scholarly journals Tetracycline-Resistant Clinical Helicobacter pylori Isolates with and without Mutations in 16S rRNA-Encoding Genes

2005 ◽  
Vol 49 (2) ◽  
pp. 578-583 ◽  
Author(s):  
Jeng Yih Wu ◽  
Jae J. Kim ◽  
Rita Reddy ◽  
W. M. Wang ◽  
David Y. Graham ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Binding Sites ◽  
Tetracycline Resistance ◽  
Nucleotide Substitutions ◽  
Single Nucleotide ◽  
Resistant Strains ◽  
H Pylori ◽  
Encoding Genes

ABSTRACT Tetracycline-resistant Helicobacter pylori strains have been increasingly reported worldwide. However, only a small number of tetracycline-resistant strains have been studied with regard to possible mechanisms of resistance and those studies have focused on mutations in the tetracycline binding sites of 16S rRNA-encoding genes. We here report studies of 41 tetracycline-resistant H. pylori strains (tetracycline MICs, 4 to 32 μg/ml) from North America (n = 12) and from East Asia (n = 29). DNA sequence analyses of 16S rRNA-encoding genes revealed that 22 (54%) of the resistant isolates carried one of five different single-nucleotide substitutions (CGA, GGA, TGA, AGC, or AGT) at the putative tetracycline binding site (AGA965-967). Single-nucleotide substitutions were associated with reduced ribosomal binding and with slightly increased tetracycline MICs (1 to 2 μg/ml). The 19 tetracycline-resistant isolates with no detectable mutations in the tetracycline binding site had normal tetracycline-ribosome binding. All tetracycline-resistant isolates, including those with and those without mutations in the tetracycline binding site, showed decreased accumulation of tetracycline. These results suggest that tetracycline resistance is multifactorial, involving alterations both in ribosomal binding and in membrane permeability.

scholarly journals 16S rRNA Mutations That Confer Tetracycline Resistance in Helicobacter pylori Decrease Drug Binding in Escherichia coli Ribosomes

2005 ◽  
Vol 187 (11) ◽  
pp. 3708-3712 ◽  
Author(s):  
Lisa Nonaka ◽  
Sean R. Connell ◽  
Diane E. Taylor
Keyword(s):  
Escherichia Coli ◽  
Helicobacter Pylori ◽  
16S Rrna ◽  
Binding Site ◽  
Tetracycline Resistance ◽  
Drug Binding ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
E Coli ◽  
H Pylori

ABSTRACT Tetracycline resistance in clinical isolates of Helicobacter pylori has been associated with nucleotide substitutions at positions 965 to 967 in the 16S rRNA. We constructed mutants which had different sequences at 965 to 967 in the 16S rRNA gene present on a multicopy plasmid in Escherichia coli strain TA527, in which all seven rrn genes were deleted. The MICs for tetracycline of all mutants having single, double, or triple substitutions at the 965 to 967 region that were previously found in highly resistant H. pylori isolates were higher than that of the mutant exhibiting the wild-type sequence of tetracycline-susceptible H. pylori. The MIC of the mutant with the 965TTC967 triple substitution was 32 times higher than that of the E. coli mutant with the 965AGA967 substitution present in wild-type H. pylori. The ribosomes extracted from the tetracycline-resistant E. coli 965TTC967 variant bound less tetracycline than E. coli with the wild-type H. pylori sequence at this region. The concentration of tetracycline bound to the ribosome was 40% that of the wild type. The results of this study suggest that tetracycline binding to the primary binding site (Tet-1) of the ribosome at positions 965 to 967 is influenced by its sequence patterns, which form the primary binding site for tetracycline.

scholarly journals Emergence of Tetracycline Resistance in Helicobacter pylori: Multiple Mutational Changes in 16S Ribosomal DNA and Other Genetic Loci

2002 ◽  
Vol 46 (12) ◽  
pp. 3940-3946 ◽  
Author(s):  
Daiva Dailidiene ◽  
M. Teresita Bertoli ◽  
Jolanta Miciuleviciene ◽  
Asish K. Mukhopadhyay ◽  
Giedrius Dailide ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Ribosomal Dna ◽  
Tetracycline Resistance ◽  
Clinical Isolates ◽  
Nucleotide Substitutions ◽  
16S Ribosomal Dna ◽  
Pcr Products ◽  
Great Genetic Diversity ◽  
H Pylori ◽  
Derivatives Of

