12B Medium Research Articles

Antibacterial, Antitubercular and Antiviral Activity Evaluations of Some Arylidenehydrazide Derivatives Bearing Imidazo[2,1-b]thiazole Moiety.

The aim of this study was to determine the probable antibacterial, antitubercular, and antiviral activities of some N2 -arylidene-(6-(4-chlorophenyl)imidazo[2,1-b]thiazol-3-yl) acetic acid hydrazides (3a-j). Further structural optimization of the identified lead structures can lead us to new more active potential antibacterial, antitubercular, and antiviral agents. Antibacterial activities of the title compounds against Staphylococcus aureus ATCC 29213, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922. These molecules were also evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) using the BACTEC 460 radiometric system and BACTEC 12B medium. Moreover, all the compounds (3a-j) were also evaluated against some DNA and RNA viruses in Madin-Darby Canine Kidney, Crandell-Rees Feline Kidney (CRFK), Vero, human embryonic lung (HEL) and HeLa cells. Among the tested compounds, 3i displayed the highest efficacy against S. aureus and E. coli. Compound 3j, 5-nitro-2-furfurylidene derivative showed the highest antituberculosis activity (IC50: 6.16 µg/mL and IC90: 14.390 µg/mL). Compound 3i showed the most potent antiviral activity against feline corona virus in CRFK cell cultures (antiviral EC50: 7.5 µM and SI>13). Furthermore, compounds 3c and 3g displayed activity against herpes simplex virus-1 and vaccinia virus in HEL cell cultures (antiviral EC50 values of 9; 16 and 20; 14 µM, respectively). On the basis of aforementioned results, it can be conluded that imidazo[2,1-b]thiazole derivatives bearing hydrazone moieties serve as promising chemical probes to design therapeutic agents with antibacterial, antitubercular, and antiviral properties.

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0–60), with negligible positive samples observed in mid (DIM 60–240) and late (DIM 240–305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd.

Short communication: Application of an N-acetyl-l-cysteine-NaOH decontamination method for the recovery of viable Mycobacterium avium subspecies paratuberculosis from milk of naturally infected cows

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne’s disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n=17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-l-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15min before culture in BACTEC 12B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold’s egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n=144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds.

Retrospective Evaluaton of Tuberculosis in Central Nervous System Enfections: Five-Years Results

In this retrospective study, we evaluate the probability of tuberculosis in central nervous system infections. Six hundred fourty one (641) central nervous system clinical samples were examined retrospectively in the Erciyes University Faculty of Medicine Hospital microbiology laboratory. Between 2006-2010 microscopic examinations after the NaOH-NALC method, all specimens except brain spinal fluid were examined for acid-fast bacillus were done with Ehrlich Ziehl Neelsen stain and all specimens were cultured on Lowenstein-Jensen (L-J) and BACTEC 12B medium. Strains identified as M. Tuberculosis as a consequence of NAP testing, were tested for sensitivity to anti-tuberculosis drugs with a BACTEC 460 TB radiometric method. The tuberculosis detection rate was 2.6% for all samples. Among culture positive samples 16 were Mycobacterium tuberculosis complex and 18 were non-tuberculous mycobacterium. Of Mycobacterium isolated samples 3 were Ziehl-Neelsen positive. Mean age of the patients with tuberculosis was 29.6±20.53. Of M. tuberculosis strains 18.7% was resistant to at least one or more of the drugs (streptomycin 12.5%, resistant to two drugs 6.2%. Tuberculosis of central nervous system has high mortality and morbidty rate among extrapulmonary tuberculosis forms. Early diagnosis and treatment is reduced mortality and morbidity rates.

