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Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge | Cabral J.,Centro Hospitalar Lisbon Central | Ramalho P.M.,Hospital Universitario Of Santa Maria | Ramalho P.M.,University of Lisbon | And 6 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2011

Objectives: The aim of this study was to prospectively assess the pattern of evolution of primary resistance to antibiotics in Helicobacter pylori strains isolated from Portuguese children over a 10 year period (2000-09). Methods: A total of 1115 H. pylori strains were tested for antibiotic susceptibility to clarithromycin, metronidazole, amoxicillin, ciprofloxacin and tetracycline. Results: H. pylori strains were isolated from children and adolescents [ages 4 months-18 years (mean age 10.17-±4.03 years)], comprising 562 (50.4%) boys and 553 (49.6%) girls. Overall, the primary resistance rate was 34.7% to clarithromycin, 13.9% to metronidazole and 4.6% to ciprofloxacin, while 6.9% were resistant to two of these antibiotics simultaneously. Resistance to amoxicillin and to tetracycline was not detected. In general, the resistance rate was not associated with gender or the children's age. European ethnicity, when compared with an African background, was associated with clarithromycin resistance [P=0.002; odds ratio (OR)=0.30; 95% confidence interval (CI) 0.14-0.66], while the inverse situation was observed for metronidazole (P<0.001; OR=3.50; 95% CI 1.90-6.45). No significant temporal trend was noticed for resistance to clarithromycin and metronidazole, whereas ciprofloxacin and double-resistance rates have significantly increased over time (P=0.004 and P=0.05, respectively). Conclusions: The primary resistance rate of H. pylori strains isolated from Portuguese children to the commonly used anti-H. pylori antibiotics used is high. Additionally, the increasing trend of ciprofloxacin-resistant and double-resistant strains may compromise H. pylori eradication in a high-prevalence population. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Vitoriano I.,Catholic University of Portugal | Vitor J.M.B.,University of Lisbon | Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge | Roxo-Rosa M.,Instituto Nacional Saude Dr Ricardo Jorge | And 2 more authors.
Journal of Applied Microbiology | Year: 2013

Aims: To understand whether the variability found in the proteome of Helicobacter pylori relates to the genomic methylation, virulence and associated gastric disease. Methods and Results: We applied the Minimum-Common-Restriction-Modification (MCRM) algorithm to genomic methylation data of 30 Portuguese H. pylori strains, obtained by genome sensitivity to Type II restriction enzymes' digestion. All the generated dendrograms presented three clusters with no association with gastric disease. Comparative analysis of two-dimensional gel electrophoresis (2DE) maps obtained for total protein extracts of 10 of these strains, representative of the three main clusters, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (P < 0·05) among clusters. Of these, 13 proteins appear to be related to the cagA genotype or gastric disease. The abundance of three protein species, DnaK, GlnA and HylB, appeared to be dictated by the methylation status of their gene promoter. Conclusions: Variations in the proteome profile of strains with common geographic origin appear to be related to differences in cagA genotype or gastric disease, rather than to clusters organized according to strain genomic methylation. Significance and Impact of the Study: The simultaneous study of the genomic methylation and proteome is important to correlate epigenetic modifications with gene expression and pathogen virulence. © 2013 The Society for Applied Microbiology.


Vale F.F.,University of Lisbon | Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge
World Journal of Gastroenterology | Year: 2014

Helicobacter pylori (H. pylori) successfully colonizes the human stomach of the majority of the human population. This infection always causes chronic gastritis, but may evolve to serious outcomes, such as peptic ulcer, gastric carcinoma or mucosa-associated lymphoid tissue lymphoma. H. pylori first line therapy recommended by the Maastricht-4 Consensus Report comprises the use of two antibiotics and a proton-pomp inhibitor, but in some regions failure associated with this treatment is already undesirable high. Indeed, treatment failure is one of the major problems associated with H. pylori infection and is mainly associated with bacterial antibiotic resistance. In order to counteract this situation, some effort has been allocated during the last years in the investigation of therapeutic alternatives beyond antibiotics. These include vaccines, probiotics, photodynamic inactivation and phage therapy, which are briefly revisited in this review. A particular focus on phytomedicine, also described as herbal therapy and botanical therapy, which consists in the use of plant extracts for medicinal purposes, is specifically addressed, namely considering its history, category of performed studies, tested compounds, active principle and mode of action. The herbs already experienced are highly diverse and usually selected from products with a long history of employment against diseases associated with H. pylori infection from each country own folk medicine. The studies demonstrated that many phytomedicine products have an anti-H. pylori activity and gastroprotective action. Although the mechanism of action is far from being completely understood, current knowledge correlates the beneficial action of herbs with inhibition of essential H. pylori enzymes, modulation of the host immune system and with attenuation of inflammation. © 2014 Baishideng Publishing Group Inc. All rights reserved.


Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge | Pelerito A.,Instituto Nacional Saude Dr Ricardo Jorge | Nogueira P.,Instituto Nacional Saude Dr Ricardo Jorge | Benoliel J.,Instituto Nacional Saude Dr Ricardo Jorge | And 7 more authors.
Helicobacter | Year: 2011

Background: Helicobacter pylori is mainly acquired in childhood. Although adult studies reported a high prevalence of H. pylori infection in Portugal, the actual rate in children remains unknown. This study aimed to determine the prevalence and the incidence of H. pylori infection in an asymptomatic pediatric population of the Lisbon area and to correlate prevalence with sociodemographic determinants. Materials and Methods: Helicobacter pylori infection was determined by stool antigen test in 844 asymptomatic children (age 0-15years; 49.4% boys). For the incidence study, H. pylori-negative children in the prevalence study were followed-up every 6months over a 3-year period. Results: The global prevalence of H. pylori infection was 31.6%, increasing with age (19.9, 37.0 and 51.5%, in age groups 0-5, 6-10, and 11-15, respectively), but was similar among genders (34.5% in boys and 28.4% in girls). Older age and attendance of nursery/kindergarten during preschool constituted independent risk factors. The overall estimated incidence was 11.6 per 100 child-years (CY). Although 47.5% of children acquired H. pylori infection before 5years of age, the mean age of acquisition was 6.3. The incidence of infection was similar among the three age groups (11.5, 13.0, and 10.5 per 100 CY, in age groups 0-5, 6-10, and 11-15, respectively). Conclusions: The prevalence of H. pylori infection in the Portuguese pediatric population is still high. Although this study confirmed that the highest acquisition rate occurs at young age, it showed that in high-prevalence populations, older children can also acquire H. pylori infection at a rate similar to that of young children. © 2011 Blackwell Publishing Ltd.


Vitoriano I.,Catholic University of Portugal | Saraiva-Pava K.D.,Catholic University of Portugal | Rocha-Goncalves A.,University of Coimbra | Santos A.,Instituto Nacional Saude Dr Ricardo Jorge | And 3 more authors.
PLoS ONE | Year: 2011

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA "on" status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors. © 2011 Vitoriano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Saraiva-Pava K.,Catholic University of Portugal | Navabi N.,Gothenburg University | Skoog E.C.,Gothenburg University | Linden S.K.,Gothenburg University | And 2 more authors.
World Journal of Gastroenterology | Year: 2015

AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori ) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Lex), Ley and Lea and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans. Entailing good gastric properties, both NCIhTERT- clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions. CONCLUSION: These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies. © The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.


Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge | Oleastro M.,French Institute of Health and Medical Research | Santos A.,Instituto Nacional Saude Dr Ricardo Jorge | Cordeiro R.,Instituto Nacional Saude Dr Ricardo Jorge | And 5 more authors.
Journal of Clinical Microbiology | Year: 2010

Helicobacter pylori is known to be a major cause of peptic ulceration. The jhp0562 gene, encoding a glycosyltransferase involved in the synthesis of the lipopolysaccharide, was associated with peptic ulcer disease (PUD) in children. The β-(1,3)-galactosyltransferase [β-(1,3)GalT] gene (jhp0563), involved in Lewis (Le) antigen expression, is highly similar to jhp0562. The clinical significance and diversity of both genes were examined by PCR and sequencing of clinical strains (n = 117) isolated from children with PUD (n = 57) and nonulcer dyspepsia (NUD; n = 60). The prevalence of the jhp0562 gene was significantly higher in strains with a more-virulent profile (strains positive for the cag pathogenicity island [PAI], vacA sl allele, babA, homB, phase-variable gene oipA "on" [i.e., functional], and hopQ I allele). The distribution of genotypes according to clinical outcome showed that the presence of jhp0562 represented one of the greatest risks for the development of PUD. Moreover, the triple-positive genotype for the cag PAI, jhp0562, and homB provided the best discriminatory model for distinguishing PUD and NUD outcomes in children. Sequence and in vitro expression analyses of jhp0562 showed the presence of a complete open reading frame, while the β-(1,3)GalT gene was shown to be a phase-variable gene. The regular presence of jhp0562 in strains with a truncated β-(1,3)GalT gene suggests that jhp0562 may also be implicated in the regulation of Le antigen expression. Overall, the results of this study suggest that the jhp0562 gene is of great clinical relevance, being a useful comarker for severe H. pylori-related disease and contributing to host adaptation. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Paulo L.,University of Beira Interior | Oleastro M.,Instituto Nacional Saude Dr Ricardo Jorge | Gallardo E.,University of Beira Interior | Queiroz J.A.,University of Beira Interior | Domingues F.,University of Beira Interior
Food Research International | Year: 2011

