Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase.

Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TlyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TlyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.

Tigecycline Is Highly Efficacious against Mycobacterium abscessus Pulmonary Disease.

Mycobacterium abscessus causes chronic pulmonary infections that are extremely difficult to cure. The currently recommended combination therapy is associated with high failure rates and relapse. Tigecycline has been explored in salvage regimens, with a response rate of 43% in those who received at least a month of therapy. We performed a dose-response study in a hollow-fiber system model of pulmonary M. abscessus infection in which we recapitulated tigecycline human pulmonary concentration-time profiles of 8 different doses for 21 days. We identified the maximal kill or efficacy in CFU per milliliter and the ratio of the 0- to 24-h area under the concentration-time curve to MIC (AUC/MIC) associated with 80% efficacy (EC80). The tigecycline efficacy was 5.38 ± 2.35 log10 CFU/ml, and the drug achieved the unprecedented feat of a bacterial level of 1.0 log10 CFU/ml below the pretreatment inoculum (1-log kill) of M. abscessus in the hollow-fiber system. The EC80 AUC/MIC ratio was 36.65, while that for a 1-log kill was 44.6. Monte Carlo experiments with 10,000 patients were used to identify the clinical dose best able to achieve the EC80 or 1-log kill. The standard dose of 100 mg/day had a cumulative fraction of response of 51% for the EC80 and 46% for 1-log kill. For both the EC80 target and 1-log kill, the optimal tigecycline clinical dose was identified as 200 mg/day. The susceptibility breakpoint was ≤0.5 mg/liter. Tigecycline is the most active single agent evaluated to date, and we propose that 200 mg/day be examined as the backbone of new combination therapy regimens to replace current treatment.

[Provincial assessment in the scope of the tuberculosis control program]. / Tüberküloz kontrol programi kapsaminda il degerlendirmesi.

INTRODUCTION: We aimed to expose the status of tuberculosis in Corum, to analyze the conducted studies about the tuberculosis control on provincial basis and contribute to future studies. MATERIALS AND METHODS: The records of the patients who were followed and treated between 2005 and 2010 at tuberculosis dispensary in Corum were respectively investigated. The statistical analyses of the data were completed as using SPSS 16.0. RESULTS: A total of 628 patients were enrolled in this study; 59.8% (n= 376) were male, 40.2% (n= 252) were female. The ratio of the pulmonary and the extrapulmonary involvement were detected as 63.7% (n= 400) and 36.3% (n= 228) respectively. The incidence of new cases was 93.5% (n= 587) whereas the percentage of previously treated cases was 6.5% (n= 41). The 0.7% (n= 4) percentage of the patients were multi-drug resistant therefore they had been treated with secondary group of drugs. 400 patients with pulmonary tuberculosis were investigated about the ratios of the following parameters; performed microscopic examination, the positivity of the microscopic examination, the performed culture examination, the positivity of the culture examination and the performed drug susceptibility test. According to this; the results were determined as 85.5% (n= 334), 44.5% (n= 178), 66% (n= 264), 35% (n= 140) and 15% (n= 60) respectively. Directly observed treatment was performed 49.7% (n= 312) of the patients by health care workers. The success of treatment for all patients with tuberculosis was determined as 93.8% (n= 576). CONCLUSION: According to the data of our study, we can conclude that, although there were some deficiencies about control of tuberculosis, the conducted control program was successful in Corum. However; having a large number of young patients with tuberculosis proved that the transmission was still going on. Besides; the examined tests like microscopy, culture and drug susceptibility were found low in rates. The practices of directly observed treatment should provide to be improved.

Detection and assessment of clarithromycin inducible resistant strains among Korean Mycobacterium abscessus clinical strains: PCR methods.

BACKGROUND: Mycobacterium abscessus group belongs to a group of rapidly growing mycobacteria (RGM) and, following Mycobacterium avium complex, is the second most common pathogen responsible for lung disease caused by nontuberculous mycobacteria (NTM). Clarithromycin is known to be the key drug in the treatment of M. abscessus group disease, but a high failure rate of treatment response is reported due to clarithromycin inducible resistance. METHODS: Using the results from a clarithromycin susceptibility test we examined the proportion of clarithromycin inducible resistant M. abscessus (sensu stricto; hereafter referred to as M. abscessus) clinical strains. Also, we attempted to detect the clarithromycin resistant strains, using the amplification refractory mutation system-PCR (ARMS-PCR) and real-time PCR methods for rapid detection of single-nucleotide polymorphisms (SNPs) at position 28 (T or C) of the erm(41) gene of M. abscessus leading to resistance to clarithromycin. RESULTS: Of the 157 M. abscessus clinical strains, clarithromycin susceptible, resistant, and inducible resistant strains accounted for 10.83% (n = 17), 22.29% (n = 35), and 66.88% (n = 105), respectively. Clarithromycin resistant strains were able to separate from clarithromycin susceptible strains by ARMS-PCR and real-time PCR identical to DNA sequence analysis. CONCLUSION: Most M. abscessus clinical strains in Korea are resistant to clarithromycin, and ARMS-PCR and real-time PCR are useful tools for the rapid detection of single-nucleotide polymorphisms (SNPs) at position 28 of the erm(41) gene.

