12B Medium Research Articles

Synthesis, Characterization, and Evaluation of Antimicrobial Activity of Some 1,2,4-Triazole Derivatives Bearing an Antipyryl Moiety

Some novel 4-[[2-[[5-(2-furanyl)-4-alkyl/aryl-4H-1,2,4-triazol-3-yl]thio]-acetyl/propionyl]-amino]-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazoles were synthesized and evaluated for in vitro antibacterial activity against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212 and antifungal activity against Candida albicans ATCC 10231, Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida parapsilosis, Trichophyton mentagrophytes var. erinacei NCPF 375, Microsporum gypseum NCPF 580, and Trichophyton rubrum using the microbroth dilution method. All of the compounds were found to be ineffective against the above bacteria within the applied MIC ranges. On the other hand, they were effective against fungi to different degrees. Three compounds showed high activity against C. parapsilosis and T. mentagrophytes var. erinacei NCPF 375 (MIC = 8 μg cm−3). The in vitro antimycobacterial activity of the new compounds was also investigated against Mycobacterium tuberculosis H37RV (ATCC 27294) in BACTEC 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA). Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. The most active compound was found with 66% inhibition at >6.25 μg cm−3.

Synthesis and Antituberculosis Activity of Cycloalkylidenehydrazide and 4‐Aza‐1‐thiaspiro[4.5]decan‐3‐one Derivatives of Imidazo[2,1‐b]thiazole.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Reliability of hsp65-RFLP analysis for identification of Mycobacterium species in cultured strains and clinical specimens

The purpose of this study was to evaluate the reliability of an amplification restriction analysis based method ( hsp65-RFLP) to detect and identify mycobacterial species in clinical samples and cultures with low number of bacilli. We examined 247 clinical specimens and 88 culture vials, comparing hsp65-RFLP results with conventional culture/biochemical tests. The analytical sensitivity of the method was assessed with cerebrospinal fluid (CSF), broncho-alveolar lavage (BAL), sputum, water, and 12B medium containing defined amounts of mycobacterial chromosome. We detected the equivalent of 10 3 cells per ml in all samples, except sputum, the most common source of clinical sample for mycobacterial testing, which presented inhibition throughout. We investigated two purification procedures to overcome inhibition of DNA amplification: DNAzol and phenol/chloroform. The former was superior, eliminating inhibition in 93.7% of the clinical samples. The technique was effective for bacterial cultures, including those with very low growth indices (GIs), substantially abbreviating time for diagnosis, but showed low sensitivity (25%) when applied to clinical samples, an issue that has never been extensively assessed by other researchers.

Antituberculous Activity of Norfloxacin Mannich Bases with Isatin Derivatives

Mannich bases of norfloxacin were synthesized by reacting them with formaldehyde and several isatin derivatives. The compounds were evaluated in vitro against Mycobacterium tuberculosis H₃₇R_v at 12.5 µg/ml in BACTEC 12B medium using the BACTEC radiometric system. Among the compounds tested, S-10 showed promising activity, with 100% inhibition at a concentration lower than 6.25 µg/ml.

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for...

Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls.

