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1.
Zhonghua Er Ke Za Zhi ; 58(1): 41-45, 2020 Jan 02.
Artigo em Chinês | MEDLINE | ID: mdl-31905475

RESUMO

Objective: To evaluate the effectiveness of eradication therapy based on Helicobacter pylori (Hp) susceptibility and CYP2C19 genotype in children with refractory Hp infection. Methods: In this prospective observational cohort study, 156 children with Hp refractory to amoxicillin+clarithromycin+omeprazole triple regimen in Baoding Children's Hospital from December 2017 to May 2018 were enrolled. Ninety-two of them underwent Hp culture and CYP2C19 detection. Seventy-five cases with positive Hp culture were defined as culture successful group and were treated according to Hp susceptibility and CYP2C19 genotype. Seventeen cases with negative Hp culture were defined as culture failed group and were treated only based on the results of CYP2C19 genotype. Sixty-four children who did not have Hp culture and CYP2C19 gene testing were defined as the empirical eradication therapy group and were treated with quadruple regimen (amoxicillin+metronidazole+omeprazole+bismuth). Bacterial resistance, CYP2C19 polymorphism and therapeutic effectiveness between the three groups were compared using chi-square test. Results: Among the 75 positive Hp culture results, 72 (96%) were resistant to clarithromycin, 3 (4%) were resistant to metronidazole, 5 (7%) were resistant to levofloxacin, 5 (7%) were resistant to rifampicin, 1 (1%) was resistant to tetracycline, and none was resistant to amoxicillin and furazolidone. The CYP2C19 polymorphism in 92 patients showed that 43 (47%) were extensive metabolizer (EM), 9 (10%) were poor metabolizer (PM), and 40 (43%) were intermediate metabolizer (IM). In terms of the effectiveness, eradication rate in the culture successful group,culture failed group and empirical eradication therapy group were 99% (74/75), 88% (15/17) and 72% (46/64), respectively (χ(2)=21.325, P<0.05). The eradication rate in the culture successful group was significantly higher than that in empirical eradication therapy group (χ(2)=21.005, P<0.05), while there was no difference between empirical eradication therapy group and culture failed group (χ(2)=1.154, P=0.283). Conclusion: Eradication regimen based on bacterial susceptibility and CYP2C19 genotype should be considered in children with refractory Hp infection.


Assuntos
Citocromo P-450 CYP2C19/genética , Quimioterapia Combinada , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/genética , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Omeprazol/uso terapêutico , Amoxicilina/uso terapêutico , Antibacterianos/uso terapêutico , Bismuto/uso terapêutico , Criança , Claritromicina/administração & dosagem , Claritromicina/uso terapêutico , Estudos de Coortes , Resistência a Medicamentos/genética , Feminino , Genótipo , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Metronidazol/uso terapêutico , Omeprazol/administração & dosagem , Polimorfismo Genético , Estudos Prospectivos , Resultado do Tratamento
2.
Int J Food Microbiol ; 312: 108363, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31669766

RESUMO

Several studies report the presence of Helicobacter pylori (H. pylori) in seawater either free or attached to planktonic organism. After considering the role played by plankton in the food chain of most aquatic ecosystems and the possible role that seafood products can assume in the transmission of H. pylori to humans, the aim of this study was to assess the survival of H. pylori in artificially contaminated Mytilus galloprovincialis (M. galloprovincialis). A traditional culture method and a reverse transcriptase-PCR (RT-PCR) assay were employed to detect the mRNA of known virulence factor (VacA) which can be considered use a marker of bacterial viability. The obtained results clearly show that H. pylori is able to survive in artificially contaminated mussels for 6 days (2 days in a cultivable form and 4 days in a non-cultivable form).


Assuntos
Helicobacter pylori/isolamento & purificação , Viabilidade Microbiana/genética , Mytilus/microbiologia , Animais , Proteínas de Bactérias/genética , Ecossistema , Helicobacter pylori/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alimentos Marinhos , Água do Mar/microbiologia
3.
BMC Evol Biol ; 19(1): 197, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675915