Development and Validation of a Liquid Medium (M7H9C) for Routine Culture of Mycobacterium avium subsp. paratuberculosis To Replace Modified Bactec 12B Medium

Liquid culture of Mycobacterium avium subsp. paratuberculosis from clinical samples, such as feces, is the most sensitive antemortem test for the diagnosis of Johne's disease in ruminants. In Australia, New Zealand, the United States, and some other countries, the Bactec 460 system with modified Bactec 12B medium (Becton, Dickinson) has been the most commonly used liquid culture system, but it was discontinued in 2012. In this study, a new liquid culture medium, M7H9C, was developed. It consists of a Middlebrook 7H9 medium base with added Casitone, albumin, dextrose, catalase, egg yolk, mycobactin J, and a cocktail of antibiotics. We found that polyoxyethylene stearate (POES) was not essential for the cultivation of M. avium subsp. paratuberculosis in either the Bactec 12B or the M7H9C medium. The limit of detection determined using pure cultures of the C and S strains of M. avium subsp. paratuberculosis was 7 bacilli per 50 μl inoculum in the two media. The new medium was validated using 784 fecal and tissue samples from sheep and cattle, >25% of which contained viable M. avium subsp. paratuberculosis. Discrepant results for the clinical samples between the two media were mostly associated with samples that contained <10 viable bacilli per gram, but these results were relatively uncommon, and the performances of the two media were not significantly different. M7H9C medium was less than half the cost of the Bactec 12B medium and did not require regular examination during incubation, but a confirmatory IS900 PCR test had to be performed on every culture after the predetermined incubation period.

Chemical Decontamination with N -Acetyl- l -Cysteine–Sodium Hydroxide Improves Recovery of Viable Mycobacterium avium subsp. paratuberculosis Organisms from Cultured Milk

Mycobacterium avium subsp. paratuberculosis is shed into the milk and feces of cows with advanced Johne's disease, allowing the transmission of M. avium subsp. paratuberculosis between animals. The objective of this study was to formulate an optimized protocol for the isolation of M. avium subsp. paratuberculosis in milk. The parameters investigated included chemical decontamination with N-acetyl-l-cysteine-sodium hydroxide (NALC-NaOH), alone and in combination with antibiotics (vancomycin, amphotericin B, and nalidixic acid), and the efficacy of solid (Herrold's egg yolk medium [HEY]) and liquid (Bactec 12B and para-JEM) culture media. For each experiment, raw milk samples from a known noninfected cow were inoculated with 10(2) to 10(8) CFU/ml of live M. avium subsp. paratuberculosis organisms. The results indicate that an increased length of exposure to NALC-NaOH from 5 to 30 min and an increased concentration of NaOH from 0.5 to 2.0% did not affect the viability of M. avium subsp. paratuberculosis. Additional treatment of milk samples with the antibiotics following NALC-NaOH treatment decreased the recovery of viable M. avium subsp. paratuberculosis cells more than treatment with NALC-NaOH alone. The Bactec 12B medium was the superior medium of the three evaluated for the isolation of M. avium subsp. paratuberculosis from milk, as it achieved the lowest threshold of detection. The optimal conditions for NALC-NaOH decontamination were determined to be exposure to 1.50% NaOH for 15 min followed by culture in Bactec 12B medium. This study demonstrates that chemical decontamination with NALC-NaOH resulted in a greater recovery of viable M. avium subsp. paratuberculosis cells from milk than from samples treated with hexadecylpyridinium chloride (HPC). Therefore, it is important to optimize milk decontamination protocols to ensure that low concentrations of M. avium subsp. paratuberculosis can be detected.

4-(3-coumarinyl)-4-thiazolin-2-one benzylidenehydrazones with antituberculosis activity.

In this study a new series of 4-(3-coumarinyl)-4-thiazoline-2-one benzylidenehydrazones (3a-v) were synthesized by condensation of 3-(omega-bromoacetyl)coumarins (1a and 1b) with 1-substituted benzylidene-4-substituted thiosemicarbazides (2a-1). Structures of the title compounds were elucidated by elemental analyses and spectrometric data (UV, IR, 1H-NMR and EIMS). These new compounds and some previously reported compounds (1a-d) were evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv. The compounds exhibited varying degrees of inhibition in the in vitro primary screening that was conducted at 12 micrograms/ml against M. tuberculosis H37Rv in BACTEC 12B medium using the BACTEC 460 radiometric system. 1b, 3b, 3e, 3h, 3o and 3p demonstrating activity in the primary screen were re-tested at lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration (MIC) in CABTEC 460 and Alamar Blue assay (MABA). The most active compound was found to be 1b. The structure-activity relationships of the derivatives were investigated.

Synthesis and antimicrobial activity of 4-carbethoxymethyl-2-[(alpha-haloacyl)amino] thiazoles and 5-nonsubstituted/substituted 2-[(4-carbethoxymethylthiazol-2-yl)imino]-4-thiazolidinones.

