greater inflammatory infiltrate of both mononuclear and polymorphonuclear cells in the antral and oxyntic gastric mucosae and degenerative and regenerative changes of the gastric mucosa. Increased cagA positivity was also associated with increased age, but no association between cagA-positive status and sex was observed.

[Human care: purification action]. / O cuidado humano: ação de purificação.

This article was originated by a request of some professors of the Masters Course in Nursing. The theme "bath", as an act of care, was developed, initially, on the meaning of moves. The esthetic of body movement, to the sound of music, was represented graphically, allowing to reach constructs as: relationship, physical contact, sharing, pleasure, satisfaction, cleanliness, reaproximation. The concept of "bath", understood through research in different bibliography and idioms, was translated as "purification". The meaning of "purification" was studied with an anthropological view. Many practices and rituals were perceived through history and human experience and bring with them tools that may help Nursing to create and recreate the experience and concept of human care. These elements and some contributions to comprehend care are shown in this study.

Differences among Helicobacter pylori strains isolated from three different populations and demonstrated by restriction enzyme analysis of an internal fragment of the conserved gene hpaA.

BACKGROUND: Our goal was to test the idea that Helicobacter pylori genotypes vary from one population to another. METHODS: Analysis of Sau3A and HinfI restriction fragment-length polymorphism (RFLP) in a 375-bp polymerase chain reaction amplicon of hpaA was used to compare 31 H. pylori isolates from a relatively small and genetically homogeneous population (Goteborg, Sweden) with those of large, genetically heterogeneous populations located in two different countries (50 isolates from Houston, TX, and 69 isolates from Minas Gerais, a state in the southeastern region of Brazil). RESULTS: Five different Sau3A and three different HinfI restriction patterns were found; different combinations of these comprise 10 different RFLP types, I through X. The RFLP types found in the United States and Brazil collections were very similar, except for two Brazil isolates belonging to type VIII and five Brazil isolates belonging to type X, neither type found in the United States. The overall profile of H. pylori isolates from Sweden was remarkably different, with 18 of 31 (58%) having a new Sau3A restriction pattern, termed gS; 10 of these 18 isolates had HinfI restriction pattern E (RFLP type VIII), and 8 had HinfI restriction pattern F (RFLP type IX). No isolates from Sweden belonged to RFLP type III or type X. CONCLUSIONS: RFLP typing of a 375-bp polymerase chain reaction-amplified DNA fragment of H. pylori hpaA revealed that H. pylori genotypes can and do vary from one population to another. We conclude that the unique RFLP profile shown by the group of H. pylori isolates from Goteborg is the result of a cohort effect in this relatively small, stable, genetically homogeneous population. Also, the overall similarity between RFLP profiles of the H. pylori isolates from Texas and Minas Gerais coincides with the fact that although geographically distanced, these populations are similar in being large, dynamic, and genetically heterogeneous.

Microbiological and histological study of the gastrointestinal tract of germ-free mice infected with Helicobacter trogontum.

Helicobacter spp. have been the focus of considerable research because of the role of this genus in gastrointestinal diseases. We infected NIH germ-free mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the distribution of this bacterium in the gastrointestinal tract and the histopathological changes it can induce in this host. Sixteen mice were challenged with a single dose of H. trogontum (test group) and killed one and six weeks after inoculation (eight animals at each point). Eight animals were challenged with 0.85% saline alone (control group) and killed at the same time points (four at each point). Fragments from the gastric and intestinal mucosa were obtained for microbiological and histological examination. H. trogontum was isolated from the cecum and colon of all test mice and also from the gastric mucosa of several of them. All infected animals presented histological changes in at least one region of the bowel. Alterations in the gastric mucosa were also observed mainly in the six-week-infected group. The predominant histological change observed was a moderate diffuse inflammatory infiltrate of mononuclear cells in the lamina propria, often accompanied by a mild infiltration of polymorphonuclear cells. Two animals presented focal infiltration of inflammatory cells in the liver, although no bacteria were found in the liver of any animal. H. trogontum is an intestinal species that is able to elicit inflammatory responses in other regions of the gastrointestinal tract such as the gastric mucosa and the liver of gnotobiotic mice.

Evaluation of enzyme-linked immunosorbent assay for the diagnosis of Helicobacter pylori infection in children from different age groups with and without duodenal ulcer.

BACKGROUND: Although enzyme-linked immunosorbent assay (ELISA) is highly sensitive and specific for the diagnosis of Helicobacter pylori infection in adults, their performance in children is still controversial. METHODS: A second-generation ELISA was used to evaluate the IgG response to H. pylori in the serum of 130 consecutive children who underwent upper gastrointestinal endoscopy. The presence of H. pylori was determined in antral biopsy specimens by culture, urease test, and histologic analysis. RESULTS: Sixty-eight children (all of the 20 who had duodenal ulcer) were H. pylori positive by microbiologic test. Immunoglobulin G antibodies to H. pylori were detected in 79.4% of the infected children and in 8.1% of the noninfected ones. The sensitivity of the test was higher in patients with duodenal ulcer (100%) than in those without (70.8%). When used in children of different ages the test also presented differences in sensitivity: 44.4% in children 2 to 6 years old; 76.7% in children 7 to 11 years old, and 93.1% in children 12 to 16 years old (p = 0.006). The serum immunoglobulin G concentration was significantly higher (p = 0.0003) in children with duodenal ulcer than in those without and was higher in older children than in younger ones without duodenal ulcer (p = 0.05). CONCLUSIONS: The accuracy of the test in children with duodenal ulcer and in children more than 12 years old was good; however, in children up to 12 years of age without duodenal ulcer, the sensitivity of the test was too low to be used for screening purposes or to rule out the presence of infection.

