7H11 Agar Research Articles

Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment

Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

Evaluation of Agar-Based Medium with Sheep Sera for Testing of Drug Susceptibility of Mycobacterium tuberculosis to Isoniazid, Rifampin, Ethambutol, and Streptomycin

The performance of sheep sera instead of sheep blood in agar-based media was investigated for susceptibility testing of Mycobacterium tuberculosis against primary drugs. The levels of agreement between agar-based medium supplemented with sheep sera and the proportion method on Middlebrook 7H11 agar as the reference method for determining susceptibility to isoniazid (INH), rifampin (RIF), ethambutol (EMB), and streptomycin (STR) were 98.4, 98.4, 95.3, and 100%, respectively.

Sensititre MYCOTB MIC Plate for Testing Mycobacterium tuberculosis Susceptibility to First- and Second-Line Drugs

For Mycobacterium tuberculosis, phenotypic methods for drug susceptibility testing of second-line drugs are poorly standardized and technically challenging. The Sensititre MYCOTB MIC plate (MYCOTB) is a microtiter plate containing lyophilized antibiotics and configured for determination of MICs to first- and second-line antituberculosis drugs. To evaluate the performance of MYCOTB for M. tuberculosis drug susceptibility testing using the Middlebrook 7H10 agar proportion method (APM) as the comparator, we conducted a two-site study using archived M. tuberculosis isolates from Uganda and the Republic of Korea. Thawed isolates were subcultured, and dilutions were inoculated into MYCOTB wells and onto 7H10 agar. MYCOTB results were read at days 7, 10, 14, and 21; APM results were read at 21 days. A total of 222 isolates provided results on both platforms. By APM, 106/222 (47.7%) of isolates were resistant to at least isoniazid and rifampin. Agreement between MYCOTB and APM with respect to susceptibility or resistance was ≥92% for 7 of 12 drugs when a strict definition was used and ≥96% for 10 of 12 drugs when agreement was defined by allowing a ± one-well range of dilutions around the APM critical concentration. For ethambutol, agreement was 80% to 81%. For moxifloxacin, agreement was 83% to 85%; incorporating existing DNA sequencing information for discrepant analysis raised agreement to 91% to 96%. For MYCOTB, the median time to plate interpretation was 10 days and interreader agreement was ≥95% for all drugs. MYCOTB provided reliable results for M. tuberculosis susceptibility testing of first- and second-line drugs except ethambutol, and results were available sooner than those determined by APM.

Mycobacterium paragordonae sp. nov., a slowly growing, scotochromogenic species closely related to Mycobacterium gordonae

A previously undescribed, slowly growing, scotochromogenic mycobacterial strain (49061(T)) was isolated from a patient with pulmonary infections during the hsp65-sequence-based identification of Korean clinical isolates. Its 16S rRNA gene sequence was unique and the phylogenetic analysis based on 16S rRNA gene sequence (1393 bp) placed the organism into the slow-growing Mycobacterium group close to Mycobacterium gordonae (99.0 % sequence similarity). Growth characteristics and acid-fastness also supported the placement of this species into the genus Mycobacterium. Phenotypically, this strain was generally similar to Mycobacterium gordonae; however, of particular interest, the optimal growth temperature of strain 49061(T) was 25-30 °C, and it was not able to grow at 37 °C on 7H10 agar slants. Unique MALDI-TOF MS profiles of lipids, phylogenetic analysis based on another two gene sequences (hsp65 and rpoB) and a low DNA-DNA relatedness (46.52±0.7) strongly supported the taxonomic status of this strain as a representative of a distinct species from M. gordonae. It was concluded that the strain represents a novel species for which the name Mycobacterium paragordonae is proposed with the type strain 49061(T) ( = JCM 18565(T) = KCTC 29126(T)).