RESUMO

BACKGROUND: Helicobacter pylori, a bacterium that infects the human stomach, has high genetic diversity. Because its evolution is parallel to human, H. pylori is used as a tool to trace human migration. However, there are few studies about the relationship between phylogeography of H. pylori and its host human. METHODS: We examined both H. pylori DNA and the host mitochondrial DNA and Y-chromosome DNA obtained from a total 119 patients in the Dominican Republic, where human demography consists of various ancestries. DNA extracted from cultured H. pylori were analyzed by multi locus sequence typing. Mitochondrial DNA and Y-chromosome DNA were evaluated by haplogroup analyses. RESULTS: H. pylori strains were divided into 2 populations; 68 strains with African group (hpAfrica1) and 51 strains with European group (hpEurope). In Y-chromosomal haplogroup, European origin was dominant, whereas African origin was dominant both in H. pylori and in mtDNA haplogroup. These results supported the hypothesis that mother-to-child infection is predominant in H. pylori infection. The Amerindian type of mtDNA haplogroup was observed in 11.8% of the patients; however, Amerindian type (hspAmerind) of H. pylori was not observed. Although subpopulation type of most hpAfrica1 strains in Central America and South America were hybrid (hspWAfrica/hpEurope), most Dominican Republic hpAfrica1 strains were similar to those of African continent. CONCLUSIONS: Genetic features of H. pylori, mtDNA, and Y haplogroups reflect the history of colonial migration and slave trade in the Dominican Republic. Discrepancy between H. pylori and the host human genotypes support the hypothesis that adaptability of hspAmerind H. pylori strains are weaker than hpEurope strains. H. pylori strains in the Dominican Republic seem to contain larger proportion of African ancestry compared to other American continent strains.


Assuntos
Helicobacter pylori/genética , Migração Humana , Adulto , Idoso , Cromossomos Humanos Y , DNA Mitocondrial/genética , República Dominicana , Feminino , Genótipo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Genética Humana , Humanos , Transmissão Vertical de Doença Infecciosa , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Filogeografia , Adulto Jovem
4.
Helicobacter ; 24(5): e12654, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31411793

RESUMO

BACKGROUND: We evaluated the efficacy of tailored therapy based on point mutation presence identified with the dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) method compared with concomitant therapy. MATERIALS AND METHODS: Subjects were randomly assigned concomitant therapy (amoxicillin 1 g, clarithromycin 500 mg, metronidazole 500 mg, and lansoprazole 30 mg twice/day for 14 days) or tailored therapy (amoxicillin 1 g, clarithromycin 500 mg, and lansoprazole 30 mg twice/day for 14 days in point mutation-negative subjects; and amoxicillin 1 g, metronidazole 500 mg, and lansoprazole 30 mg twice/day for 14 days in point mutation-positive subjects). RESULTS: A total of 397 and 352 subjects were included in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively. Point mutations were identified in 25.9% of the subjects. The overall eradication rate was not significantly different between the groups by ITT (86.2% vs 81.6%, P = .132) and PP analyses (90.2% vs 86.5%, P = .179). There was no significant difference in the eradication rates between the groups in both the point mutation-negative subjects (91.7% vs 87.3%, P = .154) and the point mutation-positive subjects (71.2% vs 64.7%, P = .312). The eradication rates were significantly lower in the point mutation-positive subjects than in the point mutation-negative subjects in both the concomitant and tailored therapy groups. CONCLUSIONS: Tailored therapy based on point mutation presence identified with the DPO-based multiplex PCR method was as effective as concomitant therapy. The eradication rates of both therapy regimens were suboptimal in point mutation-positive subjects.


Assuntos
Antibacterianos/administração & dosagem , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/genética , Mutação Puntual , Medicina de Precisão/métodos , Inibidores da Bomba de Prótons/administração & dosagem , RNA Ribossômico 23S/genética , Idoso , Farmacorresistência Bacteriana , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Testes de Sensibilidade Microbiana/métodos , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Estudos Prospectivos , Resultado do Tratamento
5.
J Med Microbiol ; 68(9): 1287-1291, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364966

RESUMO

The molecular mechanism of Helicobacter pylori resistance to tetracycline involves mutations in the primary binding site of the ribosome. A resistance or reduced susceptibility to tetracycline could be the result of single, double or triple mutations in the 16S rRNA gene of H. pylori. We investigated if the genotype was correlated to tetracycline resistance as determined phenotypically in vitro for 96 H. pylori isolates in the gastroesophageal mucosa of Venezuelan individual hosts. E-test for antimicrobial susceptibility test and real-time PCR for the detection of 16S rRNA gene mutations were performed in 96 H. pylori isolates (48 obtained from antrum, and 48 from oesophagus) from eight dyspeptic patients. In the gastric mucosa, 38 isolates were identified sensitive and 10 resistant to tetracycline by E-test, whereas 44 sensitive and 4 resistant isolates were found in the oesophagus. Real-time PCR detection of the 16S rRNA gene exhibited mutants with a single base-pair substitution (AGA926GGA) in six antrum isolates and seven oesophagus isolates, whereas only three harboured a low level of tetracycline resistance in vitro. Our results indicate that real-time PCR detection of 16S rRNA is a reliable method to classify among tetracycline-resistant genotypes and useful in patients who have experienced a first-line treatment failure with triple therapy.


Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , RNA Ribossômico 16S/genética , Resistência a Tetraciclina/genética , Tetraciclina/farmacologia , Antibacterianos/farmacologia , Mucosa Esofágica/microbiologia , Mucosa Gástrica/microbiologia , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Reação em Cadeia da Polimerase em Tempo Real
6.
PLoS Comput Biol ; 15(7): e1007208, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31335917

RESUMO

Horizontal gene transfer (HGT) has changed the way we regard evolution. Instead of waiting for the next generation to establish new traits, especially bacteria are able to take a shortcut via HGT that enables them to pass on genes from one individual to another, even across species boundaries. The tool Daisy offers the first HGT detection approach based on read mapping that provides complementary evidence compared to existing methods. However, Daisy relies on the acceptor and donor organism involved in the HGT being known. We introduce DaisyGPS, a mapping-based pipeline that is able to identify acceptor and donor reference candidates of an HGT event based on sequencing reads. Acceptor and donor identification is akin to species identification in metagenomic samples based on sequencing reads, a problem addressed by metagenomic profiling tools. However, acceptor and donor references have certain properties such that these methods cannot be directly applied. DaisyGPS uses MicrobeGPS, a metagenomic profiling tool tailored towards estimating the genomic distance between organisms in the sample and the reference database. We enhance the underlying scoring system of MicrobeGPS to account for the sequence patterns in terms of mapping coverage of an acceptor and donor involved in an HGT event, and report a ranked list of reference candidates. These candidates can then be further evaluated by tools like Daisy to establish HGT regions. We successfully validated our approach on both simulated and real data, and show its benefits in an investigation of an outbreak involving Methicillin-resistant Staphylococcus aureus data.


Assuntos
Evolução Molecular , Transferência Genética Horizontal , Metagenoma , Metagenômica/métodos , Modelos Genéticos , Biologia Computacional , Simulação por Computador , Bases de Dados Genéticas/estatística & dados numéricos , Surtos de Doenças/estatística & dados numéricos , Variação Genética , Genoma Bacteriano , Helicobacter pylori/genética , Humanos , Metagenômica/estatística & dados numéricos , Staphylococcus aureus Resistente à Meticilina/genética , Mutação , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia
7.
Helicobacter ; 24(5): e12634, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31304664

RESUMO

BACKGROUND: Detection of Helicobacter pylori in gastric biopsy is important for appropriate treatment and prevention of gastric carcinoma and lymphoma. A novel peptide nucleic acid probe (PNA)-based real-time polymerase chain reaction (PCR) method was developed for detection of H pylori and A2142G/A2143G mutation of the 23S rRNA gene, which is associated with clarithromycin resistance. METHODS: To evaluate the performance of the PNA probe-based PCR method, a total of 409 gastric biopsy samples were analyzed by PNA probe-based PCR and compared with other H pylori detection methods, including hematoxylin and eosin (HE) and Warthin-Starry (WS) staining, immunohistochemistry (IHC). A2142G/A2143G mutation of the 23S rRNA gene was tested by dual priming oligonucleotide (DPO)-based PCR and Sanger sequencing to evaluate PNA probe-based PCR. RESULTS: Among 271 cases that were positive for H pylori on IHC which was considered as a standard method, 264 cases (97.4%) and 259 cases (95.6%) were positively detected by HE/WS and PNA probe-based qPCR, respectively. Of 100 H pylori-positive patients tested by IHC, H pylori was detected in 93 cases (93.0%) by PNA probe-based PCR, 86 cases (86.0%) by DPO-based PCR, and 93 cases (93.0%) by conventional PCR. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PNA probe-based qPCR were 93.0%, 94.9%, 93.9%, 94.9%, and 93.0%, respectively, which were all higher than those of DPO-based PCR. When Sanger sequencing was determined as a standard method to detect A2142G/A2143G mutations, the sensitivity of the PNA- and DPO-based methods was 100% and 94.4%, respectively, and the specificity was 100% for both methods. CONCLUSION: PNA probe-based qPCR is an appropriate method for detecting H pylori and the clarithromycin resistance-associated mutation type.


