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Helicobacter pylori: genotypic resistance to clarithromycin, amoxicillin, metronidazole, levofloxacin, tetracyclines and rifabutin - PCR and sequencing.

 

Information 18/03/2016.

 

Helicobacter pylori is a gram - negative bacterium spiral morphology, able to colonize the human stomach by its potent urease activity allows you to create a neutral ambinte in their environment by metabolization of urea present in the mucosa (neutralofílica bacteria).

This bacterium causes a major transmissible infection that damages the gastric structure and function. It is now recognized as being responsible for peptic ulcer disease (gastric or duodenal), atrophic gastritis gastric, gastric adenocarcinoma and lymphoma associated with mucosal tissue (MALT lymphomas: Mucosa-Associated Lymphoid Tissue Lymphoma).

Gastric cancer is the second leading cause of cancer - related death, so the International Agency for Research on Cancer, part of the World Health Organization (WHO) has classified this bacterium as a type I carcinogen

Antimicrobial treatment of this infection aims to eradicate the pathogenic bacteria and thereby cure and prevent associated diseases. They are proposed various regimens that include more than one drug, and have been termed as drugs include dual therapy, triple or quadruple, and first empirical line or second line, as they are intended to be initially administered after clinical diagnosis, or treatment failure after the first group of drugs.

Most therapies against Helicobacter pylori are very effective, but the eradication rate is adversely affected by the increased incidence of antibiotic resistance of the bacteria.

One of the empirical treatment recommendations of Helicobacter pylori infections is empirical triple first line therapy including the use of proton pump inhibitors (PPIs: Proton Pumps Inhibitors), metronidazole or amoxicillin and clarithromycin as an alternative to clarithromycin. This triple therapy may be administered from the beginning with all three drugs or sequentially, initially administering PPIs for 5 days one and amoxicillin, followed by PPIs clarithromycin and metronidazole associated or tinidazole for another 5 days.

Recently the results of the efficacy of this combination have decreased, resulting in cure rates of only 70%. For this reason, it has been established the cutting point (threshold) 15 to 20% to distinguish between regions with low and high rate of resistance to clarithromycin. In regions with low resistance rate, 15-20% less, it is recommended triple therapy including clarithromycin first line, but not in those found higher rates of resistance. Proton pump inhibitor (PPIs), bismuth salts (subsalicylate), metronidazole and tetracycline: In these situations the quadruple empirical first - line therapy with four drugs can be used. In this pattern clarithromycin it is replaced by tetracycline and bismuth salt is included. Another alternative is to use an empirical triple first - line therapy in which is included levofloxacin (fluoroquinolone) instead of clarithromycin.

Nevertheless if not eradication is obtained it is recommended to use a second - line therapy, guided by susceptibility testing whenever possible. For this triple second - line therapy should be considered the following: fluoroquinolones (levofloxacin), tetracyclines, rifabutin, furazolidonas, and high doses of PPIs and amoxicillin.

Difficulties in the usual culture of this bacterium, and to determine their antimicrobial susceptibility make therapies are administered empirically, but it is recommended that after the trerapéuticos failures, sensitivity tests are performed to drugs used. Not being feasible in most Casiones, have bacteria in culture for susceptibility testing, and the slow pace of testing (10 to 14 days), it has been proposed several molecular methods that try to detect the most frequent mutations. Many laboratories use commercial methods, generally based on hybridization with specific probes to detect specific mutations. However, when there are point mutations that may be distributed throughout a gene, these methods are not available and complete amplification and sequencing of the gene is required. These methods of amplification and sequencing, each gene involved in the development of resistance, are available in IVAMI.

clarithromycin

Overall rates of resistance to macrolide clarithromycin are increasing 9% (1998) and 17.6 (2008) in Europe, and 7% (1998) and 27.7% (2008) in Japan.

Macrolides exert their action by binding to the 50S ribosomal subunit, specifically interacting with the ribosomal 23S (23S rRNA) ribonucleic acid, and inhibiting the process of the peptidyl transferase which is responsible for forming peptide bonds between amino acids that are incorporated, and ribosome moves along the messenger RNA (mRNA). When there are point mutations in the 23S rRNA in the region of the peptidyl place (23S rRNA V domain), the fixing of macrolides, such as clarithromycin, is inhibited and thus protein synthesis is not blocked.

The primary mutation is a transition (replacement of a purine nucleotide by another purine nucleotide) A to G at 2142 position (A2142G) or at position 2143 (A2143G), or a transversion (replacement of a purine nucleotide by a pyrimidine nucleotide) at the position 2142 (A2142C / T; A2143C).

We found other mutations that have been linked to low - level resistance: T2182C, T2717C and C2611A. Furthermore, other mutations described whose clinical significance has not been proven: A2115G, T2117C, G2141A, G2224A, C2245T, T2289C and.

Resistance to macrolide may be motivated by efflux pumps (efflux pumps), of which there are at least four types. These resistors have not been studied sufficiently in this bacterium.

amoxicillin

 

Amoxicillin is an antimicrobial therapies included in empirical triple first line, along with a proton-pump inhibitor (PPIs) and clarithromycin or mertronidazol. Currently they are described resistance to this antibiotic, but in general, with few exceptions, the resistance rate is less than 2%.

 

As all antimicrobial class of ?-lactams, exerts its action by inhibiting some of the proteins of the cytoplasmic membrane of the bacteria (PBPs: Penicillin Binding Proteins), involved in peptidoglycan synthesis of cell wall. Specifically they exert their action blocking those PBPs with transpeptidase activity in its C-terminal region, such as PBP1, PBP2 and PBP3.

