Mycobacterium tuberculosis complex - Cultue; Molecular diagnosis (PCR);  Antimicrobial susceptibility tests; Resistance mutations (amplification and sequencing of rpoB, katG, mabA, embB, pncA, rpsL, rrs, gyrA and tlyA genes).

 

Information 05-07-2018.

 

            Infections due to Mycobacterium species are a growing problem in many countries of the world. The species of the genus Mycobacterium has been in permanent increase, recognizing at present more than 170 species, most of which have not been related to disease. However, this genus contains several important bacteria, which do cause infections and can threaten life. Among the pathogenic species are Mycobacterium leprae and those included in the Mycobacterium tuberculosis complex, which remain a public health problem.

            The World Health Organization (WHO) for 2016 reported 10.4 million cases of tuberculosis in the world, of which 1.7 million died. However, there are many other species listed as opportunistic or pathogenic, which has been grouped under the name of atypical mycobacteria, non-tuberculous mycobacteria (NTM) or MOTT (mycobacterium other than tubercle bacilli). Non-tuberculous mycobacteria are also capable of causing serious diseases in both immunocompetent and immunocompromised individuals. Undoubtedly, NTM have been gaining more relevance in the field of public health, mainly due to the increase in their frequency as causal agents of serious pathologies that affect the lungs, lymph nodes, skin, wounds, bones, etc., representing between 0.5 and 30% of the total human diseases caused by mycobacteria. Consequently, the interest of the scientific community for these microorganisms has grown radically recently, which has allowed us to know the various aspects associated with the pathologies they cause and their virulence factors.

            The short-term therapy recommended by the World Health Organization (WHO), for infectious diseases caused by M. tuberculosis, includes; first-line drugs, more effective and less toxic, among which are: Isoniazid, rifampin, pyrazinamide, ethambutol and second-line drugs, for cases of resistance or intolerance, including streptomycin, capreomycin, ethionamide, cycloserine, PAS, and fluoroquinolones. The therapy for the active form sensitive to anti-tuberculous drugs includes a standard combination of isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol, using the DOTS strategy (direct observation treatment). This strategy is no longer the therapeutic option for patients infected with multidrug-resistant M. tuberculosis to antituberculosis drugs, defined as strains resistant to at least isoniazid and rifampicin. In the last two decades, tuberculosis with drug resistance has become a threat and a challenge for world public health. The diagnosis and treatment of these forms of tuberculosis is much more complex and the prognosis clearly worsens as the pattern of resistance increases.

            On the other hand, due to the lack of standardized or accepted criteria to define diseases caused by NTM, many cases are frequently misdiagnosed as tuberculosis and antituberculous drugs are administered, while the treatment of NTM infections is not similar to that of MTB. The new macrolides (azithromycin and clarithromycin) and the quinolones (ofloxacin and ciprofloxacin) have become key therapy for infections caused by NTM.

            Due to the crisis of drug resistance, infectious diseases caused by clinically important mycobacteria continue to be a major public health problem throughout the world.

            Bacteria can present resistance to antibiotics mainly by three mechanisms:

Mechanisms of resistance for alterations in cellular permeability

            The low permeability of the mycobacterial cell wall, with its unusual structure, is an important factor in this intrinsic resistance. The mycobacterial cell envelope consists of three main structural components: a peptidoglycan wall, the arabinogalactan polysaccharide and the long chain mycolic acids. The structure of the wall gives hydrophobic properties that generate low permeability, which has been associated with the intrinsic resistance to disinfectants and antimicrobials. The loading, size and hydrophobicity of antimicrobials determine the penetration through the cell membrane. The penetration rates of beta-lactam antibiotics through the outer cell wall of M. tuberculosis, is ten times higher than in Mycobacterium chelonae, and 100 times lower than in Escherichia coli.

            M. tuberculosis is a bacterium that is physiologically resistant to most antimicrobials, mainly due to the lipid structure of its cell wall (> 60%) that acts as a barrier only permeable to hydrophilic solutes, although these pass slowly through the cell wall due to the scarce number of porins, the low fluidity of mycolic acids and the total wall thickness of M. tuberculosis.

Mechanisms of resistance by enzymatic inactivation

            Enzymatic inactivation is another mechanism described in bacteria to neutralize the action of antimicrobials. In species of the genus Mycobacterium, beta-lactamases, methylases and acetyltransferases capable of hydrolyzing and inactivating a wide variety of antimicrobials have been found.

Mechanisms of resistance due to modifications of the molecular target

            Another of the most important factors related to resistance in the treatment schemes are the modifications of the molecular target. The bacterial ribosome is one of the targets of several antimicrobial groups, including aminoglycosides and macrolides. The action of these antibiotics is the inhibition of protein synthesis through their interaction with rRNA nucleotides, near sites that are important for their function, such as: the peptidyl transferase region of 23S rRNA, where macrolides bind and the 16S rRNA region, target for the aminoglycosides. The changes in the binding targets generate from low to high resistance to antimicrobials. The methylation of the targets of action of the macrolides is another important factor, especially in species like M. abscessus. This last mechanism explains the therapeutic failures that have been reported in patients with both lung infections and skin infections. In the case of M. tuberculosis, resistance to first-line drugs is predominantly due to alterations in the nucleotide sequence in genes that code for antibiotic targets.