Mycobacterium avium subsp. paratuberculosis has been incriminated as a cause of Crohn's disease (CD); however, studies to date have been relatively small and generally only used a single diagnostic assay. The objective of the study was to reexamine the association of M. avium subsp. paratuberculosis and CD using multiple diagnostic tests. Five methods were used to detect M. avium subsp. paratuberculosis infections in 439 inflammatory bowel disease (IBD) patients and 324 control subjects in the United States and Denmark. Most assays were adaptations of diagnostic tests for this infection performed routinely on animals. PCR for IS900, a genetic element unique to M. avium subsp. paratuberculosis, was positive significantly more often on resected bowel and lymph node tissues from CD patients (19.0%) and ulcerative colitis (UC) patients (26.2%) than from controls (6. 3%) (P < 0.05). Positive IS900 PCR results occurred more often in U. S. than in Danish IBD patients, 32.0 versus 13.3% (P = 0.025). The majority of Danish patients were bacillus Calmette-Guérin (Mycobacterium bovis BCG) vaccinated (CD, 77.5%; UC, 86.6%; controls, 83.0%) whereas none of the U.S. patients with IBD and only 2% of U. S. controls were vaccinated. Among Danish IBD patients, positive PCR findings were four times more common among subjects who were not BCG vaccinated (33.3%) than among BCG vaccinates (8.8%, P = 0.02). Culture of the same tissues tested by PCR using modified BACTEC 12B medium failed to grow M. avium subsp. paratuberculosis from patients or controls. U.S. CD patients had the highest serological evidence (enzyme-linked immunosorbent assay [ELISA] for serum antibodies) of M. avium subsp. paratuberculosis infection (20.7% of patients positive) which was higher than for all UC patients studied (6.1%) or healthy controls (3.8%, P < 0.005). Among Danish patients alone, however, no significant differences in rates of ELISA-positive results among CD, UC, or control patients were found. For 181 study subjects, both IS900 PCR and ELISA were performed. Although 11 were ELISA positive and 36 were PCR positive, in no instance was a patient positive by both tests, suggesting that these states are mutually exclusive. Evaluation of cytokine-mediated immune responses of IBD patients was complicated by the influence of immunosuppressive therapy given most IBD patients. Gamma interferon (IFN-gamma) release by peripheral blood leukocytes after M. avium purified protein derivative PPD antigen stimulation showed significantly lower responses in CD patients than in UC patients or controls in both U.S. (by ex vivo assay) and Danish (by in vitro assay) populations (P < 0.05). Interleukin-5 responses were not different among CD, UC, or control groups. Collectively, the PCR, ELISA, and IFN-gamma tests for M. avium subsp. paratuberculosis together with the unexpected observation that BCG vaccination influenced M. avium subsp. paratuberculosis detection, lead us to conclude that M. avium subsp. paratuberculosis, or some similarly fastidious mycobacterial species, infects at least a subset of IBD patients. Whether the infection is primary (causal) or secondary, it may contribute to the etiopathogenesis of IBD.

Comparison of Recoveries of Mycobacterium tuberculosis Using the Automated BACTEC MGIT 960 System, the BACTEC 460 TB System, and Löwenstein-Jensen Medium

Using two different liquid media and one conventional solid medium, a total of 57 mycobacterial isolates (Mycobacterium tuberculosis, n = 55; nontuberculous mycobacteria, n = 2) were recovered from 377 clinical specimens. The rates of recovery of M. tuberculosis were 96.4% with the BACTEC MGIT 960 liquid medium, 92.7% with BACTEC 12B liquid medium, and 81.8% with the Löwenstein-Jensen (LJ) medium. The mean time to detection of M. tuberculosis in smear-positive specimens was 12.6 days for BACTEC MGIT 960 medium, 13.8 days for BACTEC 12B medium, and 20.1 days for LJ medium, and in smear-negative specimens it was 15.8 days for BACTEC MGIT 960 medium, 17.7 days for BACTEC 12B medium, and 42.2 days for LJ medium. The rates of contamination were 3.7, 2.9, and 1.2% for the BACTEC MGIT 960, BACTEC 12B, and LJ media, respectively. In conclusion, the nonradiometric, fully automated 7-ml BACTEC MGIT 960 system can be considered a viable alternative to the semiautomated, radiometric BACTEC 460 TB system.

Evaluation of modified BACTEC 12B radiometric medium and solid media for culture of Mycobacterium avium subsp. paratuberculosis from sheep.