4-Carbethoxymethyl-2-[(chloroacetyl/alpha-chloropropionyl/al pha- bromobutyryl/alpha-chloro-(alpha-phenylacetyl)amino]thiazoles (I-IV) were synthesized by reacting 4-carbethoxymethyl-2-aminothiazole with chloroacetyl chloride, alpha-chloropropionyl chloride, alpha-bromobutyryl bromide and alpha-chloro-alpha-phenylacetyl chloride, respectively. Furthermore, I-IV were refluxed with ammonium thiocyanate to give 2-[(4-carbethoxymethylthiazol-2-yl)imino]-4-thiazolidinones (V-VIII). V was refluxed with various aromatic aldehydes to give 5-arylidene-2-[(4-carbethoxymethylthiazol-2-yl)imino]-4-t hiazolidinones (IX-XIV). The structures of synthesized compounds were confirmed by elemental analyses, hydrolysis, UV, IR, 1H-NMR and EI mass spectral data. The antimicrobial activities of the compounds were assessed by microbroth dilution technique using Mueller-Hinton broth and Mueller-Hinton Agar. In this study, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 1539, Salmonella typhi, Shigella flexneri, Proteus mirabilis and Candida albicans ATCC 10231 were used as test microorganisms. Among the tested compounds, XI and XIV showed activity against S. aureus (MIC: 78 micrograms/ml, 1.6 micrograms/ml), whereas compound V had an activity against S. flexneri (MIC: 39 micrograms/ml) and compound I against C. albicans (MIC: 125 (micrograms/ml). Compounds I, IV-XIV were also evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv using the BACTEC 460 radiometric system and BACTEC 12B medium. Only compounds I and XIV showed 86% and 67% inhibition in the primary screen.

Synthesis and antituberculosis activity of cycloalkylidenehydrazide and 4-aza-1-thiaspiro[4.5]decan-3-one derivatives of imidazo[2,1-b]thiazole.

New N2-cycloalkylidene-(6-phenyl/4-chlorophenylimidazo[2,1-b]thiazol-3-yl) acetic acid hydrazides (2a-h and 3a-b) were synthesized by reacting (6-phenyl/4-chlorophenylimidazo[2,1-b]thiazol-3-yl)acetic acid hydrazides with cyclohexanones or cyclopentanone. Furthermore, 2a-h were refluxed with thioglycolic or thiolactic acid to give 4-[[(6-phenyl/4-chlorophenylimidazo[2,1-b]thiazol-3-yl) acetyl]amino]-4-aza-1-thiaspiro[4.5]decan-3-ones (4a-h and 5a-h). The structures of the title compounds were established by spectral data (IR, 1H-NMR, 13C-NMR and EIMS (Electron Impact Mass Spectrometry)) and elemental analysis. The synthesized compounds were evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv (ATCC 27294). The compounds exhibited varying degrees of inhibition in the in vitro primary screening that was conducted at a concentration of 6.25 micrograms/ml against M. tuberculosis H37Rv (ATCC 27294) in Bactec 12B medium using the Bactec 460 radiometric system or a broth microdilution assay, the Microplate Alamar Blue Assay (MABA). Compounds 2f, 2h, 3b, 4a, 4c, 4d, 5a-e and 5h demonstrating at 1-east 90% inhibition in the primary screen were re-tested at lower concentrations against M. tuberculosis H37Rv (ATCC 27294) to determine the actual minimum inhibitory concentration (MIC) using MABA. The most active compounds were found to be 4d and 5c. The structure-activity relationships of the derivatives were investigated.

Synthesis and Preliminary Screening of Carbohydrazides and Hydrazones of Pyrrole Derivatives as Potential Tuberculostatics