            Although the concept of antimicrobial resistance is basically associated with a phenotypic characteristic (inability to grow in the presence of an antimicrobial), the technical difficulty of the phenotypic test is due to the slow growth in the culture of certain bacteria such as Mycobacterium tuberculosis, has induced since the early 90s the development of techniques that allow the detection of genes or specific sequences that code for resistance. These molecular techniques allow obtaining results in hours or days, this being a critical factor to be able to establish an adequate therapeutic guide as soon as possible. Although the culture and the tests of susceptibility to drugs cannot be ignored, however given the slowness in culture of M. tuberculosis it makes that the interest for the molecular techniques is increasing.

            These molecular tests for M. tuberculosis detect, by genetic amplification techniques, mutations in the genes that code for drug resistance, are standardized and reproducible methods. However, for other species of mycobacteria that show wide variability in their sensitivity to antimicrobials, the standardization of the methods has not been achieved, nor has it established a clear clinical correlation. There is no clear consensus on the need to perform studies in M. avium complex or other slow-growing mycobacteria. In these cases, methods such as E-test MIC, disc-plate or dilution in broth can be used.

Gene alterations associated with the development of resistance to first and second line antimicrobials for the treatment of tuberculosis

Resistance to isoniazid (INH)

            Isoniazid (nicotinic acid hydrazide or INH) has a potent bactericidal action by interfering with the biosynthesis of the mycolic acids of the mycobacteria cell wall. It is a prodrug that, when captured by the bacillus, is activated by the catalase-peroxidase system, so that the absence of catalase activity, due to mutations in the katG gene, coding for this enzyme, is one of the mechanisms of resistance for INH. Strains of M. tuberculosis with mutations in the katG gene exhibit little or no catalase activity and are highly resistant to INH. The mutations are concentrated in a coding region of the katG gene, which comprises codons 300 to 507, the most frequent being the substitutions of serine 315 by threonine (S315àT) and the residue of arginine 463 by leucine (R463àL). These mutations explain approximately 50% of the clinical isolates cases resistant to INH.

            On the other hand, the enoyl enzyme ACP reductase, involved in the steps of fatty acid elongation, encoded by the inhA gene was identified as an INH target. The INH intermediate, whose activation depends on intact catalase-peroxidase activity, inhibits the activity of the inhA enzyme and consequently the synthesis of mycolic acids. Mutations in the inhA gene induce overexpression of the inhA gene and elevated levels of the enzyme enoyl reductase in amounts that exceed the inhibitory power of INH. Mutations in inhA gene are associated with approximately 25% of cases of INH resistance, generally with low levels of resistance. Mutations in the inhA gene not only cause resistance to isoniazid, but also to ethionamide. There are two genes involved in this resistance ethionamide-isoniazid, designated as mabA and inhA to refer to ethionamide and isoniazid, respectively. The fact that clinical strains show resistance to both INH and ETH indicates a mechanism of cross-resistance. The investigation of other genes involved in resistance to INH that explain the resistance mechanism of 10-20% of strains lacking mutations in katG or inhA led to the identification of the ahpC gene, coding for the enzyme alkyl hydroperoxide reductase, involved in the response to oxidative stress. Mutations in aphC are associated with approximately 10 to 15% of clinical isolates resistant to INH. Currently, other candidate genes associated with resistance to this antimicrobial are being investigated.

Resistance to rifampicin (RIF)

            The mechanism of action of rifampicin is the antimicrobial binding to RNA polymerase, the enzyme responsible for the gene transcription process. By inhibiting the expression of genes, it leads to the death of the cell. Resistance to rifampicin is explained by mutations in the rpoB gene, which encodes the β subunit of RNA polymerase. Alterations in this subunit prevent rifampicin from interacting properly with RNA polymerase and inhibit transcription. It has been shown that resistance to rifampicin in M. tuberculosis is explained in 95% to 98% by mutations in the rpoB gene, which are generally located in a short segment of approximately 81 bp that includes codons 507 to 533 of the rpoB gene. Mutations in this region include deletions, insertions, substitutions, the most frequent being the codon mutations for asparagine 516, histidine 526 and serine 531, so that genotypic methods to detect resistance to rifampicin are based on the detection of these mutations. Mutations have been also found in other regions of the gene, but less frequently.

Resistance to pyrazinamide

            Pyrazinamide is a synthetic compound rediscovered in the 1980s that has facilitated short-term antituberculous treatment. Its mechanism of action has not been well elucidated, although the importance of the action of the enzyme pyrazinamidase has been pointed out. Strains of mycobacteria susceptible to pyrazinamide synthesize pyrazinamidase, an enzyme that transforms pyrazinamide into its active metabolite, pyrazinoic acid, which in addition to its specific activity appears to have the ability to lower the pH of the intracellular medium below the tolerance limits of the bacteria. In strains of M. tuberculosis with acquired resistance and M. bovis with constitutive resistance to pyrazinamide, interruptions have been identified in the pncA gene, encoding the enzyme pyrazinamide/nicotinamide. One of the mechanisms of resistance to pyrazinamide proposed so far is the deficiency in pyrazinamidase, with the subsequent loss of the ability to activate the antimicrobial.