Helicobacter pylori cagA status and s and m alleles of vacA in isolates from individuals with a variety of H. pylori-associated gastric diseases.

The cagA gene was detected in 100% of 16 Helicobacter pylori isolates from patients with gastric carcinoma versus 78% of 18 isolates from patients with duodenal ulcers (P = 0.344) and only 64% of 22 isolates from patients with gastritis only (P = 0.005) in Brazil. Also, there was a significant association between isolation of cagA+ s1-type vacA H. pylori in cases of stomach cancer and ulcers as opposed to cases of gastritis only (P = 0.004), but this was not true in Houston (P = 0.238), where 94% of all isolates were cagA+.

The interrelationship between Helicobacter pylori vacuolating cytotoxin and gastric carcinoma.

OBJECTIVE: We aimed to determine whether cytotoxin-positive Helicobacter pylori strains are associated with gastric carcinoma. METHODS: We studied 130 patients: 57 H. pylori-positive patients with gastric carcinoma, 53 H. pylori-positive patients without gastric carcinoma, and 20 H. pylori-negative subjects. The ability of H. pylori strains to produce vacuolating cytotoxin was tested in INT-407 and HeLa cells. The presence of antibodies to cytotoxin was investigated in blood serum from all subjects by immunoblotting. Fragments of the gastric mucosa from patients without gastric carcinoma and H. pylori-negative subjects were obtained for histopathological study. RESULTS: Considering the results as a whole, 40 (70.2%) patients with and 22 (41.5%) without gastric carcinoma were colonized by cytotoxin-positive strains. Antibodies against cytotoxin were not observed in the serum from 17 (29.8%) gastric carcinoma patients and from 31 (58.5%) patients without gastric carcinoma. H. pylori strains isolated from these patients did not produce cytotoxin in vitro. In regard to cytotoxin positivity, a significant difference was observed between patients with and without gastric carcinoma (p=0.004; odds ratio [OR]: 3.3; 95% confidence interval [CI]: 1.4-7.9). Higher scores of mononuclear (p=0.0001) and polymorphonuclear (p=0.000003) cells were observed in the antral mucosa from H. pylori-positive patients without gastric carcinoma infected by cytotoxin-positive strains than in those harboring cytotoxin-negative strains. CONCLUSION: Cytotoxin-producing H. pylori strains were more frequently observed in patients with gastric carcinoma and this aspect emphasizes the role of cytotoxin in the genesis of the tumor.

cagA-positive Helicobacter pylori and risk for developing gastric carcinoma in Brazil.

To evaluate a possible association between infection with cag A-positive strains and gastric carcinoma increased risk we studied 119 Helicobacter pylori-positive patients with gastric carcinoma and 119 matched controls. The presence of cag A gene was investigated by PCR in H. pylori isolates and in gastric biopsy specimens. A significant association was found between cag A-positive status and distal gastric carcinoma for both the intestinal and diffuse types of tumor for both males and females. On the other hand, no association was observed between cag A-positive status and proximal gastric carcinoma.

Serodiagnosis of Helicobacter pylori infection by Cobas Core ELISA in adults from Minas Gerais, Brazil

We evaluated the accuracy of a 2nd generation ELISA to detect Helicobacter pylori infection in adults from a developing country in view of variations in sensitivity and specificity reported for different populations. We studied 97 non-consecutive patients who underwent endoscopy for evaluation of dispeptic symptoms. The presence of H. pylori was determined in antral biopsy specimens by culture, by the preformed urease test and in carbolfuchsin-stained smears. Patients were considered to be H. pylori positive if at least two of the three tests presented a positive result or if the culture was positive, and negative if the three tests were negative. Sixty-five adults (31 with peptic ulcer) were H. pylori positive and 32 adults were H. pylori negative. Antibodies were detected by Cobas Core anti-H. pylori EIA in 62 of 65 H. pylori-positive adults and in none of the negative adults. The sensitivity, specificity and positive and negative predictive values of the test were 95.4, 100, 100 and 91.4 per cent, respectively. The Cobas Core anti-H. pylori EIA presented high sensitivity and specificity when employed for a population in Brazil, permitting the use of the test both to confirm the clinical diagnosis and to perform epidemiologic surveys.

Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria.

In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the ureas test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmannii" type 1), an uncultivated tightly spiralled gastric bacterium. Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation. The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears). Among the methods employed for the diagnosis of "H. heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0%) swine. Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0%) of the 50 pigs studied. Among them, 3 (6.0%) presented a positive preformed urease test. Spiral bacteria were not seen in the gastric mucosa of any control mice. These results show that the use of the mouse inoculation method improved the detection of "H. heilmannii" in swine.