Five carbohydrazides and 19 hydrazones of carboxylic acids of pyrrole were synthesized as new structural analogs of known tuberculostatics, such as isoniazid (CAS 54-85-3) and its popular hydrazones. The preliminary screening against Mycobacterium tuberculosis H37Rv (ATCC 27294) at 6.25 microg/mL in 12B medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA) registered higher inhibitory activity within the hydrazones series (up to 68 % inhibition) compared with that of the carbohydrazides (0-34 %). No simple relationship between the activity of a particular hydrazone and that of the parent carbohydrazide, or correlations with the structural features introduced by the carbonyl partner could be observed. So both most active hydrazones diethyl 5-(2-[(2,4-dimethyl-1H-3-pyrrolyl)carbonyl]hydrazonomethyl)-3-methyl-1H-2,4-pyrroledicarboxylate (22, 68 % inhibition) and ethyl 5-(4-chlorophenyl)-2-methyl-1-(2-2-1-(5-nitro-2-furyl)methylidene]hydrazino-2-oxoethyl)-1H-3-pyrrolecarboxylate (32, 55 %) originated from inactive carbohydrazides (1, 0 % and 7, 10 % respectively); ethyl 1-(2-2-[1-(3-ethoxy-4-hydroxyphenyl) methylidene] hydrazino-2-oxoethyl)-2-methyl-5-(4-nitrophenyl)-1H-3-pyrrolecarboxylate (30) showed 0 % inhibition, although derived from a hydrazide with 34 % activity. The condensation of six carbohydrazides with the same carbonyl reagent 5-nitrofurfural yielded hydrazones whose activity covered the wide range of 0-55 % inhibition.

Synthesis and Evaluation of Antimicrobial and Anticonvulsant Activities of Some New 3-[2-(5-Aryl-1,3,4-oxadiazol-2-yl/4-Carbethoxymethylthiazol-2-yl)imino-4-thiazolidinon-5-ylidene]-5-substituted/nonsubstituted 1H-indole-2-ones and Investigation of Their Structure-Activity Relationships

In the present study, 20 new compounds having 3-[2-(5-aryl-1,3,4-oxadiazol-2-yl) imino-4-thiazolidinon-5-ylidene]-5-substituted/nonsubstituted 1H-indole-2-one (I-XII) and 3-[2-(4-carbethoxymethylthiazol-2-yl)imino-4-thiazoldinon-5-ylidenel-5-substituted/nonsubstituted IH-indole-2-one (XIII-XX) systems were synthesized. The structures were confirmed by spectral methods (UV, IR, 1H-NMR, 13C-NMR, 13C-DEPT (135), electron impact mass spectrometry) and elemental analysis. All compounds were tested for in vitro antimicrobial activity against Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 4352, Pseudomonas aeruginosa ATCC 1539, Salmonella typhi, Shigella flexneri, Proteus mirabilis ATCC 14153, Candida albicans ATCC 10231, Microsporum gypseum (NCPF-580), Microsporum canis, Trichophyton mentagrophytes and Trichophyton rubrum and some of them were found to be active. Especially, compound I was more active than cefuroxime sodium (CAS 56238-63-2) which was used as a standard, and the activity of compound XII was close to that of cefuroxime sodium against Staphylococcus epidermidis ATCC 12228. Primary screening for antituberculous activity was conducted at 6.25 microg/ml against Mycobacterium tuberculosis H37Rv in BACTEC 12B medium using the BACTEC 460 radiometric system. The anticonvulsant activities of selected prototoype compounds (I, IV-VI, VIII, XI, XIII, XVI-XVIII) administered at doses of 50-200 mg/kg (i.p.) were evaluated using the pentetrazol test (PTZ) in mice.

Synthesis and in vitro Evaluation of New Hydrazones as Pyrrole Derivatives with Anti-tubercular Activity

Fourteen new hydrazones representing derivatives of pyrrole were synthesized by condensation of six carbohydrazides with ten carbonyl compounds. The new products were evaluated as potential tuberculostatics by means of two levels of high-throughput screening. The preliminary screening against <i>Mycobacterium tuberculosis</i> H37Rv (ATCC 27294) at 6.25 µg/mL in 12B medium using the Microplate Alamar Blue Assay measured the inhibitory activity within the total series in the range of 98–0%. Compound 3-i (N 4-[l-(4-acetyl-3,5-dimethyl-lH-2-pyrrolyl)-methylidene]-4-pyridinecarbohydrazide) was identified as the compound showing the highest inhibition (98%) and was subjected to level 2 screening, whereat the resulting data (IC₅₀ < 0.2 and IC₉₀ = 22.581) identified 3-i as a prospective candidate for further and more detailed evaluations. The inhibitory activity within the series was found to drop with the increase of the relevant dipole moments and all the inactive compounds distinguished themselves with a higher molecular polarity.

