Search

Tuberculosis (TB) is a worldwide burden for human health. More than 1.8 million people succumbed to TB infection in 2008 alone, and as many as two billion people may be passively infected by Mycobacterium tuberculosis, the causative agent of TB.[1] One of the challenges facing the successful treatment of M. tuberculosis infections is the development of strains that are resistant to many of the antibiotics used in the clinic. Cases of multidrug-resistant TB (MDR-TB), defined as TB caused by a M. tuberculosis strain that is resistant to both rifampin and isoniazid, have been identified in nearly every country surveyed.[1, 2] Extensively drug-resistant TB (XDR-TB), defined as MDR-TB that is also resistant to a flouroquinolone and at least one of three injectables (capreomycin, streptomycin, or amikacin), has spread to more than 45 countries, including the United States.[1, 2] The development and spread of drug-resistant strains of M. tuberculosis has put a high priority on the development of new antituberculosis drugs and the generation of derivatives of known drugs that regain their antibiotic activity against resistant strains. The tuberactinomycin family of antituberculosis drugs are important components of our drug arsenal against drug-resistant M. tuberculosis. The most prominent member of this family is capreomycin (CMN), which is a key drug in the treatment of MDR-TB based on its inclusion on the World Health Organization’s “Model List of Essential Medicines.”[3] Furthermore, if CMN is the injectable that XDR-TB infection is resistant to, it almost guarantees treatment failure.[4] Based on its level of importance in treating drug-resistant forms of TB, it is important that new CMN derivatives be developed that regain activity against resistant M. tuberculosis strains. Synthetic procedures to synthesize derivatives have been difficult.[5, 6] More success has been accomplished by semisynthetic approaches,[7–9] but the derivatives are limited by the functional groups present on the cyclic pentapeptide core. Complementing these approaches with metabolic engineering of the enzymology that produces the cyclic pentapeptide core has the potential of enabling further structural diversification. Our focus is on the metabolic engineering aspect of drug development. Harnessing the full potential of metabolic engineering to generate new drug derivatives requires a complete understanding of how the targeted drug is biosynthesized by the producing organism. To this end, we have focused on understanding tuberactinomycin biosynthesis at the molecular and biochemical level by using CMN and the structural analog viomycin (VIO) as model systems. Our focus on both CMN and VIO was based on our hypothesis that by studying the biosynthesis of two structurally related molecules (Scheme 1), we would gain considerable insights into how the tuberactinomycins are biosynthesized and structurally modified by the natural enzymology. To date, we have sequenced and annotated the CMN and VIO biosynthesis gene clusters,[10, 11] reconstituted CMN and VIO production in the heterologous host Streptomyces lividans,[12] biochemically characterized L-capreomycidine formation,[13] and identified the amino acids activated by each of the adenylation (A) domains of the CMN nonribosomal peptide synthetases (NRPS) and some of the A domains of the VIO NRPS (Scheme 2).[14] Zabriskie and colleagues have also contributed important genetic and biochemical information that enable more refined models of tuberactinomycin biosynthesis to be developed.[15–18] Open in a separate window Scheme 1 Chemical structures of viomycin (VIO), tuberactinamine A (TMN A; des-β-lysine VIO) and the four derivatives that make up capreomycin (CMN). The numbering within the cyclic pentapeptide cores of the antibiotics is used to identify positions noted in the text.

Tuberculosis (TB) remains one of the leading public health problems worldwide. Declared as a global emergency in 1993 by the WHO, its control is hampered by the emergence of multidrug resistance (MDR), defined as resistance to at least rifampicin and isoniazid, two key drugs in the treatment of the disease. More recently, severe forms of drug resistance such as extensively drug-resistant (XDR) TB have been described. After the discovery of several drugs with anti-TB activity, multidrug therapy became fundamental for control of the disease. Major advances in molecular biology and the availability of new information generated after sequencing the genome of Mycobacterium tuberculosis increased our knowledge of the mechanisms of resistance to the main anti-TB drugs. Better knowledge of the mechanisms of drug resistance in TB and the molecular mechanisms involved will help us to improve current techniques for rapid detection and will also stimulate the exploration of new targets for drug activity and drug development. This article presents an updated review of the mechanisms and molecular basis of drug resistance in M. tuberculosis. It also comments on the several gaps in our current knowledge of the molecular mechanisms of drug resistance to the main classical and new anti-TB drugs and briefly discusses some implications of the development of drug resistance and fitness, transmission and pathogenicity of M. tuberculosis.