ABSTRACT Tetracycline is useful in combination therapies against the gastric pathogen Helicobacter pylori. We found 6 tetracycline-resistant (Tetr) strains among 159 clinical isolates (from El Salvador, Lithuania, and India) and obtained the following four results: (i) 5 of 6 Tetr isolates contained one or two nucleotide substitutions in one part of the primary tetracycline binding site in 16S rRNA (AGA965-967 [Escherichia coli coordinates] changed to gGA, AGc, guA, or gGc [lowercase letters are used to represent the base changes]), whereas the sixth (isolate Ind75) retained AGA965-967; (ii) PCR products containing mutant 16S ribosomal DNA (rDNA) alleles transformed recipient strains to Tetr phenotypes, but transformants containing alleles with single substitutions (gGA and AGc) were less resistant than their Tetr parents; (iii) each of 10 Tetr mutants of reference strain 26695 (in which mutations were induced with metronidazole, a mutagenic anti-H. pylori agent) contained the normal AGA965-967 sequence; and (iv) transformant derivatives of Ind75 and of one of the Tetr 26695 mutants that had acquired mutant rDNA alleles were resistant to tetracycline at levels higher than those to which either parent strain was resistant. Thus, tetracycline resistance in H. pylori results from an accumulation of changes that may affect tetracycline-ribosome affinity and/or other functions (perhaps porins or efflux pumps). We suggest that the rarity of tetracycline resistance among clinical isolates reflects this need for multiple mutations and perhaps also the deleterious effects of such mutations on fitness. Formally equivalent mutations with small but additive effects are postulated to contribute importantly to traits such as host specificity and virulence and to H. pylori's great genetic diversity.

scholarly journals Effects of 16S rRNA Gene Mutations on Tetracycline Resistance in Helicobacter pylori

2003 ◽  
Vol 47 (9) ◽  
pp. 2984-2986 ◽  
Author(s):  
Monique M. Gerrits ◽  
Marco Berning ◽  
Arnoud H. M. Van Vliet ◽  
Ernst J. Kuipers ◽  
Johannes G. Kusters
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Base Pair ◽  
Gene Mutations ◽  
Tetracycline Resistance ◽  
Rrna Gene ◽  
H Pylori ◽  
High Level ◽  
Double Base

ABSTRACT The triple-base-pair 16S rDNA mutation AGA926-928→TTC mediates high-level tetracycline resistance in Helicobacter pylori. In contrast, single- and double-base-pair mutations mediated only low-level tetracycline resistance and decreased growth rates in the presence of tetracycline, explaining the preference for the TTC mutation in tetracycline-resistant H. pylori isolates.

scholarly journals Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline

2002 ◽  
Vol 184 (8) ◽  
pp. 2131-2140 ◽  
Author(s):  
Catharine A. Trieber ◽  
Diane E. Taylor
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Tetracycline Resistance ◽  
Clinical Isolates ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Nucleotide Substitutions ◽  
The 16S Rrna Gene

ABSTRACT Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline. Resistance to tetracycline has recently emerged in H. pylori. The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 μg/ml) were determined and compared to the consensus H. pylori 16S rRNA sequence. One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions. Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains. 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance. Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H. pylori 26695. The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates. The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype. Serial passage of several H. pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 μg/ml. These mutants all had a deletion of G942 in the 16S rRNA genes. The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H. pylori.

scholarly journals Real-Time PCR Screening for 16S rRNA Mutations Associated with Resistance to Tetracycline in Helicobacter pylori

2005 ◽  
Vol 49 (8) ◽  
pp. 3166-3170 ◽  
Author(s):  
Erik Glocker ◽  
Marco Berning ◽  
Monique M. Gerrits ◽  
Johannes G. Kusters ◽  
Manfred Kist
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
Tetracycline Resistance ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Screening ◽  
H Pylori

ABSTRACT The effectiveness of recommended first-line therapies for Helicobacter pylori infections is decreasing due to the occurrence of resistance to metronidazole and/or clarithromycin. Quadruple therapies, which include tetracycline and a bismuth salt, are useful alternative regimens. However, resistance to tetracycline, mainly caused by mutations in the 16S rRNA genes (rrnA and rrnB) affecting nucleotides 926 to 928, are already emerging and can impair the efficacies of such second-line regimens. Here, we describe a novel real-time PCR for the detection of 16S rRNA gene mutations associated with tetracycline resistance. Our PCR method was able to distinguish between wild-type strains and resistant strains exhibiting single-, double, or triple-base-pair mutations. The method was applicable both to DNA extracted from pure cultures and to DNA extracted from fresh or frozen H. pylori-infected gastric biopsy samples. We therefore conclude that this real-time PCR is an excellent method for determination of H. pylori tetracycline resistance even when live bacteria are no longer available.