Assuntos
Antibacterianos/farmacologia , Claritromicina/farmacologia , Farmacorresistência Bacteriana , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Testes de Sensibilidade Microbiana/métodos , Técnicas de Diagnóstico Molecular/métodos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Humanos , Sondas de Oligonucleotídeos/genética , Ácidos Nucleicos Peptídicos/genética , RNA Ribossômico 23S/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Temperatura de Transição
8.
Microb Pathog ; 135: 103614, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31255726

RESUMO

Helicobacter pylori is an important etiological factor involved in chronic gastritis, peptic ulcer, and gastric cancer. There are currently no optimal preventive or therapeutic interventions for H. pylori infection. H. pylori survives in the stomach by sensing and adapting to the highly acidic environment by using the two-component signal transduction system that contains the most widely known gastric acid receptor, ArsRS (which is composed of ArsS and ArsR). This study aimed to identify peptides that antagonize the acid-sensing domain of H. pylori ArsS. These peptides could be used to block the acid-sensing signal and thereby hinder H. pylori adaption to acidic environments to prevent its survival. Using proSite, the functional domains (including the N-terminal acid-sensing domain) of H. pylori J99 ArsS were predicted. The purified recombinant ArsS N-terminal acid-sensing protein (P-ArsS-A) was used as the target in a panning protocol in which peptides from the Ph.D.-7 Phage Display Peptide Library that could bind to P-ArsS-A were identified. As a result, eight phage clones that could specifically bind to P-ArsS-A were obtained and five amino acid sequences were identified, including P03 (MMSYPKH) and P06 (LTPMPNW). An in vitro minimum inhibitory concentration (MIC) evaluation showed that P03 and P06 significantly inhibited the growth of H. pylori J99. The MIC of P03 was 8 µM, and the MIC of P06 was >16 µM, indicating that P03 is a stronger inhibitor compared to P06. This was confirmed by colony counting on blood agar plates after P03 and P06 administration. Using homology modeling and molecular docking analysis, it was shown that P03 and P06 could bind to the ArsS N-terminal domain, and there were four shared binding sites: TYR25, ASN39, ARG73, and GLU74. Additionally, one hydrogen bond was found between P03 and ArsS, which is more cohesive than other forms of bonding (van der Waals force, other non-covalent bonds).


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Helicobacter pylori/metabolismo , Biblioteca de Peptídeos , Peptídeos/isolamento & purificação , Ácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Peptídeos/imunologia , Proteínas Recombinantes , Transdução de Sinais/fisiologia
9.
PLoS One ; 14(5): e0217689, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150460

RESUMO

Despite the successful application of LNA/2'OMe-FISH procedures for bacteria detection, there is a lack of knowledge on the properties that affect hybridization. Such information is crucial for the rational design of protocols. Hence, this work aimed to evaluate the effect of three essential factors on the LNA/2'OMe hybridization step-hybridization temperature, NaCl concentration and type and concentration of denaturant (formamide, ethylene carbonate and urea). This optimization was performed for 3 Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Citrobacter freundii) and 2 Gram-positive bacteria (Enterococcus faecalis and Staphylococcus epidermidis), employing the response surface methodology and a Eubacteria probe. In general, it was observed that a high NaCl concentration is beneficial (from 2 M to 5 M), regardless of the denaturant used. Urea, formamide and ethylene carbonate are suitable denaturants for LNA/2'OMe-FISH applications; but urea provides higher fluorescence intensities among the different bacteria, especially for gram-positive bacteria and for P. aeruginosa. However, a unique optimal protocol was not found for all tested bacteria. Despite this, the results indicate that a hybridization solution with 2 M of urea and 4 M of NaCl would be a proper starting point. Furthermore, a hybridization temperature around 62°C, for 14 bp probes with LNA monomers at every third position of 2'OMe and 64% of GC content, should be use in initial optimization of new LNA/2'OMe-FISH protocols.


Assuntos
Citometria de Fluxo/métodos , Hibridização in Situ Fluorescente/métodos , Oligonucleotídeos/química , RNA/genética , Citrobacter freundii/genética , Citrobacter freundii/isolamento & purificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Hibridização de Ácido Nucleico , Oligonucleotídeos/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/isolamento & purificação
10.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180067