Rifampicin reduces susceptibility to ofloxacin in rifampicin-resistant Mycobacterium tuberculosis through efflux.

Central dogma suggests that rifampicin resistance in Mycobacterium tuberculosis develops solely through rpoB gene mutations. To determine whether rifampicin induces efflux pumps activation in rifampicin resistant M. tuberculosis strains thereby defining rifampicin resistance levels and reducing ofloxacin susceptibility. Rifampicin and/or ofloxacin minimum inhibitory concentrations (MICs) were determined in rifampicin resistant strains by culture in BACTEC 12B medium. Verapamil and reserpine were included to determine their effect on rifampicin and ofloxacin susceptibility. RT-qPCR was applied to assess expression of efflux pump/transporter genes after rifampicin exposure. To determine whether verapamil could restore susceptibility to first-line drugs, BALB/c mice were infected with a MDR-TB strain and treated with first-line drugs with/without verapamil. Rifampicin MICs varied independently of rpoB mutation and genetic background. Addition reserpine and verapamil significantly restored rifampicin susceptibility (p = 0.0000). RT-qPCR demonstrated that rifampicin induced differential expression of efflux/transporter genes in MDR-TB isolates. Incubation of rifampicin mono-resistant strains in rifampicin (2 μg/ml) for 7 days induced ofloxacin resistance (MIC > 2 μg/ml) in strains with an rpoB531 mutation. Ofloxacin susceptibility was restored by exposure to efflux pump inhibitors. Studies in BALB/c mice showed that verapamil in combination with first-line drugs significantly reduced pulmonary CFUs after 1 and 2 months treatment (p < 0.05). Exposure of rifampicin resistant M. tuberculosis strains to rifampicin can potentially compromise the efficacy of the second-line treatment regimens containing ofloxacin, thereby emphasising the need for rapid diagnostics to guide treatment. Efflux pump inhibitors have the potential to improve the efficacy of anti-tuberculosis drug treatment.

Culture Phenotypes of Genomically and Geographically Diverse Mycobacterium avium subsp. paratuberculosis Isolates from Different Hosts

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

Isolation and Identification of Mycobacterium tuberculosis from AIDS Patients Attending a Rural Hospital in Central India

Aim: To isolate and characterize mycobacterium tuberculosis from clinical samples of AIDS patients. Materials and Methods: This cross -sectional observational study was conducted in department of microbiology, in a rural medical college. The inclusion criteria were adult patient of any sex, HIV seropositive, CD4 counts 200cells/cumm. Complete blood counts and CD4 counts were done. Blood, sputum, stool and other extra-pulmonary samples were processed for mycobacterial isolation. BACTEC 13A medium was used for blood samples while Lowenstein Jensen and BACTEC 12B medium was used for other samples. Smears were examined for all samples other than blood. Identification of the isolates was done by standard techniques. Results: 65% patients were TB symptomatic with cough, fever, breathlessness and loss of weight being significant clinical features. Chest X-rays finding were infiltration and air space consolidation. Cavity was found in only 3% cases. 9 mycobacterium tuberculosis isolates were isolated. Mycobacteria were isolated from various samples viz. sputum (8 M. tuberculosis), blood (1 M. tuberculosis). Conclusion: Mycobacterium tuberculosis is commonly isolated in AIDS patients.

Synthesis and antituberculosis activity of some N-pyridyl- N′-thiazolylhydrazine derivatives

In this study, new N-(1-arylethylidene)- N′-(4-arylthiazol-2-yl)hydrazine derivatives were synthesized and evaluated for their antituberculosis activity. The chemical structures of the compounds were elucidated by IR, NMR and FAB +-MS spectral data and Elemental Analyses. The initial screen was conducted against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA). The VERO cell cytotoxicity assay was done in parallel with the TB Dose Response assay. Viability was assessed using Promega's Cell Titer-Glo Luminescent Cell Viability Assay. Cytotoxicity was determined from the dose–response curve as the CC50 using a curve-fitting program. One of the compounds showed high activity with low toxicity.

