BACTEC Radiometric System Research Articles

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME NEW 1,3,4- OXADIAZOLES, 1,2,4-TRIAZOLES AND 1,3,4-THIADIAZOLES

Some new 5-[(((α-phenyl/methyl)benzylidene)amino)oxy]methyl/ethyl-2-[4-(substituted aryl)/allyl)] amino-1,3,4-oxadiazoles (4a-p), 3-[(((α-phenyl/methyl)- benzylidene) amino)oxy]methyl/ethyl-4-(4- substitutedaryl)/allyl-5-mercapto-1,2,4-triazoles (5a-p) and 5-[(((α-phenyl/methyl)-benzylidene)amino) oxy]- methyl/ethyl-2-[4-(substituted aryl)/allyl)]amino-1,3,4-thiadiazoles (6a-p) were prepared starting from α/β-[((α-(phenyl/methyl)benzylidene)amino)oxy acetic/propionic acid hydrazides (1a-d). The structures of all the compounds have been established by elemental and spectral (IR, 1HNMR and mass) analysis. All the newly synthesised compounds have been screened for their antimicrobial activity against Escherichia coli, Bacillus cirroflagellosus, Aspergillus niger and Rhizoctonia bataticola. Some of the newly synthesised compounds have been evaluated for antituberculosis activity against Mycobacterium tuberculosis H37Rv strain by BACTEC radiometric system at Southern Research Institute, Birmingham, AL and Frederick Research Centre, Frederick, MD. Significant antimicrobial activity is observed against Escherichia coli and Rhizoctonia bataticola. A few compounds also exhibited interesting antitubercular activity against Mycobacterium tuberculosis H37Rv strain.

SOME NEW 1,3,4-OXADIAZOLES AS ANTIMICROBIAL AGENTS

Some new 5-[(((α-phenyl/methyl)benzylidene)amino)oxy]methyl/ethyl-2-amino/4-substituted aryl/ aryloxymethylarmercapto/piperidino/ morpholino-1,3,4-oxadiazoles (2a-d–5a-d) with biologically active biostere methyelne amino oxy methyl/ethyl moiety, were prepared starting from α/β-[((α-(phenyl/methyl) benzylidene)amino)oxy acetic/propionic acid hydrazides (1a-d). The structures of the compounds have been characterised by elemental and spectral (IR, 1HNMR and mass) analysis. All the newly synthesised compounds have been screened for their antimicrobial activity by cup plate method, against Escherichia coli, Bacillus cirroflagellosus, Aspergillus niger and Rhizoctonia bataticola. Some of the newly synthesised compounds have been evaluated for antituberculosis activity against, Mycobacterium tuberculosis H37Rv strain as well by BACTEC radiometric system at Southern Research Institute and Frederick Research Centre, Frederick, MD.

In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance

We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. abscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.

Rapid, Standardized Method for Determination of Mycobacterium tuberculosis Drug Susceptibility by Use of Mycolic Acid Analysis

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.

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.

In vitro activity of a novel antimycobacterial compound, N-octanesulfonylacetamide, and its effects on lipid and mycolic acid synthesis.

beta-Sulfonyl carboxamides have been proposed to serve as transition-state analogues of the beta-ketoacyl synthase reaction involved in fatty acid elongation. We tested the efficacy of N-octanesulfonylacetamide (OSA) as an inhibitor of fatty acid and mycolic acid biosynthesis in mycobacteria. Using the BACTEC radiometric growth system, we observed that OSA inhibits the growth of several species of slow-growing mycobacteria, including Mycobacterium tuberculosis (H37Rv and clinical isolates), the Mycobacterium avium complex (MAC), Mycobacterium bovis BCG, Mycobacterium kansasii, and others. Nearly all species and strains tested, including isoniazid and multidrug resistant isolates of M. tuberculosis, were susceptible to OSA, with MICs ranging from 6.25 to 12.5 microg/ml. Only three clinical isolates of M. tuberculosis (CSU93, OT2724, and 401296), MAC, and Mycobacterium paratuberculosis required an OSA MIC higher than 25.0 microg/ml. Rapid-growing mycobacterial species, such as Mycobacterium smegmatis, Mycobacterium fortuitum, and others, were not susceptible at concentrations of up to 100 microg/ml. A 2-dimensional thin-layer chromatography system showed that OSA treatment resulted in a significant decrease in all species of mycolic acids present in BCG. In contrast, mycolic acids in M. smegmatis were relatively unaffected following exposure to OSA. Other lipids, including polar and nonpolar extractable classes, were unchanged following exposure to OSA in both BCG and M. smegmatis. Transmission electron microscopy of OSA-treated BCG cells revealed a disruption in cell wall synthesis and incomplete septum formation. Our results indicate that OSA inhibits the growth of several species of mycobacteria, including both isoniazid-resistant and multidrug resistant strains of M. tuberculosis. This inhibition may be the result of OSA-mediated effects on mycolic acid synthesis in slow-growing mycobacteria or inhibition via an undescribed mechanism. Our results indicate that OSA may serve as a promising lead compound for future antituberculous drug development.