Assessment of clarithromycin susceptibility in strains belonging to the Mycobacterium abscessus group by erm(41) and rrl sequencing.

Clarithromycin was the drug of choice for Mycobacterium abscessus infections until inducible resistance due to erm(41) was described. Because M. abscessus was split into M. abscessus sensu stricto, Mycobacterium massiliense, and Mycobacterium bolletii, we looked for erm(41) in the three species and determined their clarithromycin susceptibility levels. Ninety strains were included: 87 clinical strains from cystic fibrosis patients (61%) and others (39%), representing 43 M. abscessus, 30 M. massiliense, and 14 M. bolletii strains identified on a molecular basis, and 3 reference strains. Clarithromycin and azithromycin MICs were determined by broth microdilution and Etest with a 14-day incubation period. Mutations in rrl (23S rRNA gene) known to confer acquired clarithromycin resistance were also sought. erm(41) was detected in all strains but with two deletions in all M. massiliense strains. These strains were indeed susceptible to clarithromycin (MIC(90) of 1 µg/ml) except for four strains with rrl mutations. M. abscessus strains harbored an intact erm(41) but had a T/C polymorphism at the 28th nucleotide: T28 strains (Trp10 codon) demonstrated inducible clarithromycin resistance (MIC(90) of >16 µg/ml), while C28 strains (Arg10) were susceptible (MIC(90) of 2 µg/ml) except for two strains with rrl mutations. M. bolletii strains had erm(41) sequences similar to the sequence of the T28 M. abscessus group, associated with inducible clarithromycin resistance (MIC(90) of >16 µg/ml). erm(41) sequences appeared species specific within the M. abscessus group and were fully concordant with clarithromycin susceptibility when erm(41) sequencing was associated with detection of rrl mutations. Clarithromycin-resistant strains, including the six rrl mutants, were more often isolated in cystic fibrosis patients, but this was not significantly associated with a previous treatment.

[Assessment of in vitro susceptibility to antimicrobials of rapidly growing mycobacteria by E-test]. / Sensibilidad a los antimicrobianos de micobacterias de crecimiento rápido mediante el método E-test.

BACKGROUND: Rapidly growing mycobacteria (RGM) are considered opportunistic pathogens. An increasing number of post traumatic or surgical infections are caused by these microorganisms. AIM: To determine the antimicrobial susceptibility of RGM using the E-test method. MATERIAL AND METHODS: A total of 54 isolates of RGM was obtained from several clinical samples and selected for this study Strains were identified to the species level by phenotypic and biochemical characteristics, PCR-restriction enzyme analysis of the hsp65 gene (PRA) and sequencing of the 16S rRNA. Susceptibility was investigated by E-test to amikacin, cefoxitin, ciprofioxacin, clarithromycin, imipenem, quinupristin/dalfopristin, linezolid and tigecycline. RESULTS: Twelve different species of RGM were identified: Mycobacterium fortuitum (23 strains), M chelonae (11), M abscessus (10), Msenegalense (2), Malvei (1), Mbrumae (1), Mmageritense (1), mucogenicum (1), M neoaurum (1), Mperegrinum (1), M septicum (1) y M smegmatis (1). All the strains were inhibited by low concentrations of amikacin and tigecycline. Susceptibility to cefoxitin, fluoroquinolones, clarithromycin, imipenem and linezolid was variable. All but two strains were resistant to quinupristin/ dalfopristin. CONCLUSIONS: Due to the uneven antimicrobial susceptibility of different species of RGM, an antimicrobial susceptibility test is mandatory for these microorganisms. The E-test method is well suited to determine minimum inhibitory concentrations.

Sensibilidad a los antimicrobianos de micobacterias de crecimiento rápido mediante el método E-test / Assessment of in vitro susceptibility to antimicrobials of rapidly growing mycobacteria by E-test