Synergistic activity of rifampicin and ethambutol against slow-growing nontuberculous mycobacteria is currently of questionable clinical significance

A key issue in the treatment of disease caused by slow-growing nontuberculous mycobacteria is the limited association between in vitro minimum inhibitory concentrations (MICs) of rifampicin and ethambutol alone and the in vivo outcome of treatment with these drugs. Combined susceptibility testing to rifampicin and ethambutol could provide a more realistic view of the efficacy of these drugs. In this study, Mycobacterium avium (n=5), Mycobacterium chimaera (n=6), Mycobacterium intracellulare (n=4), Mycobacterium xenopi (n=4), Mycobacterium malmoense (n=3) and Mycobacterium simiae (n=2) clinical isolates were selected and the MICs of rifampicin and ethambutol alone and in combination were measured using the Middlebrook 7H10 agar dilution method. Synergy was defined as a fractional inhibitory concentration index ≤0.5. Rifampicin and ethambutol showed synergistic activity against the majority of M. avium (4/5), M. chimaera (5/6) and M. intracellulare (3/4) isolates and 1 of 2 eligible M. malmoense isolates. No synergistic activity was measured against M. xenopi and M. simiae. Synergy was neither universal for all species nor for all isolates of one species; it thus needs to be tested for rather than assumed. Even if this synergy exists in vivo, it is questionable whether the MICs to the combined drugs can be overcome by the drug exposure attained by current regimens at the recommended dosages. New dosing strategies for rifampicin and ethambutol should be studied to increase the exposure to these drugs and thus maximise their impact.

Changes in the major cell envelope components of Mycobacterium tuberculosis during in vitro growth.

One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), which causes tuberculosis. Mycobacterium tuberculosis cell envelope components such as glycolipids, lipoglycans and polysaccharides play important roles in bacteria-host cell interactions that dictate the host immune response. However, little is known about the changes in the amounts and types of these cell envelope components as the bacillus divides during in vitro culture. To shed light on these phenomena, we examined growth-dependent changes over time in major cell envelope components of virulent M.tb by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, thin-layer chromatography, mass spectrometry, immunoblotting and flow cytometry. Our studies provide evidence that major mannosylated glycoconjugates on the M.tb cell envelope change as M.tb grows in vitro on the widely used Middlebrook 7H11 agar. In particular, our compositional analyses show that from Day 9 to 28 the amounts of mannose-containing molecules, such as mannose-capped lipoarabinomannan, lipomannan and phosphatidyl-myo-inositol mannosides, change continuously in both the cell envelope and outer cell surface. Along with these changes, mannan levels on the outer cell surface also increase significantly over time. The implications of these differences in terms of how M.tb is grown for studies performed in vitro and in vivo for assessing M.tb-host recognition and establishment of infection are discussed.

Field evaluation of the direct detection of multidrug resistant Mycobacterium tuberculosis by nitrate reductase assay on 7H11 agar

In this study we evaluated the performance of colorimetric nitrate reductase assay (NRA) on Middlebrook 7H11 agar instead of Lowenstein-Jensen medium for detection of isoniazid (INH) and rifampin (RIF) resistance directly on 114 smear positive sputum specimens and compared the results with direct proportion method on LJ medium. The results of both methods were in 100% agreement for detection of RIF resistance while agreement for INH was 96.4%. The average turnaround time for NRA was 18.6 days and majority of the specimens gave positive results within 21 days. Thus direct NRA testing on smear positive sputum specimens by using 7H11 agar could be used as a fast, reliable and inexpensive method in resource starved settings.

Blood Agar Validation for Susceptibility Testing of Isoniazid, Rifampicin, Ethambutol, and Streptomycin to Mycobacterium tuberculosis Isolates

In recent studies, it was shown that blood agar can be used at least as effectively as Löwenstein-Jensen medium for growing Mycobacterium tuberculosis. It was also shown that susceptibility testing can be performed on blood agar. Additional validation of blood agar was performed on regional M. tuberculosis isolates from Turkey to determine critical concentrations of isoniazid (INH), rifampicin (RIF), ethambutol (ETM), and streptomycin (STR). In the current study, 40 M. tuberculosis clinical isolates were tested. H37Rv, which is susceptible to all antituberculosis agents, ATCC 35822 (INH-resistant), ATCC 35838 (RIF-resistant), ATCC 35837 (ETM-resistant), and ATCC 35820 (STR-resistant) quality control strains were used as control strains. Proportion method on 7H11 agar was considered as gold standard in the study. MIC values of the control strains and clinical isolates were detected on blood and 7H11 agar. Categorical agreements were 100% for each antibiotic, and essential agreements were 100%, 97.5%, 82.5%, and 95% for INH, RIF, ETM, and STR, respectively. According to the data, 0.2 µg/mL for INH, 1 µg/mL for RIF, 4 µg/mL for ETM, and 2 µg/mL for STR were appropriate breakpoint values for susceptibility testing on blood agar. Blood agar may be recommended for use in both developed and developing countries for the susceptibility testing of M. tuberculosis isolates to primary antituberculosis drugs.