Comparison of three isolation systems for the culture of mycobacteria from respiratory and non-respiratory samples

Aims—To compare the recovery of mycobacteria from clinical samples using the MB/BacT rapid culture system with that obtained using egg medium or the Bactec radiometric method. Methods—The three methods were...

Optimal detection and identification of Mycobacterium haemophilum in specimens from pediatric patients with cervical lymphadenopathy.

Acid-fast bacilli from pediatric patients with lymphadenopathy were detected in the BACTEC radiometric system and in MB Redox broth, but not on Löwenstein Jensen medium. PCR amplification identified the isolates as Mycobacterium haemophilum, which has special nutrition requirements (iron supplements) for growth. Suitable culture medium ensures optimal recovery of this microorganism, avoiding underdiagnosis.

Evaluation of a nonradiometric system (BACTEC 9000 MB) for detection of mycobacteria in human clinical samples.

This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samples processed, 364 from pulmonary sites and 415 from extrapulmonary sites, 62 (7.9%) were positive for mycobacterial isolates; of the positive samples, 59 (95.1%) were detected with the fluorescent BACTEC 9000 MB system, 57 (91.9%) were detected with the radiometric system (BACTEC 460 TB), and 43 (69.3%) were detected with LJ conventional culture. The mean times to detection of all mycobacteria with BACTEC 9000 MB and BACTEC 460 TB were similar (10.3 and 10.0 days, respectively). The results obtained indicate that the nonradiometric BACTEC (BACTEC 9000 MB) system is as efficient as Bactec 460 TB and significantly more efficient than LJ for the rapid recovery of mycobacteria from both pulmonary and extrapulmonary clinical specimens. Though the BACTEC 9000 MB system is recommended for respiratory specimens, we demonstrated that it can be successfully used also for recovery of mycobacteria from clinical specimens from various extrapulmonary sites.

Synergistic antimycobacterial activity between ethambutol and the beta-lactam drug cefepime

The activity of cefepime alone and in combination with ethambutol was assessed, using the BACTEC radiometric system, on 33 mycobacterial strains, 14 Mycobacterium avium complex (MAC), 6 isolates of M. malmoense, and 13 multidrug-resistant (MDR) isolates of M. tuberculosis. All tested mycobacteria were resistant to 8 mg/l cefepime. However, at a concentration of 32 mg/l cefepime was active on 7 13 (54%) MDR isolates of M. tuberculosis and 2 6 (33%) M. malmoense strains. All MAC strains were also resistant to this concentration. Synergistic antimycobacterial effects were seen for the combination of cefepime and ethambutol against all isolates of MAC and M. malmoense and on 4 13 (31%) MDR M. tuberculosis isolates. Moreover, this drug combination rendered 17 24 (71%) initially resistant mycobacterial strains susceptible. These promising results suggest that the antimycobacterial activity of combinations of β-lactam drugs and ethambutol should be studied further.

Tuberculosis: an update.

