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.

Identification of a new enteroinvasive Escherichia coli strain.

We isolated an enteroinvasive Escherichia coli strain which did not react with antisera raised against all known E. coli serogroups, Shigella groups A, B, C and D or provisional Shigella serotypes. We propose the provisional designation of Escherichia coli MG for this strain, until the identification of its O antigen is established.

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.

Prevalence of Helicobacter pylori in Brazilian patients with gastric carcinoma.

Investigators have speculated on the role of Helicobacter pylori in gastric carcinogenesis. The current study was performed to evaluate prospectively the prevalence of H. pylori infection in the stomachs of Brazilian patients with gastric carcinoma. Forty patients receiving gastrectomy for gastric carcinoma were studied. H. pylori was examined in four areas without atrophy or with minor atrophic changes. Mucosal fragments were obtained for microbiologic studies in 19 cases. H. pylori was detected in 82.5% of the cases. Of the cases evaluated by histologic and microbiologic methods, 94% had positive results by at least one method. In most cases there were small numbers of microorganisms. There was no correlation between the site and histologic type of neoplasia and the presence of H. pylori. The use of more than one diagnostic method improves H. pylori detection in gastric carcinoma. The small number of microorganisms observed in gastric carcinoma cases may lead to an underestimate of H. pylori in histologic sections. This might explain the variation in H. pylori infection rates reported for patients with gastric carcinoma.

Helicobacter pylori and gastric histamine concentrations.

An enzyme isotopic assay was used to determine the histamine concentration in the gastric mucosa of patients positive for Helicobacter pylori with (n = 11) and without duodenal ulceration (n = 9) and in negative controls (n = 7). A significant difference was observed when the histamine content of H pylori negative subjects was compared with that of positive patients. On the other hand, there was no significant difference in histamine concentration between H pylori positive patients with duodenal ulceration and those without duodenal ulceration. H pylori positive patients with and without duodenal ulceration had significantly lower gastric histamine concentrations than H pylori negative subjects. The lower gastric histamine concentration observed in H pylori positive patients might be due to increased histamine release which could in turn induce increased gastric acid secretion.

The inflammatory response of the gastric mucosa of mice experimentally infected with "Gastrospirillum suis".

To establish an experimental model to study gastric spiral non-cultivable bacteria, 30 4-week-old female CFW (LOB) mice were inoculated with porcine gastric mucus containing "Gastrospirillum suis" and 25 mice were inoculated with mucus without "G. suis". Mice were examined 3, 7, 14, 21, 28 and 60 days after inoculation. Fragments from the membranous, oxyntic and antral gastric mucosa and from the duodenal mucosa were obtained for histological and microbiological analysis. Tightly spiralled bacteria were seen in smears and in histological sections of the antral and oxyntic mucosa from all G. suis-infected mice. The pre-formed urease test also gave positive results in both tissues. In control mice, no tightly spiralled bacteria were seen. By 7 days after inoculation, the test animals had developed an inflammatory infiltrate of mononuclear cells, some neutrophils and a few eosinophils, mainly in the lower third of the antral and oxyntic mucosa, which persisted for the remainder of the observation period. This model can assist in the understanding of several clinical, pathological and immunological aspects of infection with spiral gastric bacteria, particularly those associated with non-cultivable spiral bacteria.

Ultrastructure of a spiral micro-organism from pig gastric mucosa ("Gastrospirillum suis").

The ultrastructural features of a helical-shaped bacterium occurring in the stomach of pigs, within the mucus on the mucosal surface of antral pits, were examined. The bacterial cell had three to eight spiral turns, flattened and truncated ends and was approximately 4.0 microns long and 0.6 microns wide. In some sections, up to six flagella, about 22 nm in diameter, were seen arising from each pole. The cytoplasm contained sparse, irregular granules, numerous ribosomes and large single-layered membrane-bound granules. In the flagella insertion area, there was a highly electron-dense component, the "polar membrane". This micro-organism differed from similar bacteria described in cats, dogs and monkeys, and may cause inflammation in the antral mucosa of pigs similar to Helicobacter pylori infection in man. Furthermore, it was morphologically similar to the spiral micro-organism distinct from H. pylori which has been described recently in human antral mucosa from patients with gastritis and may be of potential significance as a pathogen in man. The name "Gastrospirillum suis" is proposed for this bacterium.