Detection and identification of globally distributed mycobacterial fish pathogens in some ornamental fish in India

Mycobacteriosis is a progressive disease of a wide range of wild and captive, marine and freshwater fish species. Conventional detection of fish Mycobacteria is based on histopathology, culture, and biochemical characteristics. The present study analyzed the occurrence of Mycobacteria in clinically ill ornamental fish of different species, from different places of India. In first group, 60 fish were examined for presence of granulomatous inflammation and acid-fast bacteria. Thirty-eight (63.34 %) fish were positive for granulomatous inflammations. Presences of acid-fast bacteria were detected in 27 (45 %) fish having granulomatous inflammation and in two (3.33 %) fish without granulomatous inflammation. In total, AFB were found in 29 (48.34 %) of the 60 fish examined. In second group, 20 fish having granulomatous inflammation, 12 (60 %) samples were positive using Ziehl-Neelsen (Z-N) staining and 11 (55 %) of them were culture positive. Eight (40 %) samples were Z-N negative but two (10 %) of them were culture positive. In total, 13 (65 %) of the 20 examined fish were culture positive. On the basis of biochemical tests and 16S rRNA sequencing, 13 isolates were identified: five as Mycobacterium fortuitum, five as Mycobacterium gordonae, and three as Mycobacterium chelonae. In comparison of two decontamination methods, 2 % HCl treatment was better than 4 % NaOH treatment. Mycobacteria recovery from decontaminated samples was significantly high on Lowenstein-Jensen medium compared to Middlebrook 7H11 agar and Stonebrink (SB) media. The disease is transmissible from fish to fish and also from fish to human, so the significance of Mycobacteria in ornamental fish should not be overlooked.

Rapid drug susceptibility testing for Mycobacterium tuberculosis in Thin layer agar media

There is a great need to determine the susceptibility of individual Mycobacterium tuberculosis strains as rapidly as possible because emergence of multidrug-resistant and extensively drug-resistant tuberculosis in developing countries. The study was conducted to evaluate the thin layer agar (TLA) media for rapid detection of resistance of M.tuberculosis to rifampicin (RMP) and isoniazid (INH) in clinical isolates and to determine the sensitivity and time to positivity compared to the proportion method. One hundred clinical isolates of M.tuberculosis were studied. For the TLA method, three compartment Petri plate containing 7H11 agar and 7H11 agar with RMP and INH. Results were compared to the proportion method for RMP and INH. The sensitivity for INH and RMP+INH was 85.7 % and 100%. The use of a TLA plate enables the rapid detection of resistance to the two prime anti-tuberculosis drugs RMP and INH in a median time of 9.60 days. TLA was a rapid method for the detection of resistance of M.tuberculosis in the two drugs studied. This faster method is simple to perform, providing an alternative method when more sophisticated techniques are not available in low-resource settings.DOI: http://dx.doi.org/10.3329/bjmm.v7i1.19313 Bangladesh J Med Microbiol 2013; 07(01): 2-6

Identification of Growth Promoting Effect of &amp;lt;i&amp;gt;r&amp;lt;/i&amp;gt;BCG/BCG Culture Supernatant and Its Potential Applications

Background: Although BCG is the most widely administered vaccine in the world, there have never been as many cases of TB as there are now. Globally, more than 8.8 million people developed active TB and 1.4 million—many of them—died in 2010. It is estimated that half of pulmonary TB cases arise from latent Mtb infection, making the study of latency and reactivation of utmost importance. Methods: Widely administered BCG vaccines and a gene modified recombinant BCG (rBCG) strain, AERAS-422, were used as models to investigate the growth promoting function of resuscitation-promoting factors (Rpfs) in different bacilli culture phases. Different supernatant fractions were prepared by ultrafiltration, and the promoting function of each fraction containing secreted Rpf(s) was evaluated by growth curve monitoring and colony counting on 7H10 agar plates. Results: The promoting effect of culture supernatants was mainly associated with the high molecular weight fraction (>30 kDa), which stimulated bacterial growth, but did not extend the exponential phase of stimulated culture. Anti-RpfB antibody showed significant growth restriction of the tested cultures. When comparing rBCG cultures containing 7H9 medium, the 10 - 30 kDa fraction, or the >30 kDa fraction, only the >30 kDa fraction was displayed with down-regulation of the secretion of RpfC, D and E. In colony counting tests, the plates containing the >30 kDa fraction had total countable colony numbers 2 to 3 fold higher than the plates with the 10 - 30 kDa fraction, and colonies appeared one to two weeks earlier than on the regular plates. The potential applications of the prepared supernatant fractions containing RpfA and RpfB are discussed, which may include accelerating diagnosis of Mtb infection and future TB vaccine development.