1. Rosalind S. Abernathy, MD* 1. 2. *Associate Professor of Pediatrics, University of Arkansas for Medical Sciences; Pediatric Consultant, Tuberculosis, Arkansas State Department of Health, Little Rock, AR. 1. The majority of tuberculosis cases in children are from high-risk populations, that is, African-Americans, Hispanics, immigrants, and those living in poverty and in the inner cities. 2. The only acceptable tuberculin skin test is the intradermal Mantoux test with purified protein derivative (PPD), and three levels of positivity now are accepted. Children exposed to active disease who have findings suggestive of tuberculosis or who have human immunodeficiency virus (HIV) infection are positive at 5 to 9 mm; other high-risk groups are positive at 10 mm. 3. Diagnosis of childhood tuberculosis depends on a high index of suspicion coupled with suggestive epidemiologic data, tuberculin skin test results, and chest radiographic findings. Cultures are not routinely positive, and results are available only after treatment must be started. 4. Treatment is a uniform 6-month course of isoniazid, rifampin, and pyrazinamide for 2 months followed by 4 months of isoniazid and rifampin daily or twice weekly. Additional drugs are needed in cases of drug resistance. 5. Chemoprophylaxis for all children who have a positive tuberculin reaction will prevent future disease. Despite the availability of effective antituberculosis drugs since 1952, tuberculosis remains an important cause of morbidity, mortality, and health care cost in the United States. Tuberculosis case rates declined at a rate of 6% annually for more than 20 years until 1985 when they leveled off (Fig. 1⇓). Factors responsible for this include human immunodeficiency virus (HIV) infection; immigration of individuals from countries where tuberculosis rates are 10 to 30 times that of the United States; transmission of disease in hospitals, homeless shelters, and prisons; and poverty, overcrowded living, and poor access to medical care. While all this was occurring, funds for tuberculosis control by public health programs declined. Fortunately, the number of cases reported annually has decreased by 8.7% in the past 2 years, probably …

Clinical Features and Outcome of Eleven Patients with Disseminated Bacille Calmette-Guérin (BCG) Infection

Disseminated BCG infection is a very rare complication of BCG vaccination. This study presents 11 patients with such complication. The underlying disease in eight of the 11 patients was primary imm...

Mycobacterium genavense and Mycobacterium avium mixed infection in an AIDS patient

Several mixed nontuberculous mycobacterial infections have been reported in AIDS patients (l-5), and their prevalence is probably underestimated owing to the lack of differentiation of morphologically similar colonies in mixed cultures. Mixed infections are most difficult to detect when the coinfecting species, like Mycobacterium genavense, fails to grow on conventional solid media; according to Kirschner et al. (6), only the use of sequencing of PCRamplified nucleic acids can detect such mixed infections. We report, however, a case in which Mycobacterium avium and M. genavense were isolated from distinct blood samples, drawn at different times of the same day, using BACTEC 13A medium (Becton Dickinson, USA). While the isolation of M. avium was achieved in 2 wk, the recovery of M. genavense required nearly 3 mo, with the growth index starting to rise at the end of the eight week and with a further 25 d elapsing before microscopic detection. The M. avium isolate was identified by hybridization to commercial DNA probes (AccuProbe, USA), while the M. genavense was identified by E. Bottger (Medizinische Hochschule, Hannover, Germany) by means of 16S rRNA sequencing. The AIDS patient, a severely immunocompromised (20 CD4+ cells/ml) 27 yr-old homosexual man. presented with high fever, diffuse abdominal pain hepatosplenomegaly, and confusion; the case closely resembled those of two previously described patients with disseminated M. genavense infection. and the empirical therapy was based on the susceptibility patterns of those M. genavense strains (7). The regimen. a combination of amikacin. clofazimine and rifampin, produced, as in a previous case of M. genavense infection (7), a rapid improvement with cessation of fever and recovery of normal mental status. Such a prompt response to the antimicrobial therapy is very unusual in disseminated M. avium infections and suggests that M. genavense was responsible for the patients symptomatology, as in the only other published report (6). Also our M. avium isolate later was found to be resistant in vitro to clofazimine and rifampin, and only moderately susceptible to amikacin. The impact of infections due to M. genavense in HIV-infected patients is surely greater than the paucity of reported cases seem to suggest. The failure of M. genavense to grow on conventional solid media makes it undetectable in most clinical laboratories, even those using the BACTEC radiometric system, if a sufficient period of incubation is not allowed. In the case of mixed infection (most frequently with M. avium), M. genavense is missed because it is overgrown by the other species. In our case it was a matter of chance that the M. avium bacteremia was not continuous, and only the practice of performing repeated blood cultures and our previous experience with M. genavense infections allowed us to initiate a prompt and effective therapy and to isolate what appeared to be the organism mainly responsible for the clinical condition. Every effort should be made to diagnose disseminated infections by M . genavense in AIDS patients, because prompt and appropriate therapy can rapidly improve the otherwise severe and lifethreatening symptomatology caused by this organism.