The aim of this study was to clarify the conflicting data regarding cross-resistance and drug-resistance mechanisms for the cyclic peptide capreomycin and the aminoglycosides amikacin and kanamycin by comparing genotypes and phenotypes of clinical isolates and in vitro selected mutants of Mycobacterium tuberculosis.The genes rrs and tlyA and the promoter region of eis of 152 M. tuberculosis clinical isolates (including 55 capreomycin resistant) and 44 in vitro selected capreomycin-, amikacin- and kanamycin-resistant mutants were sequenced. In addition, MICs of capreomycin, amikacin and kanamycin on Middlebrook 7H10 were determined.The results clearly show major differences in genotypes and cross-resistance patterns to amikacin and kanamycin between the capreomycin-resistant clinical isolates and in vitro selected mutants. tlyA mutations were found almost exclusively among the in vitro selected capreomycin-resistant mutants, while only four were found among the clinical isolates, of which two were capreomycin susceptible. In contrast, 53 of the 55 capreomycin-resistant clinical isolates had a mutation at position 1401 in rrs and were resistant to capreomycin, amikacin and kanamycin. Low-level resistance to kanamycin was correlated to mutations in the promoter region of eis.Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.

Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis , including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis . In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed ±1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [ C max ]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis .

Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus . The basis of resistance to this drug was investigated by isolating and characterizing capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis . Colonies resistant to capreomycin were recovered from a library of transposon-mutagenized M. smegmatis . The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gen e . One capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA + strains resist capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers capreomycin resistance.

We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.

Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene ( rrs ) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.

Aqueous solutions of 0.02% isoniazid, 0.2% streptomycin, 0.2% para-aminosalicylate, and 0.5% ethambutol and ethylene glycol solutions of 0.5% ethionamide stored at 3 to 7 degrees C remained stable for 1 year, as did aqueous solutions of 0.05% ethionamide hydrochloride, 0.05% kanamycin, 0.05% viomycin, and 0.1% capreomycin stored at -20 degrees C. The ethambutol and capreomycin solutions were tested by microbiologic methods; the other solutions were tested by both spectrophotometric and microbiologic methods. Prepared susceptibility testing media made with cycloserine, rifampin, and the above solutions incorporated into Middlebrook 7H10 medium showed acceptable stability when stored at 3 to 7 degrees C for 1 month. During incubation of the test medium at 37 degrees C, approximately half of the activity of isoniazid, ethionamide, ethambutol, cycloserine, and rifampin was lost after periods ranging from 2 to 4 days for ethambutol to 2 weeks for rifampin.

Phenotypes of isolates of Mycobacterium tuberculosis H37RV showing resistance to the aminoglucoside antibiotics streptomycin, viomycin, kanamycin, capreomycin, tuberactinomycin N, lividomycin and paromomycin could be grouped into the following types: (I) resistant only to different levels of streptomycins; (2) resistant only to a low level of kanamycin; (3) triply resistant, to low levels of viomycin, tuberactinomycin N and capreomycin; (4) triply resistant, to a low level of kanamycin and high levels of lividomycin and paromomycin; (5) quadruply resistant, to a low level of capreomycin and high levels of kanamycin, lividomycin and paromomycin; (6) hextuply resistant, to high levels of viomycin, tuberactinomycin N, capreomycin, kanamycin, lividomycin, and paromomycin. Three modificatied types of the latter were also observed. Appearance rates of the six types were estimated as 10(-6) to 10(-9), 10(-6), 10(-6) to 10(-7), 10(-8), 10(-8), and 10(-8) to 10(-9), respectively, in a total viable population of the parent strain. Mutations to all phenotypes were considered to be produced by single mutations. According to cross-resistance relationships, aminoglucoside antibiotics were classified into three groups: (I) streptomycin; (II) viomycin, tuberactinomycin N and capreomycin; (III) kanamycin, lividomycin and paromomycin. No cross-resistance relationship between streptomycin and other antibiotics was observed. Resistances to viomycin, tuberactinomycin N and capreomycin occurred by single mutation to type 3. Resistances to kanamycin, lividomycin and paromomycin occurred by single mutations to types 4 and 5. Low resistance to capreomycin was produced by mutation to type 5. Therefore capreomycin was considered to be an intermediate between the second and third groups. These two groups had a close relationship, as resistance to all six agents in these groups could be produced by a single mutation to type 6 (and its modified types).

The biochemical mechanism of resistance to kanamycin, viomycin, and rifampin in five clinical isolates of Mycobacterium tuberculosis was studied. Resistance to viomycin and kanamycin was attributed to altered ribosomes, whereas resistance to rifampin was attributed to an alteration of RNA polymerase. Ribosomal resistance was, however, not the only way of expressing resistance to viomycin and kanamycin.

