Sensitivity of Mycobacterium tuberculosis to levofloxacin]

Susceptibility of 92 strains of mycobacteria to levofloxacin (5, 10 and 50 mcg/mL) was investigated by indirect method of absolute concentrations on Levenstain-Jensen media. The investigation was performed on 85 strains of Mycobacterium tuberculosis isolated from 83 patients with different types of tuberculosis and also on drug-sensitive laboratory strains of M. tuberculosis H37Rv-M, H37Rv-GISK, Academia, M. bovis-bovinus 8, M. bovis BCG and on two strains of M. fortuitum with total resistance to antimycobacterial agents. 87.8 per cent of clinical isolates were multi-drug resistant. From one patient treated with ciprofloxacin two strains of M. tuberculosis were isolated--one resistant to 5 mcg/mL of levofloxacin and second strain-resistant to 10 mcg/mL of levofloxacin. All other clinical and laboratory strains of mycobacteria (97.8 per cent) were susceptible to all three concentrations of levofloxacin.     

Antitubercular activity of trifluoperazine, a calmodulin antagonist

Trifluoperazine, a calmodulin antagonist, completely inhibited the growth of mycobacteria. The minimum inhibitory concentrations in shake cultures in a synthetic medium containing 0.2% Tween 80 were 5 and 8 micrograms/ml, respectively, for the human pathogenic strain Mycobacterium tuberculosis H37Rv and M. tuberculosis resistant to isoniazid. When added to a growing culture of M. tuberculosis H37Rv on the 10th day (mid exponential phase), trifluoperazine 50 micrograms/ml further arrested growth of this organism. It is suggested that trifluoperazine or similar calmodulin antagonists might be useful as antitubercular drugs.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Molecular epidemiology of drug-resistant tuberculosis in Sweden

Drug-resistant tuberculosis (TB), including the more severe forms of multidrug- and extensively drug-resistant forms, is an increasing public health concern globally. In Sweden the majority of patients with TB are immigrants from countries with a high incidence of TB including the drug-resistant forms. In this study, the spread of resistant TB in Sweden was investigated by molecular fingerprinting. Isolates resistant to at least one of the drugs, isoniazid, rifampicin, ethambutol or streptomycin, from 400 patients collected between 1994 and 2005, were studied by restriction fragment length polymorphism (RFLP) and by spoligotyping. Thirty-five clusters of patients infected with strains with identical RFLP and spoligotyping patterns (2-96 patients per cluster), comprising a total of 203 patients, were found. One large outbreak of isoniazid resistant tuberculosis was identified, involving 96 patients, mainly from the Horn of Africa. To identify chains of transmission, molecular epidemiological characterization of TB isolates should, if possible, be performed on isolates from all new TB patients.     

Comparative study of hepatoprotective action of remaxol, reamberin and ademethionine in liver injury induced by antituberculosis drugs (experimental study)

The hepatoprotective activity of remaxol, reamberin and ademethionine was studied on a model of the liver injury induced by antituberculosis drugs. The study included 30 male uninbred albino rats. The following antituberculosis drugs were used: isoniazid (50 mg/kg) subcutaneously + rifampicin (250 mg/kg) intragastrically + pyrazinamide (45 mg/kg) intragastically (by the procedure of Yu. I. Slivka, 1989). Remaxol, reamberin and ademethionine were administered 1.5 hour prior to the antituberculosis drugs. The treatment course was 14 days. It was shown that remaxol, reamberin and ademethionin were able to correct the structural and functional disorders in the liver due to the use of the antituberculosis drugs. By the impact on the biochemical indices, evident of the liver function condition, remaxol showed the maximum effect. The effect of ream-berin was somewhat lower and the results of the ademethionine use were less significant. Remaxol had also a distinct effect as for lowering the level of the structural injuries in the liver, evident from recovery of the organ histoarchitectonics, less extended carbohydrate, albuminous and fatty degeneration, more active intracellular regeneration. It was noted that ademethionine had an insignificant effect on necrobiosis. Moreover, there was once detected a large necrosis focus, evident of possible stimulation of the liver tissue alteration by the drug.     

