Species distribution, sequence types and antimicrobial resistance of Acinetobacter spp. from cystic fibrosis patients.

Acinetobacter spp. are important healthcare pathogens, being closely linked to antibiotic resistance and outbreaks worldwide. Although such species are rarely observed in patients with cystic fibrosis (CF), we describe the characteristics of 53 strains of Acinetobacter spp. isolated from the sputum of 39 Brazilian patients with CF. The species distribution was A. baumannii (n = 29), A. pittii (n = 13), A. nosocomialis (n = 8), A. seifertii (n = 1), A. soli (n = 1) and A. variabilis (n = 1) determined by partial rpoB gene sequencing. Sixteen strains (10 A. baumannii, 3 A. pittii and 3 A. nosocomialis) were multidrug-resistant (MDR) by disk diffusion test (30%) and eight MDR carbapenem-resistant A. baumannii strains harboured the bla OXA-23-like oxacillinase gene. Thirty-three sequence types (STs) were identified by multilocus sequence typing of which eight were novel (A. baumannii: 843, 844, 845, 847, 848; A. pitti: 643; A. nosocomialis: 862 and A. seifertii: 846); six STs (2 A. baumannii, 3 A. pittii and 1 A. nosocomialis) were found in more than one patient. Four strains of A. baumannii were assigned to two common clonal complexes (CCs), namely, CC1 (ST1, ST20 and ST160), and CC79 (ST79). This study underlines the extensive species diversity of Acinetobacter spp. strains in CF lung infections which may present difficulties for therapy due to significant antimicrobial resistance.

Identification and mapping of resistance genes to Phakopsora pachyrhizi in soybean (Glycine max L.) accession PI 594767-A.

The goal of this study was to study resistance inheritance in the soybean (Glycine max L.) accession PI 594767-A to the Phakopsora pachyrhizi isolate PPUFV02, and map the resistance gene(s) identified using microsatellite markers. Crosses between PI 594767-A and the susceptible cultivar 'Conquista' gave rise to the segregating subpopulations 26C-2 and 26C-5, which in the F2 generation were evaluated for their reactions to PPUFV02. In addition, analyses with microsatellite markers linked to the Rpp1-Rpp5 loci were also performed. The segregation pattern obtained in 26C-2 revealed that resistance was governed by a recessive gene; a 1:2:1 segregation pattern was observed in 26C-5, indicating control by a gene with partial dominance. This variability may have been caused because environmental conditions, particularly temperature, when 26C-5 was assessed were unfavorable for pathogen development, allowing the phenotypic expression of heterozygous alleles in PI 594767-A. A resistance gene was located in the soybean linkage group G, in the genomic region between Sct_187r2 and Sat_064 that contains the Rpp1 locus. Resistance in PI 594767-A is probably conferred by a new Rpp1 gene allele, because this accession has a haplotype for Sct_187r2 and Sat_064, which differs from haplotypes of accessions that also contain resistance alleles that map the Rpp1 locus. The use of Sct_187r2 and Sat_064 will facilitate the introgression of the resistance allele from PI 594767-A and its pyramiding with other resistance genes into genotypes with superior agronomic characteristics, in order to obtain cultivars with broad-spectrum resistance to P. pachyrhizi.

The role of IFN-gamma and IL-4 in gastric mucosa inflammation associated with Helicobacter heilmannii type 1 infection.

Although Helicobacter heilmannii infection is less common than H. pylori infection in humans, it is considered to be of medical importance because of its association with gastritis, gastric ulcer, carcinoma, and mucosa-associated lymphoid tissue lymphoma of the stomach. However, there have been no studies evaluating the role of the Th cell response in H. heilmannii gastric infection. We evaluated the participation of pro-inflammatory and anti-inflammatory cytokines, IFN-gamma and IL-4, in H. heilmannii gastric infection in genetically IFN-gamma- or IL-4-deficient mice. The serum IFN-gamma and IL-4 concentrations were determined by ELISA. The gastric polymorphonuclear infiltrate was higher (P = 0.007) in H. heilmannii-positive than in H. heilmannii-negative wild-type (WT) C57BL/6 mice, whereas no significant inflammation was demonstrable in the stomach of H. heilmannii-positive IFN-gamma(-/-) C57BL/6 mice. The degree of gastric inflammatory cells, especially in oxyntic mucosa, was also higher (P = 0.007) in infected IL-4(-/-) than in WT BALB/c mice. Serum IFN-gamma levels were significantly higher in IL-4(-/-) than in WT BALB/c mice, independently of H. heilmannii-positive or -negative status. Although no difference in serum IFN-gamma levels was seen between H. heilmannii-positive (11.3 +/- 3.07 pg/mL, mean +/- SD) and -negative (11.07 +/- 3.5 pg/mL) WT BALB/c mice, in the group of IL-4(-/-) animals, the serum concentration of IFN-gamma was significantly higher in the infected ones (38.16 +/- 10.5 pg/mL, P = 0.04). In contrast, serum IL-4 levels were significantly decreased in H. heilmannii-positive (N = 10) WT BALB/c animals compared to the negative (N = 10) animals. In conclusion, H. heilmannii infection induces a predominantly Th1 immune response, with IFN-gamma playing a central role in gastric inflammation.

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.

Omeprazole, clarithromycin and furazolidone for the eradication of Helicobacter pylori in patients with duodenal ulcer.

