Cheminformatics in anti-infective agents discovery.

The existing chemical data such as those created by high throughput screening (HTS), structure-activity relationship (SAR) studies are converted into information as a result of storage and registration. Accessibility, manipulation, and data mining of such information make up the knowledge for drug development. Cheminformatics, exploiting the combination of chemical structural knowledge, biological screening, and data mining approaches is used to guide drug discovery and development and would assist by integrating complex series of rational selection of designed compounds with drug-like properties, building smarter focused libraries. This paper presents cheminformatics approaches and tools for designing and data mining of chemical databases and information. Many examples of success in lead identification and optimization in the area of anti-infective therapy have been discussed.

Evaluation of SAR for Amphotericin B Derivatives by Artificial Neural Network

AIM : this study was designed to investigate the role of several descriptive structure-activity features in the antifungal drug; amphotericin B and analyze them by artificial neural networks. METHOD: Artificial neural networks (ANN) based on the back-propagation algorithm were applied to a structure-activity relationship (SAR) study for 17 amphotericin B derivatives with antifungal and membrane directed activity. A series of modified ANN architectures was made and the best result provided the ANN model for prediction of antifungal activity using the structural and biologic property descriptors. RESULTS: The best architecture; in terms of cycles of calculation was 12-15-2. Among the most important factors were biological descriptors that correlated best with the model produced by ANN. Among the chemical and structural descriptors; positive charge on Y substitution was found to be the most important; followed by lack of availability of free carboxyl and parachor. CONCLUSION: This model is found to be useful to elucidate the structural requirements for the antifungal activity and can be applied in the design and activity prediction of the new amphotericin B derivatives

Aerobic growth and toxigenicity of Clostridium botulinum types A and B.

Clostridium botulinum types A and B cultured in association with avian skin flora, had similar growth patterns under both aerobic and anaerobic conditions. The selective "C. botulinum isolation" (CBI) medium was found to be especially useful for the recovery and quantitation of small numbers of type A or type B organisms from the mixed cultures. Enzyme immunoassay in conjunction with conventional mouse bioassay provided a practical means for the quantitation of toxigenicity of C. botulinum in avian skin cultures. The amount of toxin produced by type A was always higher than that produced by type B strains. The aerobically incubated type A or type B cultures appeared to be less toxigenic than cultures incubated anaerobically.

Rapid identification of Clostridium botulinum and botulinal toxin in food.

Samples of green beans and mushrooms were inoculated with a toxigenic strain of Clostridium botulinum type A and incubated anaerobically at 37 degrees C. At various time intervals, the seeded food samples were tested for the presence of botulinal toxin and C. botulinum by an agar plating method and an enzyme-linked immunosorbent assay. C. botulinum type A that appeared as lipase-positive colonies on selective agar plates, and its elaborated toxin, were identified in all seeded food samples within 1 to 2 d. This procedure can be adapted for rapid screening of suspected food samples.

Rapid identification of Clostridium botulinum colonies by in vitro toxicity and antimicrobial susceptibility testing.

Identification of Clostridium botulinum is usually based on toxin detection of broth culture by mouse bioassay and requires 7 to 10 days to complete. Here, we describe an alternative in vitro procedure for direct identification of C. botulinum (types A and B) colonies which can be completed in 48 h. The method is based on toxigenicity of colonies demonstrable by enzyme immunoassay and resistance of C. botulinum to antimicrobial agents, sulpha-methoxazole, trimethoprim and cycloserine.

Comparative efficacy of ceftriaxone in experimental infections involving Bacteroides fragilis and Escherichia coli.

The in vivo activity of ceftriaxone was examined in an experimentally induced subcutaneous infection involving Bacteroides fragilis and Escherichia coli. Mice were challenged with 1 of 10 strains of B. fragilis or E. coli, or a dual combination of the two species. The efficacy was measured by a reduction in the count of viable organisms when antimicrobial treatment was initiated 1 h after challenge and continued for 5 days. Ceftriaxone exhibited impressive activity against E. coli but showed poor in vivo activity versus B. fragilis. The antimicrobial activity of ceftriaxone was influenced by the microbial interaction in our dual-isolate model. Pharmacokinetic studies showed that ceftriaxone penetrated into abscesses and achieved peak levels of about 40% of the peak serum levels. However, in abscesses infected with B. fragilis nearly all biological activity of ceftriaxone was lost.