Background: Rapidly growing mycobacteria (RGM) are considered opportunistic pathogens. An increasing number of post traumatic or surgical infections are caused by these microorganisms. Aim: To determine the antimicrobial susceptibility of RGM using the E-test method. Material and methods: A total of 54 isolates of RGM was obtained from several clinical samples and selected for this study Strains were identified to the species level by phenotypic and biochemical characteristics, PCR-restriction enzyme analysis of the hsp65 gene (PRA) and sequencing of the 16S rRNA. Susceptibility was investigated by E-test to amikacin, cefoxitin, ciprofioxacin, clarithromycin, imipenem, quinupristin/dalfopristin, linezolid and tigecycline. Results: Twelve different species of RGM were identified: Mycobacterium fortuitum (23 strains), M chelonae (11), M abscessus (10), Msenegalense (2), Malvei (1), Mbrumae (1), Mmageritense (1), mucogenicum (1), M neoaurum (1), Mperegrinum (1), M septicum (1) y M smegmatis (1). All the strains were inhibited by low concentrations of amikacin and tigecycline. Susceptibility to cefoxitin, fluoroquinolones, clarithromycin, imipenem and linezolid was variable. All but two strains were resistant to quinupristin/ dalfopristin. Conclusions: Due to the uneven antimicrobial susceptibility of different species of RGM, an antimicrobial susceptibility test is mandatory for these microorganisms. The E-test method is well suited to determine minimum inhibitory concentrations.

Examination of specimens for mycobacteria in clinical laboratories in 21 countries: a 10-year review of the UK National Quality Assessment Scheme for Mycobacteria Culture.

Results from clinical diagnostic microbiology laboratories taking part in the UK National Quality Assessment Service (UK NEQAS) scheme for Mycobacteria Culture between 1993 and 2003 were evaluated and assessed to determine whether the perceived increase in the use of rapid methods is improving time-to-positive reporting of results. Four simulated sputum specimens containing mycobacteria in mixed cultures with normal commensal organisms were distributed three times a year. Participating laboratories were required to report on the presence of 'mycobacteria' and on the time required to obtain a positive result. The overall level of performance with the mycobacteria culture external quality assessment specimens remained consistently high, with an average success rate of 94% over 10 years. The mean time-to-positive decreased from 24 to 17 days during the previous 8 years. A survey questionnaire, circulated in 2002, addressed the use of continuous automated mycobacterial liquid culture (CAMLiC) and molecular methods. The increase in the use of rapid culture methods for the detection of Mycobacterium tuberculosis has resulted in an overall reduction in time-to-positive data reported by participants, and has provided an indication of participants' ability to meet the 21-day target recommended by the CDC for the detection and identification of M. tuberculosis.

Antibacterials - mechanisms of action.

Recent studies on antibacterials have focused on the development of antimycobacterial agents and antibacterial peptides, and on furthering the understanding of agents that have been available for several decades, including imidazoles, beta-lactams and quinolones. New areas of research include antisense oligonucleotides, antibacterial peptides and a new class of agents, oxazolidinones.

Achievements and difficulties in maintaining the tuberculosis-free status of Hungarian cattle herds.

The tuberculosis-free status of Hungarian cattle herds in the period between 1988 and the end of 1993 is evaluated. An epidemiological analysis of tuberculin tests, laboratory assays and allergic tests yielding positive results, summarized in three tables, is given with respect to the cattle population of Hungary. The origin of positive reactions obtained in the tuberculin tests was traced in different farms of a total of 323 communities. On those farms, the diagnostic slaughter and examination of 1,851 breeding animals to exclude or confirm Mycobacterium bovis infections involved substantial economic losses. From 25 outbreaks investigated in the period of study, a total of 191 M. bovis strains were isolated from the organs of 21 cattle in 15 household stocks in 14 communities, as well as from 170 bovine organ samples from large farms of 10 communities (7 agricultural co-operatives and 3 state farms). In all of these cases, infection could be traced back to humans excreting M. bovis. Determination of the 2-thiophenecarboxylic hydrazide (TCH) resistance of the isolates facilitated the epidemiological investigation. The paper also contains some recommendations on the prevention or reduction of losses.

Assessment of mycobacterial viability by RNA amplification.

We investigated whether the presence of intact RNA is a valuable indicator of viability of mycobacteria with Mycobacterium smegmatis. M. smegmatis was exposed to various concentrations of rifampin and ofloxacin suspended in broth for different periods of time. The NASBA nucleic acid amplification system was used because of its rapid, sensitive, and specific detection of 16S rRNA. During drug exposure, the viability of the mycobacteria, expressed by the number of CFU, was compared with the presence of 16S rRNA as determined by NASBA and with the presence of DNA coding for 16S rRNA as determined by PCR. Both NASBA and PCR were shown to have a detection limit of approximately 5 x 10(2) CFU/ml. The intensity of the NASBA signal corresponded well with the number of CFU, and the lack of NASBA signal coincided with a loss of viability, which was reached after 3 days of exposure to bactericidal concentrations of both drugs. The presence of mycobacterial DNA, as determined by the intensity of the PCR signal, and the viability of M. smegmatis were not related, but an increase in the number of cells and intensity of PCR signal correlated well. Bacterial viability may thus be assessed by a rapid, sensitive, and specific, and semiquantitative technique by using NASBA. This system of viability testing provides the potential for rapid evaluation of drug susceptibility testing.