Optimisation of culture of Mycobacterium avium subspecies paratuberculosis from blood samples

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease or paratuberculosis, a chronic enteritis of ruminants, and has been suggested to play a role in Crohn's disease in humans. While disease expression is primarily in the gastrointestinal tract, isolation of MAP or MAP DNA at distant sites indicates that disseminated infections also occur. This is the first study to compare, optimise and critically evaluate different methods of concentrating MAP from spiked blood samples to enable its culture. Whole blood, erythrocytes, but not plasma, delayed the growth of MAP in BACTEC 12B medium. Culture of MAP from buffy coats (concentrated leukocytes) after lysis of erythrocytes and washing of the cells was an effective preparatory method. Several antibiotics were evaluated to reduce contamination of the slow growing MAP cultures with microbes which were derived from the skin during venipuncture, but were detrimental to the growth of MAP. However, decontamination of erythrocyte-lysed washed buffy coat samples in 0.75% hexadecylpyridinium chloride (HPC) for 72 h prior to inoculation of culture media did not inhibit the growth of MAP. The prepared samples can be stored at − 80 °C prior to batch culture. MAP was isolated from the blood of 2 of 23 sheep 20 months after experimental inoculation. The optimised method has an analytical sensitivity of at least 10 1 MAP per ml of spiked whole blood and will enable trials to determine the incidence, duration and magnitude of mycobacteraemia in infected animals and humans.

Synthesis and biological activities of new hydrazide derivatives

The synthesis of a new series of imidazo[1,2-a]pyrazine-2-carboxylic acid arylidene-hydrazides is described. The chemical structures of the compounds were elucidated by IR, 1H-NMR, FAB+-MS spectral data. Their biological activity against various bacteria, fungi species, and Mycobacterium tuberculosis was investigated. Antibacterial activity was measured against Escherichia coli (NRRL B-3704), Staphylococcus aureus (NRRL B-767), Salmonella typhimurium (NRRL B-4420), Proteus vulgaris (NRLL B-123), Enterococcus faecalis (isolated obtained from Faculty of Medicine Osmangazi University, Eskisehir, Turkey), Pseudomonas aeruginosa (NRRL B-23 27853), Klebsiella spp. (isolated obtained from Faculty of Medicine Osmangazi University, Eskisehir, Turkey), while antifungal activity was evaluated against Candida albicans (isolates obtained from Osmangazi Uni. Fac.of Medicine), Candida glabrata (isolates obtained from Osmangazi Uni. Fac.of Medicine). Compounds were also evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv using the BACTEC 460 radiometric system and BACTEC 12B medium. The compounds showed moderate inhibitor effects against human pathogenic microorganisms., whereas the preliminary results indicated that all of the tested compounds were inactive against Mycobacterium tuberculosis H37Rv.

Synthesis and antimycobacterial activity of some steroidal derivatives of tigogenin

Some 5α-type steroidal derivatives of the steroidal sapogenin tigogenin were synthesized. The structures of the synthesized derivatives were characterized by spectral methods and elemental analysis. The antituberculosis activity of the compounds toward Mycobacterium tuberculosis strain H37Rv was studied in vitro (Microplate Alamar Blue Assay) in BACTEC 12B medium and was compared with that of isoniazide. Some of the synthesized compounds exhibited high (92-98%) antimycobacterial activity.

Synthesis and Antituberculosis Activity of New Hydrazide Derivatives

The increasing clinical importance of drug-resistant mycobacterial pathogens, especially Mycobacterium tuberculosis, has lent additional urgency to microbiological research and new antimycobacterial compound development. For this purpose, new hydrazide derivatives of imidazo[1,2-a]pyridine were synthesized and evaluated for antituberculosis activity. The reaction of 2-[(2-carboxyimidazo[1,2-a]pyridine-3-yl)sulfanyl]acetic acid hydrazide with various benzaldehydes gave N-(arylidene)-2-[(2-carboxyimidazo[1,2-a]pyridine-3-yl)sulfanyl]acetic acid hydrazide derivatives. The chemical structures of the compounds were elucidated by IR,(1)H-NMR, FAB-MS spectral data and elemental analysis. Antituberculosis activities of the synthesized compounds were determined by broth microdilution assay, the Microplate Alamar Blue Assay in BACTEC 12B medium. The results were screened in vitro, using the BACTEC 460 Radiometric System against Mycobacterium tuberculosis H37Rv (ATCC 27294) at 6.25 microg/mL; the tested compounds showed significant inhibition.