Definitive diagnosis of Johne's disease in ruminants depends on confirming the presence of the causative bacterium, Mycobacterium avium subsp. paratuberculosis, in tissues of the host. This is readily achieved in most ruminant species by culture. However, culture of clinical specimens from sheep in many countries has been unrewarding. Such a culture from sheep was achieved recently in Australia by using a radiometric culture medium. The aims of the present study were to evaluate the culture of M. avium subsp. paratuberculosis from sheep by using modified BACTEC 12B radiometric medium, to determine the sensitivity of culture in relation to histopathology, and to evaluate a range of solid media. Culture of M. avium subsp. paratuberculosis from sheep with Johne's disease is a sensitive method of diagnosis: intestinal tissues from all 43 animals with multibacillary disease and all 22 animals with paucibacillary disease were culture positive, while 98% of feces from 53 animals with multibacillary disease and 48% of feces from 31 animals with paucibacillary disease were culture positive. Of sheep without histological evidence of Johne's disease from infected flocks, intestinal tissue from 32% of 41 were culture positive, while feces from 17% of 41 were culture positive. Consequently, culture is recommended as the "gold standard" test for detection of ovine Johne's disease. Of the wide range of solid media that were evaluated, only modified Middlebrook 7H10 and 7H11 agars, which were very similar in composition to modified BACTEC 12B medium, yielded growth of ovine strains of M. avium subsp. paratuberculosis. The sensitivity of detection of M. avium subsp. paratuberculosis on solid media was slightly lower than that in modified BACTEC 12B radiometric medium. Both egg yolk and mycobactin J were essential additives for growth of ovine strains of M. avium subsp. paratuberculosis in both liquid and solid media.

Cord formation in BACTEC medium is a reliable, rapid method for presumptive identification of Mycobacterium tuberculosis complex.

Serpentine cord formation in BACTEC 12B medium was evaluated as a rapid method for the presumptive identification of M. tuberculosis complex. Kinyoun acid-fast stained smears were prepared from 666 positive BACTEC 12B bottles and examined for the presence or absence of serpentine cording. Cord formation had a sensitivity, specificity, positive predictive value, and negative predictive value of 89.2, 99.2, 98.5, and 94.2%, respectively. The evaluation of the presence of cord formation in BACTEC 12B medium is reliable and permits the rapid presumptive reporting of M. tuberculosis.

Isolator component responsible for inhibition of Mycobacterium avium-M. intracellulare in BACTEC 12B medium.

We previously reported an inhibitory effect on Mycobacterium avium-M. intracellulare (MAI) when blood collected and processed with the Isolator system was placed in BACTEC 12B bottles for radiometric monitoring. We sought to identify the specific component(s) of the Isolator lysis-anticoagulant reagent (LAR) responsible for the inhibitory effect. We added the three components of the LAR, saponin, polyanetholesulfonate, and polypropylene glycol (PPG), to triplicate sets of BACTEC bottles separately and in various combinations. These bottles were then seeded with 10(2) CFU of MAI. The growth index (GI) was observed over a 42-day period and was compared with the GI for a growth control bottle set with no reagents and with the GI for a bottle set containing an equivalent volume of LAR. Growth in all growth control bottles and those bottles containing no PPG reached the maximum GI (GI = 999) within 8 to 10 days. Growth in bottles containing PPG never reached the maximum GI before day 14. In addition, the GIs of one bottle containing all three reagent components as well as all of the bottles containing actual Isolator LAR failed to reach the maximum within 42 days. Our data suggest that PPG is the main cause of the inhibitory effect, but that a secondary synergistic interaction between all three of the reagents may also be present.

Substances interfering with direct detection of Mycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serum albumin.