RESUMO

Proteins in a proteome can be identified from a sequence of K integers equal to the digitized volumes of subsequences with L residues from the primary sequence of a stretched protein. Exhaustive computations on the proteins of Helicobacter pylori (UniProt id UP000000210) with L and K in the range 4-8 show that approx. 90% of the proteins can be identified uniquely in this manner. This computational result can be translated into practice with a nanopore, an emerging technology that does not require analyte immobilization, proteolysis or labeling. Unlike other methods, most of which focus on a specific target protein, nanopore-based methods enable the identification of multiple proteins from a sample in a single run. Recent work by Kennedy, Kolmogorov and associates shows that the blockade current due to a protein molecule translocating through a nanopore is roughly proportional to one or more contiguous residues. The present study points to a modified version in which the volumes of subsequences (rather than of single residues) may be obtained by integrating the blockade current due to L contiguous residues. The advantages arising from this include lower detector bandwidth, elimination of the homopolymer problem and reduced noise. Because an identifier is based on near as well as distant (up to 2KL-L) residues, this approach uses more global information than an approach based on single residues and short-range correlations. The results of the study, which are available in a data supplement, are discussed in detail. Potential implementation issues are addressed.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Helicobacter pylori/genética , Modelos Estatísticos , Mapeamento de Peptídeos/estatística & dados numéricos , Proteoma/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos , Proteínas de Bactérias/genética , Bases de Dados de Proteínas , Helicobacter pylori/química , Nanoporos , Fragmentos de Peptídeos/análise , Mapeamento de Peptídeos/métodos , Proteoma/genética
11.
BMC Infect Dis ; 19(1): 546, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226948

RESUMO

BACKGROUND: The effectiveness of Helicobacter pylori first-line treatment has decreased drastically with the rise of strains resistant to clarithromycin. Therapy failure has also been described in patients with infections by strains with dissimilar antimicrobial susceptibilities. The present study aims to estimate the prevalence of resistance and heteroresistance to clarithromycin in H. pylori isolates from antrum and corpus of Colombian patients. METHODS: The study material included 126 isolates from antrum and corpus biopsies from 63 symptomatic patients over 18 years old who had a gastric endoscopy performed on them between June 2014 to August 2016. PCR amplification and sequencing of the H. pylori 23S rDNA gene was performed to determine the presence of mutations associated with clarithromycin resistance. Random amplified polymorphic DNA analysis was implemented in cases of resistance and heteroresistance. RESULTS: The overall frequency of resistance to clarithromycin was 38.1% (24/63 patients), of which 19 patients had resistant isolates in both stomach segments (14 with A2143G mutation and 5 with A2142G mutation), and 5 patients had a heteroresistant status. The remaining 61.9% (39/63 patients) presented only susceptible isolates. DNA fingerprinting analysis showed different patterns in 4/22 paired isolates. CONCLUSIONS: The high prevalence of H. pylori clarithromycin-resistance obtained (> 15%) constitutes an alert for gastroenterologists and suggests the need for reconsideration of the current eradication regimen for H. pylori in the studied population. The data show that heteroresistance status is an additional factor to be considered in the assessment of resistance. In consequence, it is advisable to examine at least two biopsies from different gastric segments.


Assuntos
Técnicas de Tipagem Bacteriana , Claritromicina/uso terapêutico , Farmacorresistência Bacteriana/genética , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Helicobacter pylori , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Claritromicina/farmacologia , Colômbia/epidemiologia , Feminino , Genótipo , Técnicas de Genotipagem , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase/métodos , Prevalência , Antro Pilórico/microbiologia , Antro Pilórico/patologia , Estômago/microbiologia , Estômago/patologia , Adulto Jovem
12.
PLoS Biol ; 17(5): e3000231, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31048876

RESUMO

Lifelong infection of the gastric mucosa by Helicobacter pylori can lead to peptic ulcers and gastric cancer. However, how the bacteria maintain chronic colonization in the face of constant mucus and epithelial cell turnover in the stomach is unclear. Here, we present a new model of how H. pylori establish and persist in stomach, which involves the colonization of a specialized microenvironment, or microniche, deep in the gastric glands. Using quantitative three-dimensional (3D) confocal microscopy and passive CLARITY technique (PACT), which renders tissues optically transparent, we analyzed intact stomachs from mice infected with a mixture of isogenic, fluorescent H. pylori strains with unprecedented spatial resolution. We discovered that a small number of bacterial founders initially establish colonies deep in the gastric glands and then expand to colonize adjacent glands, forming clonal population islands that persist over time. Gland-associated populations do not intermix with free-swimming bacteria in the surface mucus, and they compete for space and prevent newcomers from establishing in the stomach. Furthermore, bacterial mutants deficient in gland colonization are outcompeted by wild-type (WT) bacteria. Finally, we found that host factors such as the age at infection and T-cell responses control bacterial density within the glands. Collectively, our results demonstrate that microniches in the gastric glands house a persistent H. pylori reservoir, which we propose replenishes the more transient bacterial populations in the superficial mucosa.