Preliminary study on the In-Vitro Susceptibility of Mycobacterium tuberculosis Isolates to Virgin Coconut Oil

Background: Tuberculosis remains a major public health threat. Studies have shown that medium chain fatty acids (MCFA), such as, those found in Virgin Coconut Oil (VCO), possess activity against a wide range of microorganisms including Mycobacterium tuberculosis. The goal of the study was to determine the in-vitro susceptibility of M. tuberculosis (TB) isolates to two commercial brands of VCOs each containing different amounts of lauric acid and produced by two different extraction processes. Two brands of VCO were tested for their anti-TB actions compared to pure lauric acid and a negative control. Two replicate runs were done. VCO samples were inoculated with 0.1 mL of the H37RV strain of M. tuberculosis and incubated at 370C with shaking for 5 days. After incubation, the mixtures were diluted 10-fold up to 10-4 concentration to obtain countable colonies using Middlebrook 7H10 agar plates and Lowenstein-Jensen (L-J) slants. Colony growths of M. tuberculosis were examined in each dilution every week for three to eight weeks. Results: Both brands of VCO at increasing dilution from 1:10 to 1:10000 lowered the colony counts of M. tuberculosis by 46% to 100 % on the Middlebrook 7H10 agar. Similar results were noted on L-J slants with colony counts decreasing by 25% to 96%. Lauric acid solution showed complete inhibition of bacterial growth in both the agar plates and LJ slants. Conclusion: Different VCO preparations containing different amounts of lauric acid exhibited different degree of inhibition against M. tuberculosis. The result of the study suggests the potential of VCOs agent against the growth of M. tuberculosis Similar effect on other organisms is a possibility and should also be explored. But more studies are needed to replicate the studies using different concentrations of VCOs, lauric acid and other fatty acids in VCO such as myristic acid, caproic or caprylic acids.Key words: Lauric acid, virgin coconut oil, tuberculosis, food supplement

Improved productivity of recombinant human-like collagen ii by supplying amino acids encoded by rare codons

The spread of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis has become a challenge to the global tuberculosis control and prevention program.Nevertheless, no adequate information has been made available on the drug susceptibility status of M. tuberculosis in Ethiopia. Hence, this study aimed atevaluating the drug resistance patterns of M. tuberculosis in the three sites of Ethiopia and its association with socio-demographic factors. A total of 102 M. tuberculosis isolates obtained from tuberculosis (TB) patients from three different sites were tested for drug susceptibility against the first and second-line anti-TB drugs using the proportion method on enriched Middle Brook 7H10 agar. The result showed that 36.3% of the isolates were resistant to at least one of the first line anti-TB drugs used in this study. The proportions of mono-resistance were 29.4, 13.7, 13.7 and 23.5% to isoniazid, rifampicin, streptomycin and ethambutol, respectively. The proportion of MDR M. tuberculosis was 11.8%. Only 55% of the isolates were susceptible to all the second line anti-TB drugs used in this study. One isolate (1%) was extensively drug-resistant (XDR). The occurrence of MDR M. tuberculosisisolates was not associated with sex, age, anti-TB treatment history, HIV status andlocation. The findings of this study showed moderate number of MDR M. tuberculosis isolates in the study area, warranting the necessity of the surveillance and monitoring. Key words: Mycobacterium tuberculosis, drug resistance, Ethiopia

Synthesis and Antibacterial Activity of Some Novel 1-(4-Oxo-3-(3- methoxyphenyl)-3H-quinazolin-2-yl)-4-(substituted) thiosemicarbazides