Interfering substances have been reported to inhibit PCR assays for the direct detection of Mycobacterium tuberculosis in clinical specimens. Using an internal control, we determined that 52% of respiratory specimens interfered with our PCR assay. On the basis of these findings, we tried to circumvent the problem by simply diluting prepared sediments. With sediment from a routinely processed sputum known to be inhibitory to PCR, one aliquot was prepared in a routine manner for PCR. Remaining sediment was diluted in phosphate-buffered saline, Middlebrook 7H10 broth, or BACTEC 12B broth; an internal control was added to all reaction mixtures and controls. Internal control was detected only in the sample diluted with BACTEC 12B medium. Components of the BACTEC 12B medium including PANTA reagent (polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin), reconstituting fluid, 0.2% glycerol, 0.05% Tween 80, and 0.05% bovine serum albumin (BSA) were tested in a similar manner. Only 0.05% BSA resulted in amplification of the internal control DNA. Varying concentrations of BSA were added to 11 aliquots of a respiratory sediment known to be inhibitory to the PCR. Internal control was detected in all reaction mixtures containing 0.00038 to 0.1% BSA. To determine the ability of BSA to override inhibition, respiratory specimens were run in triplicate: undiluted, diluted 1:2 with BACTEC 12B medium, or diluted with 0.026% BSA. For 21 of 22 inhibitory specimens, BSA was able to override the presence of interfering substances. These data suggest that the presence of BSA in a PCR assay is critical for the direct detection of M. tuberculosis in respiratory specimens.

Effect of PANTA on growth of Mycobacterium kansasii in BACTEC 12B medium.

Mycobacterium kansasii isolates from two patients showed relatively slow growth in BACTEC 12B medium (12B) (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) compared with the more rapid growth of these isolates on solid media. This finding prompted an evaluation of the effect of PANTA (Becton Dickinson) on the growth rate of these isolates. Suspensions of one isolate from each of these two patients (A and B), six additional isolates from six other patients (C through H), and one M. kansasii American Type Culture Collection isolate were inoculated into 12B with PANTA, 12B with reconstituting fluid only, and Middlebrook 7H11 agar plates (Remel, Lenexa, Kans.). For the isolates from patients A and B, the average times to detection for 12B with PANTA, 12B with reconstituting fluid, and Middlebrook 7H11 agar were 12.3, 7.4, and 9.0 days, respectively. For the remaining six patient isolates and the American Type Culture Collection strain, the average times to detection for these media were 9.2, 8.1, and 9.6 days. Susceptibility tests performed with the isolates from patients A and B with the individual component antibiotics of PANTA and testing of four of the other isolates with nalidixic acid alone suggested that nalidixic acid exerts some degree of inhibition on the growth of M. kansasii. The eight patient isolates were also inoculated onto Lowenstein Jensen medium (Remel) and onto a variety of selective mycobacterial media containing nalidixic acid and other antimicrobial agents. All isolates showed some degree of inhibition on at least one of these selective media.

Inhibitory effect of the Isolator blood culture system on growth of Mycobacterium avium-M. intracellulare in BACTEC 12B bottles

The examination of 6,938 clinical specimens collected during the period January 1991 through December 1992 suggested that the Isolator blood culture system (Wampole) inhibited growth of Mycobacterium avium-M. intracellulare complex (MAC) in BACTEC 12B medium. Of 162 MAC blood culture isolates, 94% were recovered from Lowenstein-Jensen (LJ) medium, while only 50% were recovered from 12B medium. The time to detection with LJ medium was 18 days, while that with 12B medium was 24 days. In contrast, 62% of the 305 MAC nonblood culture isolates were recovered from the LJ medium, while 87% were found in the 12B medium. The time to detection for these cultures was also reversed, i.e., 28 days for LJ medium versus 15 days for 12B medium. Dilution studies using the lysis-anticoagulant reagent from Isolator tubes demonstrated inhibition of both clinical and American Type Culture Collection strains of MAC, even at low concentrations of lysis-anticoagulant reagent. Washing the Isolator blood sediment prior to inoculating the 12B bottles eliminated any growth inhibition. Clinical and experimental data suggest that the use of the Isolator blood culture tube with the BACTEC 12B medium is contraindicated for mycobacterial blood cultures.

Use of acridinium-ester-labeled DNA probes for identification of mycobacteria in Bactec 13A blood cultures

AccuProbe tests for mycobacteria (Gen-Probe) cannot be performed directly on Bactec 13A cultures because of interfering substances. This problem can be circumvented by subculturing to Bactec 12B media. Artifactual chemiluminescence greater than the positive cutoff was seen in 15 of 19 13A cultures containing Mycobacterium avium complex compared with 0 of 19 in 12B subcultures. Of the subcultures, 89% were tested after overnight incubation.