Temporal effects of tumor necrosis factor-α on intracellular survival of Mycobacterium paratuberculosis

The causative agent in Johne's disease is Mycobacterium paratuberculosis, an intracellular pathogen which causes enteritis in ruminants. Little is known about interactions between the host cell (macrophage) and M. paratuberculosis; however, this bacterium is able to evade normal host immune defenses and cause a chronic infective state. In the present study, we evaluated whether activation of a murine macrophage cell line (J774.16) by pretreatment with recombinant murine tumor necrosis factor alpha (TNF) prior to infection with M. paratuberculosis would affect their ability to restrict growth and kill the ingested bacteria. A murine cell line was utilized owing to difficulty in obtaining bovine reagents and lack of a continuous bovine macrophage cell line for repeated experimentation. After 4 h of infection, numbers of viable bacteria in cell lysates were significantly lower for macrophages pretreated with 1000 IU TNF ml −1. The rate of bacterial growth as assessed by BACTEC radiometric culture system was also reduced at this time point. Upon further extension of the infection period to 72 h, we observed that moderate doses of TNF (10–1000 IU ml −1) significantly increased the number of viable M. paratuberculosis recovered whereas the highest dose of TNF (4000 IU ml −1) effectively reduced bacterial numbers. These data indicate that TNF can either enhance or reduce macrophage mycobactericidal and mycobacteriostatic activity depending upon both the level of TNF to which cells are exposed and the duration of infection.

Comparison of the Septi-Chek AFB and BACTEC systems and conventional culture for recovery of mycobacteria

The performance of the Septi-Chek AFB system was compared with that of the BACTEC radiometric system and that of Lowenstein-Jensen agar slants (LJ) for detection of mycobacteria in clinical specimens. A total of 642 specimens were cultured; 61 (9.5%) yielded mycobacteria. Mycobacterium tuberculosis (34 isolates) and Mycobacterium avium complex (25 isolates) were the predominant species isolated. Of the 61 culture-positive specimens, 30 were smear positive and 31 were smear negative. Overall, 95% of the positive specimens were detected by Septi-Chek and BACTEC (100% of M. tuberculosis isolates) and 75% by LJ (82% of M. tuberculosis isolates). The mean times to detection were 15 days for BACTEC, 23 days for Septi-Chek, and 27 days for LJ. Of the 30 smear-positive specimens, 100% were recovered by Septi-Chek and BACTEC and 90% were recovered by LJ. Of the 31 smear-negative specimens, 90% were detected by Septi-Chek and BACTEC and 61% were detected by LJ. The Septi-Chek and BACTEC systems are superior to the conventional (LJ) mycobacterial culture method. Although Septi-Chek requires more time for the detection of mycobacteria than BACTEC, it is comparable in terms of overall recovery.

Optimum sampling time for detection of dental bacteraemia in children

A total of 458 blood cultures were taken from 229 children aged 2 to 16 yr. Each 6 ml sample was taken starting at one of the following times after the extraction of a single tooth: 10 s, 30 s, 60 s, 90 s, 120 s, 180 s, 600 s and continuously over 2.5 min. The samples were cultured in the BACTEC radiometric culture system and when positive the bacteria isolated were speciated. At 30 s 56% of the samples were positive, the highest proportion, in contrast to only 38% of positive samples at 90 s and 28% at 600 s. Over 50% of the organisms found were Viridans streptococci.

