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

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.2.578-583.2005 ◽

2005 ◽

Vol 49 (2) ◽

pp. 578-583 ◽

Cited By ~ 40

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.

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Mechanistic Understanding of the Interactions of Cationic Conjugated Oligo- and Polyelectrolytes with Wild-type and Ampicillin-resistant Escherichia coli

Scientific Reports ◽

10.1038/s41598-019-56946-2 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Ehsan Zamani ◽

Shyambo Chatterjee ◽

Taity Changa ◽

Cheryl Immethun ◽

Anandakumar Sarella ◽

...

Keyword(s):

Escherichia Coli ◽

Surface Charge ◽

Cell Envelope ◽

Drug Binding ◽

Outer Cell ◽

Binding Modes ◽

Wild Type ◽

Bacterial Surface ◽

E Coli ◽

Future Design

AbstractAn in-depth understanding of cell-drug binding modes and action mechanisms can potentially guide the future design of novel drugs and antimicrobial materials and help to combat antibiotic resistance. Light-harvesting π-conjugated molecules have been demonstrated for their antimicrobial effects, but their impact on bacterial outer cell envelope needs to be studied in detail. Here, we synthesized poly(phenylene) based model cationic conjugated oligo- (2QA-CCOE, 4QA-CCOE) and polyelectrolytes (CCPE), and systematically explored their interactions with the outer cell membrane of wild-type and ampicillin (amp)-resistant Gram-negative bacteria, Escherichia coli (E. coli). Incubation of the E. coli cells in CCOE/CCPE solution inhibited the subsequent bacterial growth in LB media. About 99% growth inhibition was achieved if amp-resistant E. coli was treated for ~3–5 min, 1 h and 6 h with 100 μM of CCPE, 4QA-CCOE, and 2QA-CCOE solutions, respectively. Interestingly, these CCPE and CCOEs inhibited the growth of both wild-type and amp-resistant E. coli to a similar extent. A large surface charge reversal of bacteria upon treatment with CCPE suggested the formation of a coating of CCPE on the outer surface of bacteria; while a low reversal of bacterial surface charge suggested intercalation of CCOEs within the lipid bilayer of bacteria.

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Antibacterial Activity of Geraniol in Combination with Standard Antibiotics against Staphylococcus aureus, Escherichia coli and Helicobacter pylori

Natural Product Communications ◽

10.1177/1934578x1801300701 ◽

2018 ◽

Vol 13 (7) ◽

pp. 1934578X1801300 ◽

Cited By ~ 1

Author(s):

Subrat Kumar Bhattamisra ◽

Chew Hui Kuean ◽

Lee Boon Chieh ◽

Vivian Lee Yean Yan ◽

Chin Koh Lee ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Helicobacter Pylori ◽

Antibacterial Activity ◽

Synergistic Effect ◽

Inhibitory Concentration ◽

Therapeutic Potential ◽

E Coli ◽

Checkerboard Assay ◽

H Pylori

The antibacterial activity of geraniol and its effect in combination with ampicillin, amoxicillin and clarithromycin against Staphylococcus aureus, Escherichia coli and Helicobacter pylori was tested. The minimum inhibitory concentrations (MICs) and combinatory effects of geraniol against the bacteria were assessed by using the modified broth microdilution and checkerboard assay, respectively. The combinatory effect is expressed as fractional inhibitory concentration index (FICI). The MIC of geraniol against S. aureus, E. coli and H. pylori was found to be 11200, 5600, and 7325 μg/mL, respectively. A significant synergistic effect was observed with geraniol and ampicillin against S. aureus with FICI in the range 0.19 to 0.32. Geraniol and ampicillin exhibited a partial synergistic effect against E. coli. A similar effect was observed with geraniol and clarithromycin against S. aureus. A partial synergistic effect was observed with clarithromycin and geraniol against H. pylori with the FICI value in the range 0.86 to 0.89. An additive effect was observed with geraniol and amoxicillin combination against H. pylori. However, the amoxicillin and clarithromycin dose was reduced by thirty-two fold when combined with geraniol against H. pylori. The anti- H. pylori effect of geraniol with clarithromycin and amoxicillin could be of potential interest in the treatment of H. pylori infection and associated ulcers in humans. Further, geraniol, in combination with other antibiotics, has substantial therapeutic potential against S. aureus and E.coli infection.

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Bacterial killing in gastric juice – effect of pH and pepsin on Escherichia coli and Helicobacter pylori

Journal of Medical Microbiology ◽

10.1099/jmm.0.46611-0 ◽

2006 ◽

Vol 55 (9) ◽

pp. 1265-1270 ◽

Cited By ~ 59

Author(s):