Histopathological study of porcine gastric mucosa with and without a spiral bacterium ("Gastrospirillum suis").

Tightly spiralled bacteria ("Gastrospirillum suis") were seen in the pyloric mucosa of the stomach of 13 (10.8%) of 120 pigs that appeared clinically healthy at slaughter and in the fundic mucosa of three (5.0%) out of 60 pigs. The spiral organism could not be cultured from any pig. Chronic gastritis was observed in the pyloric mucosa of 53 (44.2%) of 120 pigs and in the fundic mucosa of 7 (11.7%) of 60 pigs. The 13 pigs with spiral bacteria in the pyloric region comprised one animal (7.7%) with normal pyloric mucosa, two (15.4%) with "borderline gastritis", and 10 (76.9%) with chronic gastritis--in one instance accompanied by signs of activity (numerous polymorphonuclear cells). The three pigs with spiral bacteria in the fundic mucosa comprised two animals with a normal fundic region and one with "borderline gastritis". The presence of the spiral bacterium was significantly associated with pyloric gastritis (p = 0.013) and with numbers of lymphoid follicles (p = 0.014).

Experimental infection of Wistar rats with 'Gastrospirillum suis'.

In order to develop a model for the study of gastric spiral bacteria, and based on the observation that Wistar rats do not carry urease-positive spiral bacteria in their gastric mucosa, mucus from a pig naturally colonised by 'Gastrospirillum suis' (an organism with I6S rDNA 99.5% similar to that of 'G. hominis' type 1), was inoculated into 35 Wistar rats (test group). Fourteen rats were given mucus taken from 'G. suis'-negative swine (control group). Five test animals and two controls were killed 1, 2, 4, 8, 12, 26, and 52 weeks after inoculation. 'G. suis' was observed in the antral mucosa of all test rats but not in the gastric mucosa of any control animal. The number of organisms was high from the beginning of the infection and increased over the period of observation. The bacteria were seen deep in the gastric antral glands, especially in the advanced stages of infection. Histological study of two test rats killed 1 week after inoculation and of all rats killed from the second week after infection revealed the presence of a mild inflammatory response characterised by the infiltration of small numbers of mononuclear cells and scarce polymorphonuclear cells in the subglandular region of the antral mucosa. Lymphoid aggregates were observed in the antral mucosa of rats killed from 1 month onwards, and increased in size and number over the period of infection. Control animals did not have any histological changes in the gastric mucosa. The natural transmission of the bacterium from rat to rat was also investigated. Five non-inoculated animals (contact group) and rats of the test group were maintained in the same cage and killed after 12 weeks. Two animals of the contact group showed slight infiltration of mononuclear cells in the antral mucosa, although they were not colonised by 'G. suis', a finding that supports the hypothesis of faecal-oral transmission of gastric Helicobacter spp. This animal model could be used not only to understand different aspects of the relationship between spiral bacteria and the gastric mucosa but also to obtain large numbers of the organism, free from other spiral bacteria to study some of its properties.

Ultrastructure of Helicobacter trogontum in culture and in the gastrointestinal tract of gnotobiotic mice.