There is considerable interest in alternative approaches for the eradication of Helicobacter pylori using biologically active compounds including antioxidants from a wide range of natural sources. In this work we have investigated the antibacterial properties of resveratrol towards different H. pylori strains. In addition we studied the inhibition of H. pylori urease by resveratrol and red wine. In those assays, resveratrol inhibited the growth of all the 17 H. pylori strains tested, with inhibition diameters ranging from 16 to 28 mm and minimum inhibitory concentration values varying from 25 to 100. μg/mL, confirming its antimicrobial properties. Moreover, resveratrol and red wines showed an inhibitory effect on H. pylori urease activity, which is considered a virulence factor of this organism and essential for colonization and establishment of the infection. Further kinetic analysis revealed that inhibition occurred in a non-competitive and concentration-dependent manner. Overall, the results suggest that resveratrol and red wine may have potential for new therapy schemes that include natural products as an alternative therapeutic approach. © 2011 Elsevier Ltd.


PubMed | Instituto Nacional Saude Dr Ricardo Jorge
Type: Journal Article | Journal: Helicobacter | Year: 2011

Helicobacter pylori is mainly acquired in childhood. Although adult studies reported a high prevalence of H. pylori infection in Portugal, the actual rate in children remains unknown. This study aimed to determine the prevalence and the incidence of H. pylori infection in an asymptomatic pediatric population of the Lisbon area and to correlate prevalence with sociodemographic determinants.Helicobacter pylori infection was determined by stool antigen test in 844 asymptomatic children (age 0-15 years; 49.4% boys). For the incidence study, H. pylori-negative children in the prevalence study were followed-up every 6 months over a 3-year period.The global prevalence of H. pylori infection was 31.6%, increasing with age (19.9, 37.0 and 51.5%, in age groups 0-5, 6-10, and 11-15, respectively), but was similar among genders (34.5% in boys and 28.4% in girls). Older age and attendance of nursery/kindergarten during preschool constituted independent risk factors. The overall estimated incidence was 11.6 per 100 child-years (CY). Although 47.5% of children acquired H. pylori infection before 5 years of age, the mean age of acquisition was 6.3. The incidence of infection was similar among the three age groups (11.5, 13.0, and 10.5 per 100 CY, in age groups 0-5, 6-10, and 11-15, respectively).The prevalence of H. pylori infection in the Portuguese pediatric population is still high. Although this study confirmed that the highest acquisition rate occurs at young age, it showed that in high-prevalence populations, older children can also acquire H. pylori infection at a rate similar to that of young children.


PubMed | Instituto Nacional Saude Dr Ricardo Jorge
Type: Journal Article | Journal: The Journal of antimicrobial chemotherapy | Year: 2011

The aim of this study was to prospectively assess the pattern of evolution of primary resistance to antibiotics in Helicobacter pylori strains isolated from Portuguese children over a 10 year period (2000-09).A total of 1115 H. pylori strains were tested for antibiotic susceptibility to clarithromycin, metronidazole, amoxicillin, ciprofloxacin and tetracycline.H. pylori strains were isolated from children and adolescents [ages 4 months-18 years (mean age 10.174.03 years)], comprising 562 (50.4%) boys and 553 (49.6%) girls. Overall, the primary resistance rate was 34.7% to clarithromycin, 13.9% to metronidazole and 4.6% to ciprofloxacin, while 6.9% were resistant to two of these antibiotics simultaneously. Resistance to amoxicillin and to tetracycline was not detected. In general, the resistance rate was not associated with gender or the childrens age. European ethnicity, when compared with an African background, was associated with clarithromycin resistance [P=0.002; odds ratio (OR)=0.30; 95% confidence interval (CI) 0.14-0.66], while the inverse situation was observed for metronidazole (P<0.001; OR=3.50; 95% CI 1.90-6.45). No significant temporal trend was noticed for resistance to clarithromycin and metronidazole, whereas ciprofloxacin and double-resistance rates have significantly increased over time (P=0.004 and P=0.05, respectively).The primary resistance rate of H. pylori strains isolated from Portuguese children to the commonly used anti-H. pylori antibiotics used is high. Additionally, the increasing trend of ciprofloxacin-resistant and double-resistant strains may compromise H. pylori eradication in a high-prevalence population.

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