 

Resistance to amoxicillin is motivated by mutations in the binding site of ?-lactams to PBPs, and confer resistance due to reduction in binding of the antimicrobial to them, so that their action is not inhibited. These mutations occur in genes encoding the said PBP: pBP1 gene for the enzyme PBP1; PBP2 gene for PBP2 and PBP3 gene for FTSI. Only found a strain whose resistance is due to the production of ?-lactamase.

 

Some of the mutations found are: Val16Ile; Val45Ile; Ser414Arg; Asn562Tyr; Thr593Ala; Gly595Ser; and Ala599Thr.

 

When mutations occur in the gene and PBP3 PBP1 resistance is higher than when mutations occur only in the PBP1 gene, or genes and PBP2 PBP1. When mutations occur in the genes of the three PPBs mentioned, the MIC for amoxicillin is increased to 256 times.

           

metronidazole

 

Metronidazole (or equivalent tinidazole) has been recommended as part of empiric therapy frontline triple or quadruple therapy for first line.

 

In addition to resistance to clarithromycin, metronidazole resistance is increasingly common. The prevalence of resistance to metronidazole is between 8 and 80% depending on the country, it is higher in developing countries.

 

This drug, actually, is a prodrug that must be activated by reduction of the nitro group attached to the imidazole ring. By reducing nitroso hydroxylamine groups or DNA damaging generated. To be reduced metronidazole an oxygen-resisente NADPH nitroreductase (rdxA), a NADPH oxidoreductase type flavin (FrxA) and a ferredoxin (FrxB) is required.

 

Metronidazole resistance may be due to the existence of mutations in rdxA (insertions, deletions, nonsense mutations, ...), frxA, frxB (point mutations) or RPSU genes.

 

The rdxA gene encoding NADPH nitroreductase resistant oxygen. The frxA and frxB genes encode an enzyme similar to ferredoxin, NADPH oxidoreductase flavin. Mutations in recent increase resistance genes generally when coexist with rdxA gene mutations, but has also been found that mutations in the gene frxA alone can cause resistance to metronidazole. Also it found in strains resistant mutations in the gene RPSU, which could possibly lead to resistance.

 

levofloxacin

 

Primary fluoroquinolone resistance is between 2 and 22% according to the regions studied.

 

Fluoroquinolones act by inhibiting the function of enzyme DNA gyrase. The function of this enzyme is essential to maintain the supercoiled helix structure of DNA in bacteria, and is also involved in DNA replication and transcription unrolling sequentially molecule will occur when these processes. DNA gyrase is a tetramer comprised of two A subunits and two B subunits, respectively coded by gyrA and gyrB genes of. The A subunit of DNA gyrase is responsible for cutting and joining of DNA strands during unwinding / supercoiling.

 

Mutations are clustered generally in a part of the gene sequence called QRDR (Quinolones Resistance Detemining Region), which when mutated change the amino acid sequence of the enzyme. These changes prevent joining it fluoroquinolones resulting resistance.

 

In other bacteria there are other genes (parC and parE) encoding other enzymes, called topoisomerase IV, but these genes do not exist in this bacterium.

 

Point mutations in QRDRs of DNA gyrase are mainly: Asn87Lys / Tyr (C261A; C261G); Asp91Gly / Asn / tyr (A272G, G271A, G271G).

 

It has been noted that a mutation in the gyrB gene in position 463 could be a new mechanism of resistance.

           

tetracyclines

 

Tetracyclines inhibit bacterial growth by blocking protein synthesis in bacteria. For this, these antimicrobials bind to the 30S subunit interacting with 16S ribosomal (16S rRNA) ribonucleic acid. When there are mutations in some 16S rRNA genes fixing the aminoacyl-tRNA, the transfer ribonucleic acid (tRNA), carrying an amino acid to be incorporated into the protein being synthesized on the ribosome is blocked.

 

Most isolates of Helicobacter pylori are sensitive to tetracyclines with a lower minimum inhibitory concentration (MIC) of 1 mg / L. However, the incidence of resistance is increasing in regions where the drug can be obtained without a prescription, so the rate generally continuous tetracyclines resistance remains low (about 2%). Some of the rates of resistance found are: America (2.7%), Europe (2.1%), Asia (2.4%), Africa (43.9%) and Iran (9%).

 

The high - level resistance produced by nucleotide substitution in the triplet 926-928 (AGA926-928TTC) of rrnA / 16S rRNA genes B. Found resistors low substitution one or two nucleotides in the same gene: A926G; A928C; or AG926-927GT. In isolates with triplet AGA926-928 MIC is 0.016 to 0.5mg / L, while if AGC GGA or MIC is 0.75 to 1.5 mg / L.

 

rifabutin

 

Rifabutin is a derivative of rifamycin S, with a similar molecular structure to rifampicin. This antimicrobial exerts its action by inhibiting the beta-subunit of RNAP dNAD (RNA polymerase, DNA dependent), encoded by the rpoB gene. By inhibiting this enzyme, DNA transcription and mRNA formation (mRNA) it is prevented.

 

The average rate of resistance is low and is 1.3%. When studied in patients not exposed to this drug it is less (0.6%). However, when studied in patients who had been treated with rifampicin resistance rate is high.

 

Strains of Helicobacter pylori resistant have mutations in some of the following codons: 149, 525-545, or 586.

 

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