Direct, automated detection of rifampin-resistant Mycobacterium tuberculosis by polymerase chain reaction and single-strand conformation polymorphism analysis

A rapid screening test was recently established for the detection of mutations in the rpoB gene of Mycobacterium tuberculosis, a region identified as the locus for rifampin resistance (Rifr). The detection method involved the amplification by polymerase chain reaction (PCR) of the Rifr region and the identification of mutations by single-strand DNA conformation polymorphism analysis (SSCP) of the amplification products. Experience using two different PCR-SSCP formats for the evaluation of BACTEC cultures and sputum is presented here; the previously described manual procedure for the detection for the detection of radiolabelled amplification products and an automated SSCP by which fluorescein-labelled products were detected on a Pharmacia DNA sequencer apparatus. All 17 different Rifr mutations known to date were consistently detected. PCR-SSCP could be used for the evaluation of minimally grown cultures (BACTEC 12B medium with a growth index of < or = 100) and for direct screening of microscopically positive sputa with greater than 10 organisms per field (magnification, x250). Implementation of this technique could result in rapid detection of rifampin resistance in M. tuberculosis, a marker of multidrug-resistant tuberculosis.

Use of polymerase chain reaction for early identification of Mycobacterium tuberculosis in positive cultures.

To develop a readily applicable polymerase chain reaction (PCR) based technique which would permit the identification of Mycobacterium tuberculosis complex isolates from Bactec phials at an earlier stage than currently available methods. Mycobacterial cells cultured in Bactec 12B medium were harvested by centrifugation. The cells were lysed by heating in distilled water. Oligonucleotide primers based on the sequence of the gene coding for the immunogenic protein MPB64 were then used to amplify a 240 base pair fragment of DNA directly from the crude cell lysate. The PCR product was visualised under ultraviolet light following electrophoresis of an aliquot in an agarose gel containing ethidium bromide. The sensitivity of the PCR was adjusted so that about 600 cfu of M tuberculosis gave a positive result. The lowest growth index at which this method of identification might be applied to Bactec phials was determined and a number of routine cultures giving a positive growth index examined. M tuberculosis was positively identified at the lowest growth index, as determined by the Bactec system. Of 45 routine cultures examined, with growth indexes ranging from 6 to 999, the 15 confirmed by conventional means to contain M tuberculosis were correctly identified from 1 ml of culture medium. The method described can be used to identify M tuberculosis isolates cultured in the Bactec system at the earliest detectable rise in growth index. It may therefore allow cultured mycobacteria to be identified at an earlier stage than conventional methods or the commercially available DNA probes adapted for use with the Bactec system.

Role of solid media when used in conjunction with the BACTEC system for mycobacterial isolation and identification

This study evaluated the necessity and the contribution of solid media when used in conjunction with radiometric Middlebrook 7H12 (BACTEC 12B; Becton Dickinson, Towson, Md.) medium for recovery and complete identification of mycobacteria. Each of 1,184 digested, decontaminated respiratory specimens was inoculated into one BACTEC 12B vial, one 7H11 plate, and two Lowenstein-Jensen (LJ) slants. When the 12B vial was smear positive for acid-fast bacilli, the organisms were subcultured onto LJ slants and the BACTEC p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test system was inoculated with the organisms. Niacin tests were performed by using the growth from the original LJ slants and organisms from the LJ slants subcultured from 12B or 7H11 medium. The times to achieve definitive NAP and niacin test results were recorded. Recovery of all 143 isolates found in this study could not be achieved with a single medium. Among the three media, the highest percentage (92.8%) of Mycobacterium tuberculosis isolate recovered was with BACTEC 12B. The use of either 7H11 medium or LJ slants along with a 12B vial increased by 4 to 6% the total percentage of M. tuberculosis organisms that were isolated. Isolation of the M. tuberculosis complex and NAP differentiation in 12B medium were completed in an average of 17 days. On average, isolation and definitive niacin test results for M. tuberculosis cultures were obtained in 39.3 days by a conventional procedure and in 36.3 days when 12B subcultures were used. These results support the conclusion that LJ slants contribute 4 to 6% increased recovery of M. tuberculosis when used in conjunction with 12B medium. Additionally, a subculture onto LJ slants from 12B medium yielded sufficient growth for niacin testing earlier than an original LJ slant did.

