Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pylori clinical isolates

BACKGROUND: In this study, we evaluated the prevalence of primary resistance of Brazilian H. pylori isolates to metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone. In addition, the vacA, iceA, cagA and cagE genotypes of strains isolated from Brazilian patients were determined and associated with clinical data in an effort to correlate these four virulence markers and antibiotic resistance. METHODS: H. pylori was cultured in 155 H. pylori-positive patients and MICs for metronidazole, clarithromycin, amoxicillin, tetracycline, and furazolidone were determined by the agar dilution method. Genomic DNA was extracted, and allelic variants of vacA, iceA, cagA and cagE were identified by the polymerase chain reaction. RESULTS: There was a strong association between the vacA s1/cagA -positive genotype and peptic ulcer disease (OR = 5.42, 95% CI 2.6–11.3, p = 0.0006). Additionally, infection by more virulent strains may protect against GERD, since logistic regression showed a negative association between the more virulent strain, vacA s1/cagA-positive genotype and GERD (OR = 0.26, 95% CI 0.08–0.8, p = 0.03). Resistance to metronidazole was detected in 75 patients (55%), to amoxicillin in 54 individuals (38%), to clarithromycin in 23 patients (16%), to tetracycline in 13 patients (9%), and to furazolidone in 19 individuals (13%). No significant correlation between pathogenicity and resistance or susceptibility was detected when MIC values for each antibiotic were compared with different vacA, iceA, cagA and cagE genotypes. CONCLUSION: The analysis of virulence genes revealed a specific association between H. pylori strains and clinical outcome, furthermore, no significant association was detected among pathogenicity and resistance or susceptibility

Envolvimento dos genes rdxA e frxA de Helicobacter pylori na resistencia aos antibioticos metronidazol e furazolidona

Orientador: Ronilson Agnaldo MorenoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: A infecção por Helicobacter pylori é uma das mais comuns em todo o mundo estando relacionada ao câncer gástrico, gastrite crônica, úlceras, adenocarcinomas e linfomas gástricos. O metronidazol foi muito usado como componente de terapias contra H. pylori. No entanto metronidazol é um composto mutagênico e a resistência a esse antimicrobiano é muito comum. Isso estimulou o interesse em antimicrobianos que tivessem resistência incomum para H. pylori. No Brasil, onde o índice de resistência para metronidazol foi determinado em 42%, a furazolidona passou a ser usada nas terapias de erradicação. A furazolidona e o metronidazol, são compostos classificados como nitro-heterocíclicos ou nitroaromáticos e possuem mecanismos de ação similares. O mecanismo de resistência da H. pylori ao metronidazol foi relacionado com alterações nos genes NAD(P)H nitroredutase oxigênio insensível (rdxA) e NAD(P)H flavina oxidorre- dutase (fixA) produtores de nitroredutases que reduzem o metronidazol gerando produtos intermediários os quais tem ação tóxica. Devido a algumas linhagens apresentarem resistência simultânea para metronidazol e furazolidona foi sugerido que os mecanismos de resistência para essas drogas poderiam ser os mesmos. Entretanto linhagens.construídas em laboratório com os genes rdxA e frxA inativados não apresentaram resistência a furazolidona. Nesse estudo foi utilizado o método de transformação natural, feitos com os produtos de PCR dos genes rdxA e frxA de oito amostras brasileiras resistentes simultaneamente aos dois antimicrobianos para verificar o envolvimento dos genes rdxA e fdxA com a resistência ao metronidazol e a furazolidona. Das oito amostras utilizadas foram obtidos foram obtidos seis transformantes de diferentes produtos de PCR do gene rdxA, resistentes ao metronidazol. Não foram obtidos transformantes com o gene fixA resistentes ao metronidazol, nem transformantes resistentes a furazolidona com nenhum dos dois genes. Os resultados .obtidos nesse trabalho indicam que à resistência ao metronidazol esta relacionada ao gene rdxA , mas não ao frxA, e sugere que o gene rdxA influencia a concentração inibitória mínimade furazolidona, mas não confere resistênciaAbstract: Helicobacter Pylori infection is one of the most common infections worldwide and recognized as the major cause of peptic ulcer disease, risk factor for gastric adenocarcinoma and primary gastric Iymphoma. Metronidazole has often used in combination therapies against H. pylori. However, metronidazole is highly mutagenic and resistance to this drug is very common.These stimulated the use the altematives drugs that resistance was uncommon in H. pylori. In Brazil, where the levei of resistance for metronidazole was detected in 42%, the nitrofuran furazolidone passed to be one of the components therapies. Metronidazole and furazolidone are classified as nitroeterocyclic and nitroaromatic compounds, hence the modes of drug action are similar and nitroreduction is required for activation these. The mechanism of metronidazole resistance among H. pylori strains has been reIated to alterations in gene products having metronidazole nitroreductase activity, like the oxygen insensitiveNAD(p)H nitroredutase (RdxA), and NAD(p)H flavin oxidoredutase (FrxA). Due some strains present simultaneous resistance for furazolidone and metronidazole suggests that the resistance mechanismfor these drugs may be the same. However, frxA and rdxA knockout mutations constructed in laboratory reference strains are not resistant to furazolidone, which indicates that mutations in others genes were also need for resistance. We used natural transformation with PCR products of eigth brazilian clinical isolates, simultaneous resistant to this these two drugs, for verifify the involvement of the genes rdxA and ftxA with resistance to metronidazole and furazolidone drugs. Our results showed that metronidazole resistance are associated with rdxA gene, but not fixA, and suggest the involvement of rdxA gene in increased leveI of furazolidone minimum inhibitoryconcentration , but not coffer resistanceMestradoMestre em Farmacologi