scholarly journals 16S rRNA Mutation-Mediated Tetracycline Resistance in Helicobacter pylori

2002 ◽  
Vol 46 (9) ◽  
pp. 2996-3000 ◽  
Author(s):  
Monique M. Gerrits ◽  
Marcel R. de Zoete ◽  
Niek L. A. Arents ◽  
Ernst J. Kuipers ◽  
Johannes G. Kusters
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
Resistance Genes ◽  
Base Pair ◽  
Tetracycline Resistance ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Tetracycline Resistance Genes ◽  
Base Pair Substitution ◽  
H Pylori

ABSTRACT Most Helicobacter pylori strains are susceptible to tetracycline, an antibiotic commonly used for the eradication of H. pylori. However, an increase in incidence of tetracycline resistance in H. pylori has recently been reported. Here the mechanism of tetracycline resistance of the first Dutch tetracycline-resistant (Tetr) H. pylori isolate (strain 181) is investigated. Twelve genes were selected from the genome sequences of H. pylori strains 26695 and J99 as potential candidate genes, based on their homology with tetracycline resistance genes in other bacteria. With the exception of the two 16S rRNA genes, none of the other putative tetracycline resistance genes was able to transfer tetracycline resistance. Genetic transformation of the Tets strain 26695 with smaller overlapping PCR fragments of the 16S rRNA genes of strain 181, revealed that a 361-bp fragment that spanned nucleotides 711 to 1071 was sufficient to transfer resistance. Sequence analysis of the 16S rRNA genes of the Tetr strain 181, the Tets strain 26695, and four Tetr 26695 transformants showed that a single triple-base-pair substitution, AGA926-928→TTC, was present within this 361-bp fragment. This triple-base-pair substitution, present in both copies of the 16S rRNA gene of all our Tetr H. pylori transformants, resulted in an increased MIC of tetracycline that was identical to that for the Tetr strain 181.

scholarly journals Genotyping pattern of the vacuolating cytotoxin A and cytotoxin associated gene A of the Helicobacter pylori strains detected in fecal samples of household dogs

2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Asieh Bolandi ◽  
Saam Torkan ◽  
Iman Alavi
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Fecal Samples ◽  
The Status ◽  
Cytotoxin Associated Gene A ◽  
H Pylori

In despite of the high clinical impact of Helicobacter pylori, its exact sources and routes of transmission are unknown. Dogs may play an imperative role in the transmission of H. pylori to humans. The current investigation was done to study the status of vacA and cagA genotypes in the H. pylori strains of dogs. One-hundred and fifty fecal samples were collected from healthy and complicated household dogs. Genomic DNA was extracted from fecal samples and presence of 16S rRNA gene was studied using the PCR amplification. Distribution of vacA and cagA genotypes were studied by the multiplex PCR. Thirteen out of 150 fecal samples (8.66%) were positive for H. pylori 16S rRNA gene. Prevalence of H. pylori in healthy and complicated dogs were 5.55% and 8.57%, respectively. Male had the higher prevalence of H. pylori (P=0.038). The most commonly detected genotypes among the H. pylori strains were vacAs1A (61.53%), cagA (38.46%), vacAm1a (38.46%), vacAs2 (30.76%) and vacAm2 (30.76%). The most commonly detected combined genotypes were s1aCagA (30.76%), s1am1a (23.07%), s2m1a (23.07%) and s2CagA (23.07%). Iranian household dogs harbor H. pylori in their fecal samples similar in genotypes of the vacA and cagA alleles which suggest that complicated and even healthy dogs may be the latent host of the H. pylori and its genotypes. However, supplementary studies are required to found the exact role of dogs as a definitive host of the H. pylori.

scholarly journals In Vitro Analysis of Protein-Operator Interactions of the NikR and Fur Metal-Responsive Regulators of Coregulated Genes in Helicobacter pylori

2005 ◽  
Vol 187 (22) ◽  
pp. 7703-7715 ◽  
Author(s):  
Isabel Delany ◽  
Raffaele Ieva ◽  
Alice Soragni ◽  
Markus Hilleringmann ◽  
Rino Rappuoli ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Binding Site ◽  
Gene Networks ◽  
Iron Homeostasis ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Target Sequence ◽  
Gene Promoters ◽  
Direct Role ◽  
H Pylori