Comparison of improved BACTEC and Lowenstein-Jensen media for culture of mycobacteria from clinical specimens

A 4-month trial involving 2,563 routine clinical specimens was conducted to compare the improved BACTEC TB system (12B medium) with the conventional Lowenstein-Jensen (LJ) media for the isolation, identification, and susceptibility testing of mycobacteria. One hundred sixty-two mycobacterial isolates were recovered, 147 (91%) with BACTEC and 118 (73%) with LJ media. Of these, 62 were Mycobacterium tuberculosis complex strains, 59 (95%) of which were isolated with BACTEC and 54 (87%) of which were isolated with LJ media. Of the remaining 100 isolates, which were mycobacteria other than tuberculosis (MOTT), BACTEC and LJ media detected 88 and 64%, respectively. The contamination rate was significantly higher in BACTEC (5%) than in LJ media (3.3%). The mean isolation time for M. tuberculosis complex with BACTEC was 15.5 days, compared with 25.6 days with LJ. For MOTT, the mean isolation times were 5.8 and 21.4 days, respectively. Identification of 32 M. tuberculosis complex isolates and 38 isolates of MOTT by the BACTEC NAP (p-nitro-alpha-acetylamino-beta-hydroxypropiophenone) inhibition test gave 100% agreement with conventional biochemical identifications. The results of susceptibility testing of 18 M. tuberculosis complex isolates with BACTEC agreed completely with those obtained by the resistance ratio method.

Comparative in vitro activities of 20 fluoroquinolones against Mycobacterium leprae

The in vitro activities of 20 fluoroquinolones against Mycobacterium leprae were evaluated by using the BACTEC 460 system. M. leprae was incubated in BACTEC 12B medium at 33 degrees C under reduced oxygen for 2 to 3 weeks in the presence of fluoroquinolones at 0.31 to 5 micrograms/ml. Activity was determined by a reduction in 14CO2 evolution compared with that of drug-free controls. Of the commercially available agents, ofloxacin was most active, while enoxacin and norfloxacin were inactive. However, a number of newer fluoroquinolones (AT-4140, OPC-17100, OPC-17066, PD-117596, PD-124816, PD-127391, and WIN-57273), all containing a cyclopropyl group at R-1 and, with the exception of WIN-57273, either a halogen or methyl group at R-8, were more active than ofloxacin in vitro. Further in vivo evaluations of these agents should help determine their potential for use against leprosy.

Drug susceptibility testing of Mycobacterium leprae in the BACTEC 460 system.

The susceptibility of Mycobacterium leprae to clinical and experimental antileprosy agents was assessed in the BACTEC 460 system. Nude-mouse-derived M. leprae (10(7) cells), incubated in BACTEC 12B medium at 33 degrees C under reduced oxygen, maintained a fairly constant growth index (14CO2 evolution) for 2 to 3 weeks. At concentrations ranging from 0.031 to 2.0 micrograms/ml, dapsone, rifampin, clofazimine, ethionamide, ofloxacin, clarithromycin, and minocycline all effected reductions in the growth index within 1 to 2 weeks, the extent of inhibition increasing with the incubation time. An in vivo rifampin-resistant isolate displayed markedly reduced susceptibility to rifampin compared with an in vivo-susceptible strain. This system appears to be highly suitable for in vitro drug susceptibility testing of M. leprae.