Assuntos
Mucosa Gástrica/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Microscopia Confocal/métodos , Animais , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Contagem de Colônia Microbiana , Feminino , Mucosa Gástrica/efeitos dos fármacos , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Especificidade da Espécie , Linfócitos T/efeitos dos fármacos
13.
Microbiol Immunol ; 63(6): 199-205, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31045263

RESUMO

Success in eradication of Helicobacter pylori is declining globally because H. pylori has developed resistance against most of the antibiotics proposed for eradication regimens, mainly through point mutations. The present study included 200 patients with dyspepsia attending Taif Hospital. Gastric biopsies were obtained during gastroscopy and subjected to rapid urease testing. Molecular methods were used to confirm diagnoses of H. pylori infection and to identify resistance gene variants of four antibiotics; namely, clarithromycin, metronidazole, fluoroquinolones and tetracycline (23S rRNA, gyrA, rdxA and 16S rRNA respectively). Of all investigated patients, Molecular diagnoses were made in 143 of all investigated patients; thus, the prevalence was .5%. The overall rate of resistance to clarithromycin among the H. pylori-positive patients was high (39.9%) and the rate of resistance significantly greater (48.2%) among the secondary resistance group, secondary resistance being defined as resistance as a result of previous exposure to the relevant antibiotic. The rate of resistance to fluoroquinolones was considered moderate; the difference in rate of resistance between the primary and secondary resistance groups (8.4% and 9.5%, respectively) was not significant Also, there was a low prevalence of both primary and the secondary tetracycline resistance in the study cohort. In contrast, the prevalence of metronidazole resistance was considered high with no significant difference between the two resistance groups. H. pylori showed an increased prevalence of resistance to all four of the commonly used therapeutic agents. Thus, eradication therapy should be based on the regional results of susceptibility testing. Moreover, treatment tailored according to individually determined H. pylori susceptibility may be a reasonable future goal.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Patologia Molecular , Adulto , Idoso , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Claritromicina/farmacologia , Estudos de Coortes , DNA Girase/genética , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Fluoroquinolonas/farmacologia , Infecções por Helicobacter/epidemiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Humanos , Masculino , Metronidazol/farmacologia , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Epidemiologia Molecular , Nitrorredutases/genética , Prevalência , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Arábia Saudita/epidemiologia , Tetraciclina/farmacologia , Adulto Jovem
14.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31061142

RESUMO

Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.


Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter pylori/citologia , Helicobacter pylori/crescimento & desenvolvimento , Estômago/patologia , Animais , Aderência Bacteriana , Doença Crônica , Feminino , Infecções por Helicobacter/patologia , Helicobacter pylori/genética , Humanos , Camundongos Endogâmicos C57BL , Antro Pilórico/microbiologia , Antro Pilórico/patologia , Estômago/microbiologia
15.
Helicobacter ; 24(4): e12587, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31062466

RESUMO

BACKGROUND: Helicobacter pylori represents an interesting model of bacterial pathogenesis given that most infections are asymptomatic, while a minority of infections cause severe gastric disease. H pylori strain B128 7.13 is used extensively to understand H pylori pathophysiology. Due to extensive restriction-modification systems, the fact that only some H pylori strains are naturally transformable, the inability of common plasmid and transposon vectors to replicate in this bacterium, as well as the limited number of antibiotic cassettes that are functional in H pylori, there are relatively few genetic tools for the mutagenesis of this bacterium. MATERIALS AND METHODS: Here, we use PacBio and Illumina sequencing to reveal the complete genome sequence of H pylori B128 7.13. Furthermore, we describe a system to generate markerless and scarless mutations on the H pylori chromosome using the counter-selection marker, galactokinase from Escherichia coli. RESULTS: We show that this mutagenesis strategy can be used to generate in-frame insertions, gene deletions, and multiple independent mutations in B128 7.13. Using the closed genome as a reference, we also report the absence of second site chromosomal mutations and/or rearrangements in our mutagenized strains. We compare the genome sequence of H pylori B128 7.13 with a closely related strain, H pylori B8, and reveal one notable region of difference, which is a 1430 bp insertion encoding a H pylori-specific DUF874 family protein of unknown function. CONCLUSIONS: This article reports the closed genome of the important H pylori B128 7.13 strain and a mutagenesis method that can be adopted by researchers as an alternative strategy to generate isogenic mutants of H pylori in order to further our understanding of this bacterium.