Helicobacter trogontum is a micro-aerophilic urease-positive bacterium that has recently been isolated from the intestinal mucosa of rats. The purpose of this investigation was to study the ultrastructural details of this micro-organism in both pure culture and in the gastrointestinal tract of germ-free mice infected with H. trogontum. The micro-organism was a fusiform to slightly spiral gram-negative cell, 4-6 microm long and 0.6-0.7 microm wide, with four to seven bipolar sheathed flagella. The cytoplasm presented several irregular and also globular granules. On each side of the polar regions of the cells, there was a highly electron-dense band, the 'polar membrane'. Coccoidal forms were seen in old cultures. H. trogontum showed several ultrastructural characteristics of the Helicobacter genus and much resemblance to H. rappini and H. bilis. H. trogontum mainly colonised the large bowel of the gnotobiotic mice where it could be seen in the lumen and also inside the enterocytes. Vacuolation of the ileal epithelial cells, loss of microvilli and pronounced desquamation of the enterocytes of the caecum were observed in the bowel colonised by the bacterium. These observations raise the possibility that H. trogontum could cause some harm to the host at least in particular circumstances such as when it colonises the gastrointestinal tract of a germ-free host.

A spiral microorganism in the stomach of pigs.

Light and electron microscopic and microbiologic evaluations were performed on mucosa of stomachs from 120 healthy slaughtered pigs. Helicobacter pylori was not found, but a tightly spiralled bacterium, not previously described, was seen in histological sections and/or in carbol fuchsin stained smears in 13 (10.8%) stomachs. In paraffin sections stained with carbol fuchsin, the bacteria were seen in the mucus of the lumen of the antral pits and in the mucosa surface within and beneath the mucus. In this sections of Polilyte embedded tissue the bacteria had three to eight spiral turns per cell (mean = five), flattened ends, a Gram-negative cell-wall structure and a sheathed flagella. The urease test was positive in gastric mucosa of 13 bacteria-positive pigs (10.8%). The microorganism was not cultured and did not cross-react with polyclonal antibodies raised in rabbits against H. pylori. Superficial chronic gastritis and "borderline" gastritis were observed in antral mucosa of 10 (76.9%) and of two (15.4%) spiral bacteria-positive pigs, respectively.

Immunocytochemical identification of Campylobacter pylori in gastritis and correlation with culture.

Endoscopic biopsy specimens of antral mucosa from 25 patients presenting with gastric complaints were obtained for culture and histologic and immunocytochemical studies. The histopathologic study revealed chronic gastritis in 22 patients and borderline chronic gastritis in three patients. The unlabeled-antibody peroxidase-antiperoxidase (PAP) method was applied for the detection of Campylobacter pylori, and its results were compared with those obtained with the culture technique. Strongly positive immunoperoxidase staining was localized in spiral, curved bacteria that were present in the mucus layer adjacent to the gastric epithelial cell surface. The microorganisms were frequently congregated in clumps and were sectioned in several directions. The PAP stains were positive in 19 specimens (76%), and the cultures were positive in 20 (80%). All results negative by culture were also negative by PAP method. Compared with the cultures, the sensitivity and positive predictive value of the PAP method for identification of C pylori in antral mucosa obtained from endoscopic biopsy specimens were 95% and 100%, respectively.

Diagnosis and distribution of Helicobacter pylori in the gastric mucosa of symptomatic children.

1. The demonstration of the association of H. pylori with gastritis and peptic ulcer has been of increasing interest to gastroenterologists, microbiologists, and histopathologists. 2. In this study, the presence of H. pylori in the gastric mucosa of children was investigated by culture, preformed urease test, and carbolfuchsin staining of biopsy smears. 3. The organism was detected in 44.9% of the children studied, and found to be distributed equally on the antral and fundic mucosa. 4. Compared to culture, the urease test and carbolfuchsin staining proved to be of higher sensitivity and specificity in detecting H. pylori.

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.

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.

Comparison of the urease test and of direct smear examination in the control of treatment of Helicobacter pylori-induced infection.

The sensitivity and specificity of the preformed urease test and of carbolfuchsin-stained smears for the diagnosis of the presence of Helicobacter pylori in gastric mucosa were evaluated before and after antimicrobial treatment. The results obtained by culture were used as the reference point. We studied 41 patients with endoscopically diagnosed duodenal ulcer. Twenty-five of these were treated with furazolidone (100 mg t.i.d.), amoxicillin (500 mg t.i.d.) and metronidazole (250 mg t.i.d.) for 5 days and then with only furazolidone (100 mg t.i.d.) for an additional 25 days. The 16 control patients were treated with cimetidine (800 mg, 4 times a day). The sensitivity of the urease test and of direct smear examination was 100% before treatment and 84.6% and 92.3%, respectively, after treatment. We conclude that the urease test and carbolfuchsin-stained smears, which are highly sensitive for H. pylori diagnosis, present reduced sensitivity when they are employed for the follow-up of patients treated with antimicrobials.