H. Zhu ◽

C. A. Hart ◽

D. Sales ◽

N. B. Roberts

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Gastric Juice ◽

Bacterial Killing ◽

E Coli ◽

Test Organisms ◽

H Pylori ◽

Ph Range ◽

K 12 ◽

Rough Mutant

The susceptibility of Escherichia coli and Helicobacter pylori to pH and the effect of pepsin-mediated proteolysis were investigated. This was to establish the relative importance of their bacterial killing properties in gastric juice. Solutions in the pH range 1.5–7.4 with or without pig pepsin A were used, together with seven gastric juice samples obtained from patients undergoing routine gastric collection. Escherichia coli C690 (a capsulate strain), E. coli K-12 (a rough mutant) and Helicobacter pylori E5 were selected as the test organisms. Suspensions of bacteria (1×106 E. coli ml−1 and 1×108 H. pylori ml−1) were pre-incubated with test solutions at 37 °C for up to 2 h, and then cultured to establish the effect on subsequent growth. Survival of bacteria was diminished at pHs of less than 3.5, whereas killing required a pH of less than 2.5. Pre-incubation with pig pepsin at 0.5, 1.0 and 2.0 mg ml−1 at pH 3.5 reduced viable counts by 100 % for E. coli 690 and E. coli K-12 after 100 min incubation. With H. pylori, the viable counts decreased to 50 % of the control after 20 min incubation in 1 mg pepsin ml−1 at pH 2.5, 3.0 and 3.5. The gastric juices showed bactericidal activity at pH 3.5, and the rate of killing was juice dependent, with complete death of E. coli 690 occurring between 5 and 40 min post-incubation. Thus, killing of E. coli and H. pylori occurs optimally at pHs of less than 2.5. At pH 3.5, little effect is observed, whereas addition of pepsin alone or in gastric juice causes a marked increase in bacterial susceptibility, suggesting an important role for proteolysis in the killing of bacteria.

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Effect of Host Species on RecG Phenotypes in Helicobacter pylori and Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.186.22.7704-7713.2004 ◽

2004 ◽

Vol 186 (22) ◽

pp. 7704-7713 ◽

Cited By ~ 21

Author(s):

Josephine Kang ◽

Don Tavakoli ◽

Ariane Tschumi ◽

Rahul A. Aras ◽

Martin J. Blaser

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Replication Forks ◽

E Coli ◽

Repeat Sequences ◽

Dna Repeat ◽

H Pylori ◽

Dna Repeat Sequences ◽

Prokaryotic Evolution ◽

Fundamental Mechanism

ABSTRACT Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.

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Helicobacter pylori DnaB helicase can bypass Escherichia coli DnaC function in vivo

Biochemical Journal ◽

10.1042/bj20050062 ◽

2005 ◽

Vol 389 (2) ◽

pp. 541-548 ◽

Cited By ~ 28

Author(s):

Rajesh K. Soni ◽

Parul Mehra ◽

Gauranga Mukhopadhyay ◽

Suman Kumar Dhar

Keyword(s):

Escherichia Coli ◽

Helicobacter Pylori ◽

Temperature Sensitive ◽

Chromosomal Dna ◽

E Coli ◽

Dnab Helicase ◽

H Pylori

In Escherichia coli, DnaC is essential for loading DnaB helicase at oriC (the origin of chromosomal DNA replication). The question arises as to whether this model can be generalized to other species, since many eubacterial species fail to possess dnaC in their genomes. Previously, we have reported the characterization of HpDnaB (Helicobacter pylori DnaB) both in vitro and in vivo. Interestingly, H. pylori does not have a DnaC homologue. Using two different E. coli dnaC (EcdnaC) temperature-sensitive mutant strains, we report here the complementation of EcDnaC function by HpDnaB in vivo. These observations strongly suggest that HpDnaB can bypass EcDnaC activity in vivo.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.9.2984-2986.2003 ◽

2003 ◽

Vol 47 (9) ◽

pp. 2984-2986 ◽

Cited By ~ 46

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.

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Metronidazole Activation Is Mutagenic and Causes DNA Fragmentation in Helicobacter pylori and inEscherichia coli Containing a Cloned H. pylori rdxA+ (Nitroreductase) Gene

Journal of Bacteriology ◽

10.1128/jb.182.18.5091-5096.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5091-5096 ◽

Cited By ~ 65

Author(s):

Gary Sisson ◽

Jin-Yong Jeong ◽

Avery Goodwin ◽

Louis Bryden ◽

Norma Rossler ◽

...

Keyword(s):

Helicobacter Pylori ◽

High Sensitivity ◽

Wild Type ◽

Gene Encoding ◽

E Coli ◽

Evolution Of Virulence ◽

Rifampin Resistance ◽

H Pylori ◽

Emergence Of Resistance ◽

Forward Mutation

ABSTRACT Much of the normal high sensitivity of wild-type Helicobacter pylori to metronidazole (Mtz) depends on rdxA(HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance inrdxA + (Mtzs) and also inrdxA (Mtzr) H. pylori strains, and that expression of rdxA in Escherichia coliresults in equivalent Mtz-induced mutation. A reversion test using defined lac tester strains of E. coli carryingrdxA + indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in H. pylori and in E. coli carryingrdxA +, suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtzs H. pylori with E. coli (highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in E. coli, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected withH. pylori may stimulate mutation and recombination inH. pylori, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.

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Function of the ςE Regulon in Dead-Cell Lysis in Stationary-Phase Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.182.18.5231-5237.2000 ◽

2000 ◽

Vol 182 (18) ◽

pp. 5231-5237 ◽

Cited By ~ 35

Author(s):

Takeshi Nitta ◽

Hiroshi Nagamitsu ◽

Masayuki Murata ◽

Hanae Izu ◽

Mamoru Yamada

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Cell Lysis ◽

Dead Cell ◽

Multicopy Plasmid ◽

Wild Type ◽

E Coli ◽

Intracellular Proteins ◽

In The Wild ◽

Cloned Gene

ABSTRACT Elevation of active ςE levels in Escherichia coli by either repressing the expression of rseAencoding an anti-ςE factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase. The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene. In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed. These results suggest that dead cell lysis in stationary-phase E. coli occurs in a ςE-dependent fashion. We propose there is a novel physiological function of the ςE regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

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