AIM: To evaluate the efficacy of omeprazole plus clarithromycin and furazolidone in Helicobacter pylori eradication and duodenal ulcer healing in Brazilian patients. METHODS: Forty H. pylori-positive patients with duodenal ulcer were randomized to receive 20 mg omeprazole o.m. or b.d. for 1 month plus 500 mg clarithromycin (b.d. ) and 200 mg furazolidone (b.d.) for 1 week. RESULTS: Three months after the end of the treatment the eradication rates were 90% by intention-to-treat analysis, and 97% by per protocol analysis. Mild side-effects were observed in 25 patients, none of whom abandoned the protocol. No difference was observed between the 20 mg and 40 mg omeprazole daily doses. Cure or significant improvement of the symptoms and of the histological alterations were observed after H. pylori eradication. CONCLUSION: Our results demonstrate that clarithromycin and furazolidone in combination with omeprazole are a good alternative for H. pylori eradication in Brazilian patients with duodenal ulcer.

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 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.

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 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).

Seroconversion for Helicobacter pylori in adults from Brazil.

We studied, prospectively, seroconversion for Helicobacter pylori in adults from a developing country and investigated risk factors for the acquisition of the microorganism in this population. A group of 213 volunteers of low socioeconomic level from a district in the metropolitan area of Belo Horizonte, south-east Brazil was evaluated. Anti-H. pylori IgG antibodies were measured by ELISA using Cobas Core anti-H. pylori EIA (Roche) in serum samples collected in 1992 and in 1997. The subjects were interviewed and sociodemographic data were collected. A total of 174 (81.7%) subjects presented anti-H. pylori antibodies on the occasion of the first visit. During 56 months of follow-up, 2 of 39 seronegative adults converted to seropositive with an annual infection rate of 1.1%, and 2 of 174 seropositive subjects reverted to seronegative (0.2%/year). The prevalence of infection increased significantly with age and an inverse association was observed between prevalence of infection and educational level. In conclusion, the results of the present study demonstrate that in a developing country there is a low but continuous risk of H. pylori infection in adulthood.

Antimicrobial susceptibility test of Helicobacter pylori isolated from Jos, Nigeria.

Fifty-five strains of Helicobacter pylori isolated from November 1997 until October 1998 from 33 female and 22 male adults attending for endoscopy at the Evangel Hospital, Jos, Nigeria were assayed for antibiotic susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline by the E-test strip method. Minimum inhibitory concentration (MIC) within the attainable peak serum concentrations for each drug was used as the parameter to determine the susceptibility of H. pylori. The results showed 100% susceptibility for amoxycillin, 89.0% for tetracycline, 87.3% for clarithromycin and 60% for metronidazole. The MIC50 and MIC90 values were: 0.016 microgram/mL and 0.75 microgram/mL for amoxycillin, 0.016 microgram/mL and 2 micrograms/mL for clarithromycin, 0.094 microgram/mL and 12 micrograms/mL for tetracycline, and 2 micrograms/mL and > 48 micrograms/mL for metronidazole. The MIC90 values for metronidazole (> 48 micrograms/mL) and tetracycline (12 micrograms/mL) were in each case higher than the break-point value (peak serum concentrations) of 8 micrograms/mL for metronidazole and 3 micrograms/mL for tetracycline. This pattern of resistance to metronidazole and tetracycline has to be considered when therapeutic regimens against H. pylori contain either or both drugs.

Prevalence and risk factors associated with Helicobacter pylori infection in native populations from Brazilian Western Amazon.

We evaluated the prevalence of, and factors associated with, Helicobacter pylori infection in 222 subjects from 3 distinct communities of native populations (Uru-Eu-Wau-Wau Indians and 2 riverine communities living on the banks of the Machado river and in Portuchuelo) living in isolation in the rainforest of Brazilian Western Amazon. The overall prevalence was 78.8% (95% CI 72.7-83.9). The prevalence was higher in the Machado river community compared with Portuchuelo (chi2 = 3.84, P = 0.05), but no significant difference was observed between the Machado river community and the Uru-Eu-Wau-Wau Indians. Logistic regression showed that residential crowding and age were factors associated with the presence of H. pylori infection. Acquisition of the bacterium started early in life and by the age of 2 years 50% of children were infected. The prevalence increased with age, reaching near universal levels during adulthood (97.9%). Residential crowding was high with a global index of 3.3 persons/room (SD = 1.8), varying significantly between the 3 communities (P = 0.001). These data provide further evidence supporting direct person-to-person spread of the bacterium.

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.

Hepatic changes in mice chronically infected with Helicobacter trogontum.

We infected NIH germ-free female mice with Helicobacter trogontum, a recently described intestinal bacterium of rats, in order to study the lesions it induced in the liver of this host. Fifteen mice were challenged with a single dose of H. trogontum (test group) and killed 6, 12 and 18 months after inoculation (5 animals/group). Nine animals were challenged with 0.85% saline alone (control group) and killed at the same times. Fragments from the liver, cecum and colon were obtained for microbiologic and histologic examination. Stool samples were also collected. H. trogontum was detected in the cecum, colon and/or stool samples of all test mice. As expected, the bacterium was not isolated from any specimen obtained from the control animals. On the other hand, although we could not cultivate the bacterium from the liver, 13 test animals (86.7%) presented histological changes in this organ. The 6-month group presented infiltration of mononuclear and polymorphonuclear cells in the hepatic parenchyma and the two other groups presented foci of mononuclear cells. The results suggest that H. trogontum can elicit a hepatic inflammatory response in mice since the only difference between control and test animals was the presence of H. trogontum in the latter. This result, together with the growing number of related reports in the literature, reinforces the possible role of Helicobacter infection in the pathogenesis of hepatobiliary diseases.

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.

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.