Histology of the mucosa of gastric antrum and body before and after eradication of Helicobacter pylori.

1. Helicobacter pylori status and the histology of the antral and oxyntic mucosa were evaluated in 25 patients with duodenal ulcer treated with a triple schedule of furazolidone, metronidazole and amoxicillin, and in 16 patients treated only with cimetidine. 2. Before treatment, H. pylori was detected in all patients. One month after treatment with the antimicrobial agents, H. pylori was not found in 18 (72.0%) of 25 patients treated with the triple schedule. In the patients treated with cimetidine (N = 16) the H. pylori tests continued to be positive after treatment. 3. Inflammatory activity and intensity of gastritis were significantly reduced in patients treated with the antimicrobial agents but not in cimetidine-treated patients. Three patients who had negative cultures and improvement of gastritis 1 month after treatment became H. pylori positive again within 2 months, with concomitant reappearance of gastritis. 4. This study provides additional evidence that histological gastritis observed in H. pylori-positive patients with duodenal ulcer is due to the presence of the microorganism.

Indirect immunofluorescence determination of the frequency of anti-H. pylori antibodies in Brazilian blood donors.

1. The accuracy of an indirect immunofluorescence (IIF) test for the serodiagnosis of Helicobacter pylori infection was evaluated in adult patients and compared with culture, preformed urease test and smears stained with carbolfuchsin. 2. We studied 80 consecutive patients submitted to gastroduodenoscopy (17 of whom were found to have duodenal ulcer) plus 57 patients with duodenal ulcer. 3. H. pylori was identified by microbiological methods in 65 of the 80 consecutive patients and in all 57 patients with duodenal ulcer. 4. Among the 74 patients with duodenal ulcer, 71 presented antibody titers > or = 1:20 and 46 of the 48 H. pylori-positive patients without duodenal ulcer presented antibody titers > or = 1:20. 5. Thirteen of the 15 H. pylori-negative patients presented antibody titers < or = 1:10. 6. The sensitivity, specificity and positive predictive value of the IIF test were 95.9%, 88.8% and 98.4%, respectively. 7. The seroprevalence of H. pylori in 380 asymptomatic Brazilian blood donors was also studied by the IIF test. The presence of IgG antibodies against H. pylori was observed in 62.1% of the individuals. The prevalence of H. pylori infection increased with age and no difference was observed between males (60.3%) and females (66.6%).

Chicken carcasses as a source of Campylobacter jejuni in Belo Horizonte, Brazil.

The presence of Campylobacter jejuni was investigated in stool specimens from chicken meat workers and in ready-for-market chicken carcasses from one industrial and nine non industrial slaughters in Belo Horizonte. In the latter C. jejuni was isolated from 19 (38.0%) of the 50 chicken carcasses and from 2 (13.3%) of the stool specimens obtained from 15 chicken meat workers. In the industrial slaughter it was found in only 1 (2.0%) of the 50 chicken carcasses and it was not isolated from any of the 40 stool specimens. There was a significant difference between industrial and non industrial slaughter in regard to the frequency of C. jejuni isolation from carcasses (p = 0.000002), probably due to the low hygiene conditions present in non industrial slaughters. The results of antimicrobial susceptibility tests, SDS gel electrophoresis and biotyping of the strains isolated from stool specimens obtained from chicken meat workers were similar to those observed in strains isolated from chicken carcasses which suggest that chicken could be the source of C. jejuni for the workers and both, chicken and workers, could be implicated in the transmission of C. jejuni infection in Belo Horizonte.