Effect of altered headspace atmosphere on yield and speed of detection of the Oxoid Signal blood culture system versus the BACTEC radiometric system

The one-bottle Oxoid Signal blood culture system altered to provide a more aerobic bottle headspace was reassessed in a comparative study versus the two-bottle BACTEC radiometric system in 5,426 blood cultures. The BACTEC system detected more microorganisms (P less than 0.02), particularly anaerobes (P less than 0.05) and fungi (P less than 0.05).

The BACTEC Radiometric System for Detection and Rapid Identification of Mycobacteria

We have found the current BACTEC system using 12B medium to be a rapid, reliable method for the detection and differentiation of mycobacteria species. We evaluated the results of 1,081 mycobacterial cultures using a combination of BACTEC andhowenstein-Jensen medium. Twenty-nine cultures (2.68%) were positive for clinically significant isolates with either BACTEC, Lowenstein-]ensen, or both. BACTEC had an overall sensitivity of 89.6% for both Mycobacterium tuberculosis and other mycobacteria compared to 69% for recovery on Lowenstein-Jensen medium. The combined mean time to detection was greatly reduced with BACTEC compared to Lowenstein-] ensen medium, 12.4 vs24.3 days. Rapid differentiation between M tuberculosis complex and other mycobacteria was achieved with an overall mean time of4.4 days. Preliminary identification using the NAP differential test was confirmed with conventional tests in 22 of 24 cases. Two preliminary identifications were incorrect—one was M tuberculosis read as mycobacteria other than tuberculosis and one was M kansasii read as M tuberculosis complex. Suggestions are made for stricter interpretationof the NAP differential growth pattern to avoid such incorrect preliminary identification.

1,25(OH)2-Vitamin D3Synergizes with Pyrazinamide To Kill Tubercle Bacilli in Cultured Human Macrophages

Pyrazinamide (PZA) is believed to be mycobactericidal in vivo. Because it is ineffective at neutral pH in vitro, it is thought to owe its in vivo activity at least partly to acting upon tubercle bacilli (TB) in the helpfully low pH of macrophage (MP) phagolysosomes. However, when it was tested in TB-infected cultured human MP, it was not bactericidal and was able only to slow intra-MP bacillary growth. Recent evidence has suggested that human MP need hormonal support from certain vitamin D metabolites to resist TB. This support was not provided in the culture medium of the earlier experiments in which the PZA was relatively ineffective. Here, PZA has been retested in MP cultured in medium supplemented with the hormonally active metabolite of vitamin D, 1,25(OH)2-vitamin D3 (1,25D3). The MP were infected with virulent TB and incubated in various concentrations of PZA. 1,25D3 was added to postinfection medium at 4 micrograms/ml. Inhibition or killing of intracellular TB was quantitated by counts of culturable TB from samples of lysed MP taken at zero, 4, and 7 days after MP infection. Previous evidence for the protectiveness of 1,25D3 alone for human MP against TB was confirmed. The weak inhibition of TB in MP by PZA alone also was confirmed. The two used together synergized to decrease concentrations of PZA which were inhibitory and to switch the action of PZA from weakly inhibitory to bacteriostatic or mildly bactericidal. 1,25D3 had no direct anti-TB effect, and it did not synergize with PZA in the absence of MP, as determined with acidified bacteriologic culture medium in the BACTEC radiometric system.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved detection of Mycobacterium avium complex with the bactec radiometric system

A reconsideration of the laboratory methods used for primary isolation of mycobacteria other than Mycobacterium tuberculosis is needed due to the increasingly recognized importance of such mycobacterial infections in immunocompromized patients. One example of this is the severe opportunistic infections caused by Mycobacterium avium complex among AIDS patients. In this study, the Bactec radiometric system was compared to conventional culture on solid medium for the detection of M. avium complex in 3,612 selected clinical specimens, mainly of extrapulmonary origin. Of a total number of 63 M. avium complex isolates, the Bactec system detected 58 (92%), compared to 37 (59%) for conventional culture. A much more rapid detection was attained with radiometric technique than with conventional culture. The mean detection time for the cultures positive with both methods was 7.1 and 28.3 days, respectively. The Bactec radiometric system achieves a rapid and significantly more sensitive detection and seems to be an excellent complement to conventional culture in the laboratory diagnosis of infections with the M. avium complex.