A new series of novel 1-(4-oxo-3-(3-methoxyphenyl)-3H-quinazolin-2-yl)-4-(substituted) thiosemicarbazides were synthesized by the reaction of 2-hydrazino-3-(3-methoxyphenyl) quinazolin-4(3H)-one with various methyl esters of dithiocarbamic acid. When tested for their in vitro antitubercular activity using H37Rv strain on Middle brook 7H11 agar slants with OADC growth supplement, all the test compounds inhibited the growth of Mycobacterium tuberculosis at microgram concentration. Among the test compounds, three compounds 1-(4-oxo-3-(3-methoxy phenyl)-3H-quinazolin-2-yl)-4-(thiazol- 2-yl) thiosemicarbazide (A1), 1-(4-oxo-3-(3-methoxyphenyl)-3H-quinazolin-2-yl)-4-(4-chlorophenyl) thiosemi-carbazide (A6) and 1-(4-oxo-3-(3-methoxyphenyl)-3H-quinazolin-2-yl)-4-(4-nitrophenyl) thiosemicarbazide (A7) were inhibited at the 6 μg/mL concentration. The title compounds are also screened for the antimicrobial activity against some other gram positive and gram negative bacteria by agar dilution method, compounds A6 and A7 were emerged as the most active compounds of the series. Compound A6 showed most potent activity against E. coli, K. pneumoniae and B. subtilis.

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose-1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α(2)-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.

High Prevalence of Primary Multidrug Resistant Tuberculosis in Persons with No Known Risk Factors

IntroductionIn high multidrug resistant (MDR) tuberculosis (TB) prevalence areas, drug susceptibility testing (DST) at diagnosis is recommended for patients with risk factors for MDR. However, this approach might miss a substantial proportion of MDR-TB in the general population. We studied primary MDR in patients considered to be at low risk of MDR-TB in Lima, Peru.MethodsWe enrolled new sputum smear-positive TB patients who did not report any MDR-TB risk factor: known exposure to a TB patient whose treatment failed or who died or who was known to have MDR-TB; immunosuppressive co-morbidities, ex prison inmates; prison and health care workers; and alcohol or drug abuse. A structured questionnaire was applied to all enrolled participants to confirm the absence of these factors and thus minimize underreporting. Sputum from all participants was cultured on Löwenstein-Jensen media and DST for first line drugs was performed using the 7H10 agar method.ResultsOf 875 participants with complete data, 23.2% (203) had risk factors for MDR-TB elicited after enrolment. Among the group with no reported risk factors who had a positive culture, we found a 6.3% (95%CI 4.4–8.3) (37/584) rate of MDR-TB. In this group no epidemiological characteristics were associated with MDR-TB. Thus, in this group, multidrug resistance occurred in patients with no identifiable risk factors.ConclusionsWe found a high rate of primary MDR-TB in a general population with no identifiable risk factors for MDR-TB. This suggests that in a high endemic area targeting patients for MDR-TB based on the presence of risk factors is an insufficient intervention.

Nasal Fish Tank Granuloma: An Uncommon Cause for Epistaxis

An immunocompetent 28-year-old male aquarium store employee presented with 1 month of intermittent epistaxis. A bleeding granulomatous lesion was identified over the right nasal septum (Figure 1). Histopathology from the incisional biopsy revealed granulomatous inflammation with the presence of acid-fast bacilli (Figure 2). Culture of the homogenized specimen on Middlebrook 7H11 agar after 14 days displayed white clones of non-tuberculous Mycobacterium (NTM) that turned brilliant yellow upon light exposure (Figure 3). Polymerase chain reaction (PCR) amplification of the 65-kDa heat shock protein gene with restriction enzyme analysis using BstE II and Hae III confirmed the diagnosis of Mycobacterium marinum infection. The lesion regressed completely with 3 months of doxycycline treatment. Figure 1. Anterior rhinoscopy revealed a granulomatous lesion with active oozing (arrow) on the right nasal septum. Inferior turbinate was identified as an asterisk. Figure 2. Histopathology of the specimen disclosed acid-fast bacilli (arrow) with the granulation inflammation. Figure 3. Mycobacterium marinum clones on Middlebrook 7H11 agar plates showed the photochromogenic characteristic, which are white when grown in the dark (A) and turn a brilliant yellow soon after exposure to light (B). Fish tank granuloma is a rare granulomatous skin infection caused by Mycobacterium marinum, an opportunistic NTM, commonly found in aquatic environments.1 Extremities are most frequently affected; nasal cavity infection is seldom reported. The diagnosis is generally based on a history of aquatic exposure (occupational or recreational), histopathology of granulomatous inflammation with or without the presence of acid-fast bacilli, and a slow-growing photochromogenic NTM incubated at low temperature (28–30°C). Furthermore, PCR facilitates an early identification of Mycobacterium marinum infection. Superficial lesion sometimes resolves spontaneously, but several months of antimicrobial drug are usually necessary. Various monotherapies, including doxycycline, are effective.2