Mechanism for the pyridoxal neutralization of isoniazid action of Mycobacterium tuberculosis

In Sauton's synthetic liquid medium, 10 mug of pyridoxal per ml completely protected Mycobacterium tuberculosis (H37R(a)) from the effects of a minimal inhibitory concentration of isoniazid (0.01 mug/ml). (14)C-labeled isoniazid was employed to study the nature of this protective effect. Uptake of the drug by cells in a Sauton environment containing 0.01 mug of (14)C-isoniazid per ml was inhibited 20 to 40% by 10 mug of pyridoxal per ml during the early hours of drug exposure. A stronger inhibition of uptake resulted when labeled isoniazid and pyridoxal were increased to 0.1 mug/ml and 50 to 100 mug/ml, respectively. Further studies revealed that certain Sauton nutrients are required to achieve this effect. When l-asparagine or salts (MgSO(4) and ferric ammonium citrate) or both were deleted from the menstruum, pyridoxal did not inhibit isoniazid incorporation by the tubercle bacilli. Pyridoxal also failed to inhibit uptake when (NH(4))(2)SO(4) was substituted for l-asparagine. Growth experiments in Sauton's medium modified to contain (NH(4))(2)SO(4) instead of l-asparagine were consistent with the latter finding. Pyridoxal did not prevent isoniazid growth inhibition in this medium. It is postulated that a large excess of pyridoxal in Sauton's medium protects tubercle bacilli from the effects of isoniazid through formation of an extracellular complex involving drug, vitamin, and certain medium constituents, thereby reducing the level of isoniazid available to the cells.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

Detection of mutations associated with multidrug-resistant Mycobacterium tuberculosis clinical isolates

Antimicrobial resistance was studied in 100 Mycobacterium tuberculosis strains selected randomly from sputum cultures of newly diagnosed tuberculosis patients. Resistance of the isolates to rifampicin, isoniazid, and ethambutol was tested by both drug susceptibility testing (DST) and allele-specific PCR (AS-PCR). A total of 19 (19%) isolates were found resistant to at least one of the antituberculosis drugs investigated by PCR compared with 14 (14%) resistant isolates detected by DST. Eleven mutations were detected by AS-PCR in the rpoB gene (codons 516, 526, and 531), associated with rifampicin resistance, a marker of multidrug-resistant tuberculosis (MDR-TB), 14 mutations in the katG gene codon 315 that confers resistance to isoniazid, and nine mutations in the embB gene codon 306 that confers resistance to ethambutol. Mutations in the six multidrug-resistant isolates were confirmed by DNA sequencing. Results were compared with phenotypic DST data. Nineteen different mutation types to at least one of the drugs were found; six isolates (6%) were classified as MDR-TB, defined as resistance to at least rifampicin and isoniazid. The rates of concordance of the PCR with the phenotypic susceptibility test were 71.4, 54.5, and 44.4 for isoniazid, rifampicin, and ethambutol, respectively. These results highlight the importance of molecular epidemiology studies of tuberculosis in understudied regions with a tuberculosis burden to uncover the true prevalence of the MDR-TB.     

Evaluation of susceptibility of Mycobacterium bovis to antituberculous drugs by radiometric BACTEC 460TB system

Susceptibility of Mycobacterium bovis strains to antituberculous drugs (isoniazid and rifampin) was detected by radiometric BACTEC 460TB system. M.bovis strains were isolated from tissue samples showing tuberculous lesions collected at an abbattoir from cattle belonging to 47 tuberculosis outbreaks occurring in Northern Italy in 1995-1999. Forty-six out of 61 strains (75.4%) resulted susceptible to both isoniazid and rifampin. Thirteen strains (21.3%) were resistant to isoniazid only. No strains showed resistance to rifampin only. Two strains (3.3%) resulted resistant to both drugs, showing antituberculous multidrug-resistance. Given the compulsory eradication program of bovine tuberculosis by elimination of infected animals and the ban on antituberculous drug treatments in animals, detection of resistant M. bovis strains appears of great interest.     

Increasing antituberculosis drug resistance in the United Kingdom: analysis of national surveillance data