Assuntos
Técnicas Genéticas , Genoma Bacteriano , Helicobacter pylori/genética , Sequência de Bases , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Helicobacter pylori/isolamento & purificação , Humanos , Mutagênese , Mutação , Sequenciamento Completo do Genoma
16.
Acta Med Indones ; 51(1): 34-41, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31073104

RESUMO

BACKGROUND: early detection of H. pylori is essential to prevent the development of infections into gastric malignancies. The coccoid form of H. pylori is difficult to detect either by culture or histopathology; however, it can be detected using molecular methods, such as real-time PCR. The study was expected to provide new information on the development of H. pylori detection. METHODS: a cross-sectional study was conducted at the Gastrointestinal Endoscopy Center of Cipto Mangunkusumo Hospital between October 2016 and August 2017. The sampling method used was consecutive sampling. Biopsy from gastric antrum and corpus were performed in 64 patients. We collected 2 specimens from each site to be examined using real-time PCR and histopathology. Initially, we conducted real-time PCR optimization followed by application of clinical samples from gastric biopsy. Data analysis using McNemar's χ2 and Kappa tests. RESULTS: the real-time PCR showed 25% positivity, while the positive proportion of histopathological examination was 14%. Real-time PCR has a sensitivity and specificity 88.9% dan 85.5%, respectively. The McNemar's χ2 test showed that there is significantly different (p=0.039) between the two tests; kappa value (p=0.561).  Conclusion: the real-time PCR examination is more sensitive than histopathology. This technique can improve diagnosis by 11% compared to histopathological examination.


Assuntos
Dispepsia/microbiologia , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/genética , RNA Ribossômico 16S/genética , Adulto , Idoso , Estudos Transversais , Feminino , Gastroscopia , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Estômago/microbiologia , Estômago/patologia , Adulto Jovem
17.
Helicobacter ; 24(4): e12597, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111585

RESUMO

BACKGROUND: Since "Helicobacter pylori (H. pylori) infection" was set as the indication in the Japanese Society for Helicobacter Research (JSHR) Guidelines 2009, eradication treatment for H. pylori gastritis is covered under insurance since 2013 in Japan, and the number of H. pylori eradication has rapidly increased. Under such circumstances, JSHR has made the third revision to the "Guidelines for diagnosis and treatment of H. pylori infection" for the first time in 7 years. METHODS: The Guideline Committee held 10 meetings. Articles published between the establishment of the 2009 Guidelines and March 2016 were reviewed and classified according to the evidence level; the statements were revised on the basis of this review. After inviting public comments, the revised statements were finalized using the Delphi method. RESULTS: There was no change in the basic policy that H. pylori infectious disease is an indication for eradication. Other diseases presumed to be associated with H. pylori infection were added as indications. Serum pepsinogen level, endoscopic examination, and X-ray examination were added to the diagnostic methods. The effects of 1-week triple therapy consisting of potassium-competitive acid blocker (P-CAB), amoxicillin, and clarithromycin have improved, and high eradication rates can also be expected with proton pump inhibitors (PPI) or P-CAB combined with amoxicillin and metronidazole. If the susceptibility test is not performed, the triple PPI or P-CAB/amoxicillin/metronidazole therapy should be chosen, because the PPI/amoxicillin/metronidazole combination demonstrated a significantly higher eradication rate than PPI/amoxicillin/clarithromycin. In the proposal for gastric cancer prevention, we divided gastric cancer prevention measures by age from adolescent to elderly, who are at an increased gastric cancer risk, and presented measures for gastric cancer prevention primarily based on H. pylori eradication. CONCLUSION: We expect the revised guidelines to facilitate appropriate interventions for patients with H. pylori infection and accomplish its eradication and prevention of gastric cancer.


Assuntos
Antibacterianos/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Guias de Prática Clínica como Assunto , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Amoxicilina/uso terapêutico , Farmacorresistência Bacteriana , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/fisiologia , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Quinolonas/uso terapêutico , Adulto Jovem
18.
Helicobacter ; 24(4): e12595, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111610