Prevalence of Helicobacter pylori infection in a rural area of the state of Mato Grosso, Brazil.

The prevalence of Helicobacter pylori infection was evaluated by ELISA in 40 children and teenagers and in 164 adults from a rural area of the State of Mato Grosso, Brazil. Antibodies to H. pylori were detected in the serum of 31 (77.5%0 children and teenagers and in 139 (84.7%) adults. The prevalence of infection increased with age (x2 for trend, p < 0.01) even though no variations occurred in the region in the present century in terms of living conditions or sanitation, economical development and migratory influx supporting the hypothesis that the infection is also acquired during later life in developing countries. An inverse correlation was observed between the prevalence of infection and annual family income (x2 for trend, p < 0.013). There was no correlation between type of system for sewage disposal and prevalence of infection (p = 0.8). In conclusion, the prevalence of H. pylori infection in Nossa Senhora do Livramento, a rural area from Brazil, is very high and similar to that observed in other developing countries. Furthermore, the increase in the prevalence of infection with age observed in this population seems to be due to both, cohort effect and acquisition of the infection during later life.

Diversity in the variable region of Helicobacter pylori cagA gene involves more than simple repetition of a 102-nucleotide sequence.

CagA, a product of cytotoxin-associated gene A cagA, is an important virulence-related antigen of Helicobacter pylori (HP). CagA varies in size from 128 kDa to about 140 kDa and this variation is believed to be generated by a 102-nucleotide (NT) repeat sequence in the so-called variable region of cagA. However, this explanation has not previously been tested by comparing the NT sequences of cagA derived from a number of different isolates of HP. In this study we first compared the size of PCR products obtained from 54 different isolates of HP with oligonucleotide primers designed to amplify a cagA fragment predicted to be at least 1059 NT, including the variable region of the gene. As expected, the size of the PCR products varied considerable, from 1110 to 1822 NT, but the majority (50 of 54) measured 1335 NT or less. The deduced amino acid (AA) sequences of 9 representative amplicons and 4 other known sequences were compared with the following result: Within the variable region of cagA there are actually two adjacent variable regions, which we label as proximal and distal. The proximal-variable region of CagA extends from a motif of 4 to 6 asparagine residues to a 7-AA repeat sequence (KIDQLNQ); the distal-variable region is confined between KIDQLNQ and a well-conserved duplicate, KIDNLNQ. Despite these shared features CagA of every HP strain examined to date has a variable region with a unique AA sequence.

Mouse inoculation for the detection of non-cultivable gastric tightly spiralled bacteria

In the present study we compared the inoculation of swine gastric mucus into the stomach of mice, the urease test and carbolfuchsin-stained smears for the diagnosis of the infection with "Gastrospirillum suis" ("Helicobacter heilmmannii" type 1), an uncultivated tightly spiralled gastric bacterium. Fragments obtained from the antral and oxyntic mucosa of the stomach of 50 slaughtered pigs were used for urease test, for carbolfuchsin-stained smears and for obtaining scrapings of mucus for mouse inoculation. The mice were killed by spinal dislocation 10 days after inoculation and fragments of the antral and oxyntic mucosa were used for spiral bacterium identification (urease test and carbolfuchsin-stained smears).Among the method employed for the diagnosis of "H. heilmannii" infection, the inoculation of gastric mucus into the stomach of mice was the most sensitive and demonstrated bacterial positivity in 31 (62.0 percent) swine. Direct examination showed tightly spiralled bacteria in the gastric mucosa of only 4 (8.0 percent) of the 50 pigs studied. Among them, 3 (6.0 percent) presented a positive preformed urease test. Spiral bacteria were not seen in the gastric mucosa of any control mice. These results show that the use of the mouse inoculation method improved the detection of "H. heilmannii" in swine.

Serodiagnosis of Helicobacter pylori infection by Cobas Core ELISA in adults from Minas Gerais, Brazil.

We evaluated the accuracy of a 2nd generation ELISA to detect Helicobacter pylori infection in adults from a developing country in view of variations in sensitivity and specificity reported for different populations. We studied 97 non-consecutive patients who underwent endoscopy for evaluation of dispeptic symptoms. The presence of H. pylori was determined in antral biopsy specimens by culture, by the preformed urease test and in carbolfuchsin-stained smears. Patients were considered to be H. pylori positive if at least two of the three tests presented a positive result or if the culture was positive, and negative if the three tests were negative. Sixty-five adults (31 with peptic ulcer) were H. pylori positive and 32 adults were H. pylori negative. Antibodies were detected by Cobas Core anti-H. pylori EIA in 62 of 65 H. pylori-positive adults and in none of the negative adults. The sensitivity, specificity and positive and negative predictive values of the test were 95.4, 100, 100 and 91.4%, respectively. The Cobas Core anti-H. pylori EIA presented high sensitivity and specificity when employed for a population in Brazil, permitting the use of the test both to confirm the clinical diagnosis and to perform epidemiologic surveys.