Objective To identify recent trends in, and factors associated with, resistance to antituberculosis drugs in England, Wales, and Northern Ireland.Design Cohort of tuberculosis cases reported to the enhanced tuberculosis surveillance system matched to data on drug susceptibility and national strain typing data.Setting England, Wales, and Northern Ireland 1998-2005.Main outcome measures Unadjusted and adjusted odds ratios for drug resistance and associated factors. Proportion of multidrug resistant tuberculosis cases clustered.Results 28 620 culture confirmed cases were available for analysis. The proportion of cases resistant to isoniazid increased from 5% to 7%. Rifampicin resistance increased from 1.0% to 1.2% and multidrug resistance from 0.8% to 0.9%. Ethambutol and pyrazinamide resistance remained stable at around 0.4% and 0.6%, respectively. Regression analyses showed a significant increase in isoniazid resistance outside London (odds ratio 1.04, 95% confidence interval 1.01 to 1.07, a year, associated with changes in age (0.98, 0.98 to 0.99, a year), place of birth (1.49, 1.16 to 1.92), and ethnicity (P<0.05). In London, the rise (1.05, 1.02 to 1.08, a year) was related mainly to an ongoing outbreak. Increases in rifampicin resistance (1.06, 1.01 to 1.11, a year) and multidrug resistance (1.06, 1.00 to 1.12, a year) were small. A fifth of patients with multidrug resistant tuberculosis in 2004-5 had indistinguishable strain types, and one case was identified as extensively drug resistant.Conclusions The rise in isoniazid resistance reflects increasing numbers of patients from sub-Saharan Africa and the Indian subcontinent, who might have acquired resistance abroad, and inadequate control of transmission in London. The observed increases highlight the need for early case detection, rapid testing of susceptibility to drugs, and improved treatment completion.     

广泛耐药结核分枝杆菌耐药机制及其疾病诊断的研究进展

自20世纪90年代以来,全球结核病疫情回升,结核分枝杆菌耐药是其中的一个重要原因.广泛耐药结核病是指在耐多药结核病(即同时对异烟肼和利福平耐药的结核分枝杆菌引起的结核病)的基础上,还对氟喹诺酮类药物和至少3种二线静脉用抗结核药物(卷曲霉素、卡那霉素、阿米卡星)中的1种耐药的结核分枝杆菌引起的结核病.我国是结核病高流行国...     

High Isoniazid Resistance Rates in Rifampicin Susceptible Mycobacterium tuberculosis Pulmonary Isolates from Pakistan

Background

Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential.

Objective

To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan.

Materials and Methods

Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009–2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin.

Results

Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains.

Conclusions

Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.     

Drug resistance of strains of Mycobacterium tuberculosis isolated in Brazil.

Tuberculosis remains a serious public health problem, worsened by an increased frequency of multidrug-resistant Mycobacterium tuberculosis. We report here a retrospective study of resistance to antituberculosis drugs of 170 strains of M. tuberculosis isolated from the state of Rio Grande do Sul, Brazil. The frequency of resistance to at least one drug was 34%, while 22% were resistant to more than one drug. Among the strains isolated from patients without a history of previous treatment for tuberculosis, patients with positive serology for HIV and patients with previous treatment for tuberculosis, the resistance to at least one drug was 14, 27 and 73%, respectively. Multidrug-resistant tuberculosis, defined as resistant to at least rifampicin (RMP) and isoniazid (INH), was found in the groups of patients without previous treatment, HIV co-infected and with previous treatment for tuberculosis at 10, 17 and 44%, respectively. With the purpose of evaluating whether the sensitivity test to INH and RMP would be a good marker to indicate resistance to other antituberculosis drugs, sensitivity tests were performed with four more drugs in 32 strains, initially classified as resistant to INH, RMP or both. Of 18 strains resistant to INH and RMP simultaneously, 89% showed resistance to four more drugs.     

Complex of recombinant heat shock protein with lipopolysaccharide induces rapid protection of mice against Salmonella typhimurium and avirulent for humans avian influenza virus H5N2

Protective effect of immunization with heat shock protein (HSP) against bacterial and viral infections in mice was studied. Recombinant HSP 70 kDa of Mycobacterium tuberculosis contaminated with lypopolysaccharide (0.185 mcg/ml) was used for experiments. One intraperitoneal injection of 100 or 400 mcg of HSP induced rapid protection against intraperitoneal challen e with 125 LD50 of Salmonella typhimurium (on 3rd-6th day) and against intranasal challenge with 10 LD50 of avirulent for humans avian influenza virus H5N2 (A/ mallard/Pennsylvania/10218/84) (on 5th-8th day). Three daily injections with 10 mcg of HSP induced rapid, significant and long-term protection against S. typhimurium. Immunization with HSP protected 100% of mice during 3 days after the challenge, 50% of immunized animals survived during 21 days (duration of the study). All nonimmunized mice died on 6th day.     

Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides

A pyrazinamidase (PZase)-deficient pncA mutant of Mycobacterium tuberculosis, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 microg/ml), in accordance with the well-established role of pncA in the PZA susceptibility of M. tuberculosis (A. Scorpio and Y. Zhang, Nat. Med. 2:662-667, 1996). Integration of the pzaA gene encoding the major PZase/nicotinamidase from Mycobacterium smegmatis (H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809-5814, 1998) or the M. tuberculosis pncA gene into the pncA mutant complemented its PZase/nicotinamidase defect. In both pzaA- and pncA-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The pzaA-complemented strain was hypersensitive to PZA (MIC, /=20 microg/ml) and was also sensitive to benzamide (MIC, 20 microg/ml), unlike the wild-type and pncA-complemented mutant strains, which were highly resistant to this amide (MIC, >500 microg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on M. smegmatis (MIC, 150 microg/ml in all cases) and rendered Escherichia coli hypersensitive for growth at low pH.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献   

The effect of fractionated exposure to unequal concentrations of chemical mutagens

The effect of unequally fractionated concentrations of dipin and thiophosphamid on chromosomes of human lymphocytes was investigated at Go phase. There were used five low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) mcg/ml and one high concentration 20 mcg/ml by which cells have been treated. Decrease of chromosome breaks and exchanges were observed, but the level of the aberration cell was stable. The "protective" levels for dipin were in concentrations of 0.2, 2.10(-2), 2.10(-3) mcg/ml. Only one "protective" concentration was 2.10(-2) mcg/ml.     

Unclassified Mycobacteria Isolated from Human Suspect Tuberculosis Cases in Newfoundland: Preliminary Studies on Fifteen Strains

Unclassified mycobacteria were isolated from 36 of 35,555 clinical specimens cultured for M. tuberculosis. The majority of isolations were from patients suspected of having tuberculosis and from whom repeat attempts at culture failed to yield typical tubercle bacilli. Fifteen strains thus far studied were not capable of causing generalized tuberculosis in guinea pigs, and all were highly resistant to the commonly employed antituberculous therapeutic agents. Eleven of the 15 strains were resistant to 100 or more μg./ml. of streptomycin; 12 strains were resistant to 25 or more μg./ml. of para - aminosalicylic acid; and all 15 showed growth in the presence of 50 or more μg./ml. of isoniazid. All strains were niacin-negative and catalase-positive. In the absence of other cultural findings, isolation of anonymous mycobacteria poses a major problem, especially in those cases in which the clinical and radiographic findings are typically those of tuberculosis.     

In Vitro Chemotherapeutic Combinations Against Isoniazid-Resistant Mycobacterium tuberculosis and Mycobacterium fortuitum

It is an acceptable medical practice to use second-line antimycobacterial drugs in combination with isoniazid in treatment of isoniazid-resistant tuberculosis. Recent investigations have demonstrated the importance of determining chemotherapeutic interaction in instances of multiple antibiotic use. We studied the inhibitory effect of combinations of isoniazid with ethambutol, rifampin, ethionamide, cycloserine, viomycin, and kanamycin against three isoniazid-resistant strains of Mycobacterium tuberculosis and three strains of M. fortuitum. The isobologram technique with drug concentrations of 0.4 to 100 mug/ml was used. With the exception of single instances in which kanamycin plus isoniazid (M. tuberculosis strain 9999) and ethionamide plus isoniazid (M. fortuitum strain 2080) seemed to have a synergistic effect, neither synergy nor antagonism was noted for any of the combinations. These studies show that the combined use of isoniazid and a second line antimycobacterial agent results in vitro in indifferent inhibitory activity.     

高分辨率熔解曲线分析技术检测结核分枝杆菌的耐药性

目的:探讨高分辨率熔解曲线分析(High resolution melting,HRM)技术检测结核分枝杆菌耐药突变位点的可行性。方法:对218株结核分枝杆菌进行利福平(RFP)和异烟肼(INH)的药物敏感性测定,并进行耐药基因位点的PCR扩增和测序,同时采用HRM方法检测RFP和INH耐药基因位点情况,分析HRM的敏感性和特异性。结果:218株结核分枝杆菌药敏试验结果显示,有106株(48.6%)对RFP耐药,100株(45.9%)对INH耐药,81株(37.4%)对RFP和INH均耐药。测序发现,101株(46.3%)存在RFP耐药基因的突变,107株(49.1%)存在INH耐药基因的突变。HRM检测结果显示,100株(45.9%)存在RFP耐药基因的突变,103株(47.2%)存在INH耐药基因的突变。分别以药敏试验和测序结果为标准,HRM检测RFP耐药的敏感性为94.3%(100/106)和99.0%(100/101);特异性为97.3%(109/112)和100%(117/117);INH耐药的敏感性为97.0%(97/100)和98.1%(103/105);特异性为97.3%(109/112)和100%(113/113)。结论:HRM快速检测结核分枝杆菌耐药具有较高的特异性和灵敏度,能够满足临床需求。