Mutations in therrsA1401G Gene and Phenotypic Resistance to Amikacin and Capreomycin inMycobacterium tuberculosis

The aminoglycosides amikacin (AMK)/kanamycin (KAN) and the cyclic polypeptide capreomycin (CAP) are important injectable drugs in the treatment of multidrug-resistant tuberculosis. Cross-resistance among these drug classes occurs and information on the minimum inhibitory concentrations (MICs), above the normal wild-type distribution, may be useful in identifying isolates that are still accessible to drug treatment. Isolates from the Eastern Cape Province of South Africa were subjected to DNA sequencing of the rrs (1400-1500 region) and tlyA genes. Sequencing data were compared with (i) conventional susceptibility testing at standard critical concentrations (CCs) on Middlebrook 7H11 agar and (ii) MGIT 960-based MIC determinations to assess the presence of AMK- and CAP-resistant mutants. Isolates with an rrs A1401G mutation showed high-level resistance to AMK (>20 mg/L) and decreased phenotypic susceptibility to CAP (MICs 10-15 mg/L). The MICs of CAP were below the bioavailability of the drug, which suggests that it may still be effective against multi- or extensively drug resistant tuberculosis [M(X)DR-TB]. Agar-based CC testing was found to be unreliable for resistance recognition of CAP in particular.

In vitro antimycobacterial activities of a new quinolone, balofloxacin

Balofloxacin (BLFX), a newly developed fluoroquinolone, was studied for its in vitro antimycobacterial activity by the agar dilution method with 7H11 agar medium. The MIC90s were as follows: 0.39 microgram/ml for M. tuberculosis, > 50 micrograms/ml for M. avium, > 50 micrograms/ml for M. intracellulare, 0.39 microgram/ml for M. kansasii, 0.39 microgram/ml for M. fortuitum, > 50 micrograms/ml for M. abscessus, and 50 micrograms/ml for M. chelonae. The antimycobacterial activity of BLFX was comparable or slightly inferior to that of levofloxacin (LVFX). Considering the present findings and pharmacokinetics of BFLX, it appears that BFLX may achieve favorable outcome in the treatment of patients with infection due to M. tuberculosis, M. kansasii, or M. fortuitum similar to that of ofloxacin or LVFX.

Interrelationship among colonial morphology, susceptibility to antituberculosis drugs, growth rate, and viability in Dubos liquid medium in Mycobacterium avium complex strains

Interrelationship among colonial morphology, susceptibility to antituberculosis drugs, growth rate, and viability in Dubos liquid medium were studied in Mycobacterium avium complex strains, which were not exposed to any antituberculosis drugs. All five strains that were relatively susceptible to the majority of antituberculosis drugs formed smooth, opaque, domed colonies (SmD) on 7H10 agar medium, and all five strains that were naturally resistant to those drugs formed smooth, transparent, flat colonies (SmT). The SmD colony-forming strains showed small, domed, smooth colonies (DS-type) on Ogawa egg medium, and the SmT colony-forming strains usually large, flat, wrinkled colonies (FW-type) on Ogawa egg medium. However, there was one exceptional strain (11004) that formed DS-type colonies on Ogawa egg medium and SmT-type on 7H10 agar medium. Accordingly, the SmD morphology correlated with drug susceptibility and the SmT colonial morphology with drug resistance. All six DS-colony-forming strains had slow growth rates on Ogawa egg medium, whereas all four FW-colony-forming strains had rapid growth rates on Ogawa egg medium. However, such correlation was not observed in Dubos liquid medium. When the viability was defined as a ratio of the number of colony-forming units contained in 1 mg wet weight of bacteria, the five drug-susceptible strains usually showed low viability and the five drug-resistant strains showed high viability in the Dubos liquid medium. The above four characteristics, colonial morphology, drug susceptibility, growth rate on Ogawa egg medium, and viability in Dubos liquid medium, usually correlated with each other.(ABSTRACT TRUNCATED AT 250 WORDS)