Resistance to aminoglycosides (streptomycin, kanamycin, amikacin)

            Streptomycin is a bactericidal aminoglycoside antibiotic that acts on ribosomes by inhibiting protein synthesis through binding to the 30S subunit of the bacterial ribosome, which causes misinterpretation of the mRNA message during translation. The site of action of streptomycin is the 30S subunit of the ribosome in the ribosomal protein S12 and the 16S rRNA.

            In most strains of M. tuberculosis resistant to streptomycin mutations have been found in the rpsl gene that encodes the ribosomal protein S12. Another identified, but less frequent mutation is located in the rrs gene that encodes 16S ribosomal RNA in a region that interacts with the S12 protein. Mutations in the rpsL and rrs genes are the main mechanism of resistance to streptomycin totaling approximately 50% and 20%, respectively, of the resistant strains.

            Kanamycin and its derivative, amikacin, are also inhibitors of protein synthesis through the modification of ribosomal structures in 16S rRNA. Mutations at position 1400 of the 16S rRNA (rrs) are associated with a high level of resistance to both antimicrobials. It was shown that a gene called tlyA that encodes the methyltransferase of rRNA is involved in resistance to capreomycin in strains of M. tuberculosis.

Resistance to ethambutol

            Ethambutol is a synthetic compound that acts as a bacteriostatic, whose mechanism of action is to inhibit the synthesis of components of the mycobacterial wall at the usual doses. The gene alterations identified so far are concentrated in a region designated as embCAB, which includes genes coding for arabinosyltransferases, enzymes that participate in the synthesis of unique components of the mycobacterial cell wall. Mutations in the emb region are associated with high levels of resistance and have been identified in approximately 65% ​​of the clinical isolates resistant to ethambutol.

Resistance to fluoroquinolones 

            DNA topoisomerases are a set of several essential enzymes responsible for maintaining chromosomes in a proper topological state. These drugs inhibit the action of topoisomerases that are responsible for the supercoiling of DNA to facilitate its location within the bacterium, as well as the unwinding to facilitate its replication and transcription, acting mainly on DNA-gyrase enzymes, mainly encoded by the gyrA gene. Resistance to quinolones in the course of treatment appears frequently and is associated with point mutations in the gyrA gene. They are considered second-line drugs in antituberculous treatment, although the increase in resistance to first-line drugs has increased their use in the case of resistant strains.

Resistance to ethionamide / protionamide and thioamides

            Ethionamide (2-ethylisonicotinamide) is a derivative of isonicotinic acid and is bactericidal only against M. tuberculosis, M. avium-intracellulare and M. leprae. Like isoniazid, ethionamide is also a prodrug that is activated by EtaA / EthA (a monooxygenase) and inhibits the same target as isoniazid, the InhA of the mycolic acid synthesis pathway.

            Prothionamide (PTH, 2-ethyl-4-pyridinecarbothioamide) has a structure and activity almost identical to those of ethionamide. EtaA or EthA is a flavin adenine dinucleotide (FAD) that contains the enzyme that oxidizes ethionamide to the corresponding sulfur oxide, which is subsequently oxidized to 2-ethyl-4-amidopyridine, presumably via the unstable oxidized sulphonic acid intermediate. EtaA also activates thioacetazone, thiocaride, thiobenzamide and, perhaps, other drugs belonging to thioamides, which explains the cross-resistance between ethionamide and thioacetazone, thiocaride and other thioamides and thioureas. Mutations in the enzyme EtaA / EthA activator of the antimicrobial cause resistance to ethionamide and other thioamides. In addition, mutations in the InhA target confer resistance to both ethionamide and isoniazid.

Tests carried out in IVAMI:

• Molecular diagnosis (PCR) for the genes that determine resistance (rpoB, katG, mabA, embB, pncA, rpsL, rrs, gyrA, tlyA) and subsequent sequencing.

• Culture and sensitivity testing to first line drugs in MGIT 960 equipment.

Recommended sample:

• Respiratory samples (sputum, bronchial aspirate, bronchoalveolar lavage, gastric juice), sterile fluids (CSF, joints, peritoneal, pleural, pericardial, etc.), tissues (biopsies) and urine (it is advisable to collect three samples (minimum of 40 ml) during three consecutive days).

• strain from culture.

Conservation and shipment of the sample:

• Refrigerated (preferred) for less than 2 days.

• Paraffin-embedded biopsies can be stored and sent at room temperature or refrigerated.

• Fresh biopsies should be stored preferably frozen.

Delivery of results:

• Molecular diagnosis (PCR) for the genes that determine resistance and subsequent sequencing: 3-5 days.

Cost of the test:

• Molecular detection (PCR) for the genes that determine resistances and subsequent sequencing: consult ivami@ivami.com.