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.

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-g 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.

Phenotypic study of peripheral blood lymphocytes and humoral immune response in Helicobacter pylori infection according to age.

There are differences between children and adults in certain aspects of the Helicobacter pylori (HP) infection, among them the lower titre of IgG antibodies anti-HP in the former group. Thus, we investigated by means of flow cytometry CD4+/CD3+ (CD4+T), CD8+/CD3+ (CD8+T) and CD19+/CD3- (B) cells, activation/co-stimulatory markers (CD4+/HLA-DR+, CD4+/CD28+, CD8+/HLA-DR+ and CD8+/CD28+) and by means of ELISA IgG anti-HP antibodies in the peripheral blood from HP-positive and -negative children and adults. An increased CD4+/CD28+ and CD8+/CD28+ percentage and number of CD4+/CD3+ cells were seen in infected adults. Conversely, no difference was observed between infected and noninfected children, but when they were stratified by age, an increased CD4+/CD28+ cell percentage was seen in the HP-positive group older than 10 years. The mean level of IgG anti-HP was lower in younger infected children, increased with age and correlated with CD4+ cells. Our data suggest that the immune response to HP infection vary according to the age. Low percentage of activated CD4+ cell may contribute to the lower level of serum IgG anti-HP observed in younger infected children. In addition, the CD4+ cell participation during the infection seems to begin after 10 years old, when the immune response becomes similar to that seen in adults.

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 percent 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 percent) 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

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.

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.

Cellular immune responses in Helicobacter heilmannii infection: evaluation of the role of the host and the bacterium.

We evaluated some aspects of the immune response to Helicobacter heilmannii in two mouse strains. Gastritis that was more severe in infected C57BL/6 mice. A proliferative response to H. pylori antigens was observed in splenocytes from H. heilmannii-positive and -negative mice, similar in the positive- and negative-BALB/c mice, but lower in the positive- than in the negative-C57BL/6 animals. A decrease in B cells and an increase in CD4+ cells after stimulation with type I H. pylori antigen and an increase in CD8+ cells after stimulation with type I and II antigens was observed in infected C57BL/6 mice. Conversely, the percentage of CD4+, CD8+, and B cells was similar in positive- and negative-BALB/c mice. These results demonstrated that the immune response is similar in H. heilmannii and H. pylori infection and strengthened the importance of host and bacterial virulence markers in the immune response to gastric Helicobacter infections.

Anti-CagA antibodies in Helicobacter pylori-positive patients and blood donors from Nigeria.

The prevalence of antibodies to CagA protein was evaluated in 174 Helicobacter pylori-positive. subjects: 110 patients submitted to upper gastrointestinal endoscopy and 64 male blood donors. The patients were from different regions of Nigeria: Jos, North, Benue,West and East and the blood donors were from Jos. Sera were assayed for anti-CagA antibodies using Helicobacter p120, CagA ELISA (Viva Diagnostika, Hürth, Germany). Anti-CagA antibodies were detected in 104 (94.5%) patients and in 61 (95.3%) blood donors (P=1.0). Patients from the Benue region presented a lower frequency of anti-CagA antibodies than patients from the other regions (P=0.0004). When the subjects were stratified by age, a significant increase in IgG concentration was observed among the men (P=0.03) but not among the women (P=0.57) or among the blood donors (P=0.83). In conclusion, similarly toAsian countries, CagA-positive H. pylori infection is highly frequent in Nigeria.

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.

Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer.

Several studies have demonstrated that enzyme-linked immunosorbent assay is not a sensitive and specific method to diagnose Helicobacter pylori infection in children, especially in the younger ones. Since serum immune response can also be determined by immunoblotting and it permits the detection of antibodies to virulence factors such as CagA and VacA, we evaluated the accuracy of a commercial immunoblotting test to diagnose H. pylori infection and to assess the humoral immune response to different H. pylori antigens in 122 children who underwent upper gastrointestinal endoscopy. The presence of H. pylori was determined in antral biopsy specimens by culture, preformed urease test, and histological analysis. H. pylori was identified by microbiological and histopathological methods in 66 children (including all of the 21 who had duodenal ulcer). Antibodies to H. pylori were detected in 63 infected children and in 8 noninfected ones. The sensitivity, specificity, and positive and negative predictive values of the immunoblotting test were 95.5, 85.7, 88.7, and 94.1%, respectively. The number of immunoreactive bands increased with age (P = 0.003), and the bands of 35 kDa (P = 0.013); 89 kDa, the VacA antigen (P = 0.001); and 116 kDa, the CagA antigen (P = 0.00004) were more frequently observed in older children. The frequency of the bands of 89 kDa (P = 0.001) and 116 kDa (P = 0.03) was higher in children with duodenal ulcer than in H. pylori-positive children without the disease. In conclusion, the immunoblotting test appears to be useful for the diagnosis of H. pylori infection in children, even in the younger ones.

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.

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.

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.

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.