ABSTRACT Two important metal-responsive regulators, NikR and Fur, are involved in nickel and iron homeostasis and controlling gene expression in Helicobacter pylori. To date, they have been implicated in the regulation of sets of overlapping genes. We have attempted here dissection of the molecular mechanisms involved in transcriptional regulation of the NikR and Fur proteins, and we investigated protein-promoter interactions of the regulators with known target genes. We show that H. pylori NikR is a tetrameric protein and, through DNase I footprinting analysis, we have identified operators for NikR to which it binds with different affinities in a metal-responsive way. Mapping of the NikR binding site upstream of the urease promoter established a direct role for NikR as a positive regulator of transcription and, through scanning mutagenesis of this binding site, we have determined two subsites that are important for the binding of the protein to its target sequence. Furthermore, by alignment of the operators for NikR, we have shown that the H. pylori protein recognizes a sequence that is distinct from its well-studied orthologue in Escherichia coli. Moreover, we show that NikR and Fur can bind independently at distinct operators and also compete for overlapping operators in some coregulated gene promoters, adding another dimension to the previous suggested link between iron and nickel regulation. Finally, the importance of an interconnection between metal-responsive gene networks for homeostasis is discussed.

scholarly journals Vertical Helicobacter pylori transmission from Mongolian gerbil mothers to pups

2009 ◽  
Vol 58 (5) ◽  
pp. 656-662 ◽  
Author(s):  
Ichiro Oshio ◽  
Takako Osaki ◽  
Tomoko Hanawa ◽  
Hideo Yonezawa ◽  
Cynthia Zaman ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Gastric Mucosa ◽  
16S Rrna ◽  
Real Time ◽  
Mongolian Gerbil ◽  
Rt Pcr ◽  
Antibody Titres ◽  
Mother's Milk ◽  
Mother’S Milk ◽  
H Pylori

To identify the time frame and route of mother-to-child Helicobacter pylori infection, a Mongolian gerbil model was used. Four-week-old female Mongolian gerbils were infected with H. pylori, and then mated with uninfected males 2 months after infection. The offspring were sacrificed weekly after birth, and then serum, mother's milk from the stomach and gastric tissues were obtained from pups. Anti-H. pylori antibody titres were measured in sera and maternal milk using an ELISA. The stomach was cut in two in the sagittal plane, and then H. pylori colonization in mucosa was confirmed by culture and real-time RT-PCR in one specimen and by immunochemical staining in the other. Faeces and oral swabs were obtained from infected mothers, and H. pylori 16S rRNA was measured using real-time RT-PCR. H. pylori was not identified in cultures from the gastric mucosa of pups delivered by infected mothers, but H. pylori 16S rRNA was detected from 4 weeks after birth, suggesting that Mongolian gerbil pups become infected via maternal H. pylori transmission from 4 weeks of age. The anti-H. pylori antibody titre in sera of pups from infected mothers was maximum at 3 weeks of age and then rapidly decreased from 4 weeks of age. High antibody titres in mother's milk were detected during the suckling period, and GlcNAcα was detectable at 2–4 weeks of age, but disappeared as the offspring aged. Thus H. pylori seems to infect Mongolian gerbil pups from 4 weeks of age, in parallel with decreasing GlcNAcα expression in the gastric mucosa. These results suggested that H. pylori infection of Mongolian gerbil pups occurs via faecal–oral transmission from an infected mother.

scholarly journals Lack of Association between Helicobacter pylori Infection and Biliary Tract Diseases

2012 ◽  
Vol 61 (4) ◽  
pp. 319-322 ◽  
Author(s):  
SOMAYEH JAHANI SHERAFAT ◽  
ELAHE TAJEDDIN ◽  
MOHAMMAD REZA SEYYED MAJIDI ◽  
FARZAM VAZIRI ◽  
MASOUD ALEBOUYEH ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Biliary Tract ◽  
Helicobacter Pylori Infection ◽  
Rrna Gene ◽  
Biliary Tract Diseases ◽  
Hepatobiliary Diseases ◽  
H Pylori ◽  
Bile Samples

There are ambiguous results about the involvement of Helicobacter species in production of hepatobiliary diseases. This study was aimed to investigate any possible association between the presences of Helicobacter spp., their genotypes and occurrence of different biliary diseases. Cultures of 102 bile samples for Helicobacter spp. did not show any growth, but the presence of Helicobacter genus specific DNA (16s rRNA gene) was detected in 3.92% of them. No significant association was found between development of the diseases and presence of the bacteria. All the Helicobacter genus positive samples belonged to H. pylori species and showed vacA+ (s1/m2), cagA- genotypes.

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