Drug resistant mutants to streptomycin, kanamycin, viomycin, capreomycin, and rifampicin were isolated from four strains of Mycobacterium tuberculosis. The mutants isolated from each parent were then tested for evidence of development of cross-resistance to other drugs. There was no cross-resistance between either streptomycin or rifampicin and any of the other drugs. Complete cross-resistance between viomycin and capreomycin was found. Cross-resistance between kanamycin and capreomycin, and kanamycin and viomycin was variable. A review of the medical histories of 27 patients with kanamycin-resistant tubercle bacilli indicated that cross-resistance with capreomycin and viomycin occurs, but is unpredictable. Because of this variability in cross-resistance and the fact that kanamycin is a more toxic drug than capreomycin, it is suggested that capreomycin be used in the first retreatment regimen for tuberculosis when streptomycin resistance has been demonstrated.

Palmitoyltuberactinamine N (Pal-Tua N) and di-beta-lysylcapreomycin IIA (di-beta-Lys-Cpm IIA), which are synthetic derivatives of the antituberculous agent tuberactinomycin (Tum) and capreomycin (Cpm) respectively, were tested for anti-bacterial activity. Pal-Tua N inhibited not only tuberactinomycin-resistant Mycobacterium smegmatis but also Escherichia coli, Corynebacterium diphtheriae, Staphylococcus aureus, Streptococcus pyogenes, although it has lost activity against Mycobacterium tuberculosis. Di-beta-Lys-Cpm IIA inhibited the growth of laboratory-derived Tum-resistant M. smegmatis and M. tuberculosis as well as Tum-resistant M. tuberculosis from patients with one exceptional case.

In vitro susceptibility testing to streptomycin, kanamycin and enviomycin was carried out in nontuberculous mycobacteria and the results were compared with those of M. tuberculosis strains. Among nontuberculous mycobacteria tested, only M. xenopi strains exhibited similar minimal inhibitory concentration (MIC) values to streptomycin with M. tuberculosis strains. On the other hand, M. xenopi, M. malmoense and M. marinum strains showed the same MIC values to kanamycin with the MIC values for M. tuberculosis strains. The MICs of enviomycin for M. kansasii, M. malmoense, M. szulgai, M. xenopi and M. marinum strains were similar to those for M. tuberculosis strains. Out of 64 strains of M. avium complex, the MIC value similar to that for M. tuberculosis strains was shown in 16% of strains to streptomycin, in 42% of strains to kanamycin, and in 50% of strains to enviomycin.

Data on the experimental and clinical study of capreomycin in the treatment of tuberculosis are presented. It was shown that capreomycin had low activity with respect to the sensitive strain of Mycobacterium tuberculosis H37 Rv in vitro and the respective infection caused by it in mice. The activity of capreomycin in vitro with respect to streptomycin resistant strains was the same as that with respect to the sensitive strains, while in vivo it increased 3 times. Capreomycin showed a tendency to decreasing its activity with respect to strains highely resistant to canamycin only in vitro. The effect of capreomycin on tuberculosis infection caused by strains resistant to different concentrations of canamycin was the same as that on tuberculosis infection caused by sensitive strains. Cross resistance between florimycin (viomycin) and capreomycin was shown. Clinical trails of capreomycin revealed its moderate therapeutic efficiency, relatively low toxicity and an allergenizing effect on the host. Transient ventibulopatia without pronounced signs of ototoxic action was observed. The nephrotoxic effect was moderate and transient. It was observed predominantly at the peak of the allergic reactions to the antitubercle drugs. The data obtained during the study allow recommendation of capreomycin use in clinics as reserve drug when the causative agent is not resistant to florimycin. The drug should be used under regular control of the blood picture, electrolyte metabolism, state of the kidneys, auditory and vestibular apparatus.

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10−5-10−6, 10−5-10−6, and 10−6-10−7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.

In the present report evaluation of the possible metabolic changes in partially revertants of M. tuberculosis var. hominis, reisolated from strains at high levels of drug-resistance after treatment with mutagenic agents, was reported. Based on the data presented here, it must be concluded that no significant biological and biochemical differences in mycobacterial cells revertant from high mutants mono-resistant to four aminoglycosides (Sm, Kana, Vio, Neo) and p-aminonasalicylic acid (PAS) are to be found.

The main characteristics of Mycoplasma ulcerans are its remarkably distinct drug sensitivity pattern and its rather poor enzymatic activity. M. microti is clearly distinguishable from M. bovis and M. tuberculosis. Some of the M. buruli strains appear to be identical with M. ulcerans, Whereas others show similarity either to M. microti or to M. tuberculosis. It seems that M. buruli is not a legitimate species.