RESUMO

BACKGROUND: Antimicrobial resistance is a global public health problem, particularly in low- and middle-income countries (LMICs), where antibiotics are often obtained without a prescription. H. pylori antimicrobial resistance patterns are informative for patient care and gastric cancer prevention programs, have been shown to correlate with general antimicrobial consumption, and may guide antimicrobial stewardship programs in LMICs. We report H. pylori resistance and antimicrobial utilization patterns for western Honduras, representative of rural Central America. METHODS: In the context of the western Honduras gastric cancer epidemiology initiative, gastric biopsies from 189 patients were studied for culture and resistance patterns. Antimicrobial utilization was investigated for common H. pylori treatment regimens from regional public (7 antimicrobials) and national private (4 antimicrobials) data, analyzed in accordance with WHO anatomical therapeutic chemical defined daily doses (DDD) method and expressed as DDD/1000 inhabitants per day (DID) and per year (DIY). RESULTS: H. pylori was successfully cultured from 116 patients (56% males, mean age: 54), and nearly all strains were cagA+ and vacAs1m1+ positive (99% and 90.4%, respectively). Unexpectedly, high resistance was noted for levofloxacin (20.9%) and amoxicillin (10.7%), while metronidazole (67.9%) and clarithromycin (11.2%) were similar to data from Latin America. Significant associations with age, gender, or histology were not noted, with the exception of levofloxacin (28%, P = 0.01) in those with histology limited to non-atrophic gastritis. Total antimicrobial usage in western Honduras of amoxicillin (17.3 DID) and the quinolones had the highest relative utilizations compared with other representative nations. CONCLUSIONS: We observed significant H. pylori resistance to amoxicillin and levofloxacin in the context of high community antimicrobial utilization. This has implications in Central America for H. pylori treatment guidelines as well as antimicrobial stewardship programs.


Assuntos
Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Adulto , Idoso , Amoxicilina/uso terapêutico , América Central , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Levofloxacino/uso terapêutico , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade
19.
Acta Microbiol Immunol Hung ; 66(3): 399-411, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096759

RESUMO

The aim of this study was to evaluate the prevalence of Helicobacter pylori genotypes (vacA and cagPAI) directly in gastric biopsy specimens in patients with gastric diseases in Belarus. Gastric biopsies were collected from 461 patients with different gastrointestinal disorders: superficial gastritis (287 subjects), atrophy gastritis (59 subjects), erosive gastritis (47 subjects), duodenal ulcer disease (54 subjects), and stomach ulcer (14 subjects). PCR-based genotyping was used to detect s1a, s1b, s2, m1a, m1b, m2, cagM, cagA, and cagT genes. Overall prevalence of vacA s1a allele was 60.5% followed by m2 (47.1%) and m1a (37.5%). The analysis of data showed that genotype s1a/m1a was significantly more prevalent in patients with duodenal ulcer (21.4% vs. 45.1%, OR = 3.0, 95% CI = 1.5-6.1). The cagA gene was found with a high incidence in most patients with inflammatory diseases of stomach and duodenum. There was a significant increase in the frequency of cagT in patients with duodenal ulcer as compared to superficial gastritis. A high cagM prevalence was found in patients with atrophy gastritis and duodenal ulcer disease. All three island genes of pathogenicity of cagPAI are more often detected in patients with duodenal ulcer, which increases the risk of developing duodenal ulcer by 4.5 times.


Assuntos
Duodenopatias/microbiologia , Genótipo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/classificação , Helicobacter pylori/genética , Gastropatias/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Duodenopatias/epidemiologia , Duodenopatias/patologia , Feminino , Técnicas de Genotipagem , Infecções por Helicobacter/epidemiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prevalência , República de Belarus/epidemiologia , Gastropatias/epidemiologia , Gastropatias/patologia , Resultado do Tratamento , Fatores de Virulência/genética , Adulto Jovem
20.
Nat Commun ; 10(1): 2273, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118420

RESUMO

The human pathogen Helicobacter pylori displays extensive genetic diversity. While H. pylori is known to evolve during infection, population dynamics inside the gastric environment have not been extensively investigated. Here we obtained gastric biopsies from multiple stomach regions of 16 H. pylori-infected adults, and analyze the genomes of 10 H. pylori isolates from each biopsy. Phylogenetic analyses suggest location-specific evolution and bacterial migration between gastric regions. Migration is significantly more frequent between the corpus and the fundus than with the antrum, suggesting that physiological differences between antral and oxyntic mucosa contribute to spatial partitioning of H. pylori populations. Associations between H. pylori gene polymorphisms and stomach niches suggest that chemotaxis, regulatory functions and outer membrane proteins contribute to specific adaptation to the antral and oxyntic mucosa. Moreover, we show that antibiotics can induce severe population bottlenecks and likely play a role in shaping the population structure of H. pylori.


Assuntos
Adaptação Biológica/genética , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Taxa de Mutação , Adulto , Idoso , Biópsia , Quimiotaxia/genética , Mucosa Gástrica/patologia , Genoma Bacteriano/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Humanos , Pessoa de Meia-Idade , Filogenia , Polimorfismo Genético
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