A simple procedure for identification ofClostridium botulinum colonies.

For direct identification of toxigenic colonies ofClostridium botulinum type E, suspected colonies are uniformly suspended in a phosphate buffer containing 0.5% (w/v) gelatin and 0.05% (w/v) Tween 20. After centrifuging, the supernatant is tested for botulinal toxin by an enzyme-linked immunosorbent assay (ELISA). The assay is specific for this type as it did not react with culture filtrates of otherClostridium species, including non-toxigenic 'E-like' organisms.

Identification of toxigenic Clostridium botulinum type E by enzyme immunoassay.

A sensitive double-sandwich enzyme immunoassay for identification of toxigenic Clostridium botulinum type E was developed with a type-E-specific antitoxin. The antitoxin was produced in a rabbit, following inoculation of minute quantities of type-E toxoid. The toxoid was prepared from a commercially available toxin by using a detoxification method. Cross-reactivity of the antitoxin with C. botulinum types A and B was avoided by absorption of the serum with alum-precipitated toxoids A and B. The immunoassay proved to be specific for C. botulinum type E because it did not react with culture filtrates of various Clostridium species, including nontoxigenenic "E-like" organisms and Clostridium sporogenes.

Kinetics study of immunological response to Clostridium botulinum toxin.

A double-antibody enzyme-linked immunosorbent assay (ELISA) for detection of humoral antibody to type A botulinal toxin was developed. This assay was used to study the kinetics of antibody response of a volunteer to botulinal toxoid. The circulating type A antitoxin was first detected by the ELISA 2 weeks after the first booster injection of the toxoid. The antibody titer stayed level until the second booster at 12 weeks. The titer then continued to rise throughout the remaining study period. The neutralizing antibody to type A toxin was detected by mouse assay 15 weeks after detection of antitoxin by the ELISA.

Effects of irradiation on growth and toxigenicity of Clostridium botulinum types A and B inoculated onto chicken skins.

This study was conducted to examine the effects of 0.3-Mrad irradiation on growth and toxigenicity of Clostridium botulinum types A and B on chicken skins. Irradiation followed by aerobic or anaerobic incubation at 30 degrees C extended the shelf life of skin samples and delayed growth and toxin production by C. botulinum. During 2 weeks of incubation at 10 degrees C, the irradiated and nonirradiated C. botulinum spores failed to grow or produce toxin.

Kinetics of growth and toxigenicity of Clostridium botulinum in experimental wound botulism.

An animal model of wound botulism was developed in mice using an inoculum of Clostridium botulinum type A spores. The number of C. botulinum in infected wounds was quantitated by culturing on egg yolk agar, and the level of C. botulinum toxin in infected wound tissue was measured by a bioassay in mice and by an enzyme-linked immunosorbent assay. All infected mice receiving no further treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca. 48 h, and all of these animals died. Serotherapy with C. botulinum type A antitoxin initiated 24 h postchallenge reduced the mortality rate to 5%. Treatment with metronidazole 2 to 24 h postchallenge resulted in recovery rates of 40 to 91%.

Detection of Clostridium botulinum type B toxin in the presence of a lethal substance interfering with toxin neutralization.

A low molecular weight substance that induced seizures and death in mice was present in the stool of a child with infant botulism. This interfered with mouse bioassay used for detecting Clostridium botulinum toxin. After removal of the interfering substance by a prolonged dialysis with buffered saline, in vivo neutralization of botulinal toxin was readily achieved. Alternatively, the botulinal toxin was demonstrated in stool of the infant by an enzyme-linked immunosorbent assay (ELISA), using antibodies produced in immunologically tolerant rabbits.

Selective isolation and rapid identification of Clostridium botulinum types A and B by toxin detection.

A simple procedure for rapid identification of Clostridium botulinum type A and B colonies from cultures and stool samples from infants with botulism was devised. The stool samples were directly streaked on C. botulinum isolation medium containing selective inhibitory agents. Typical lipase-positive colonies that appeared within 24 to 48 h were examined for the presence of botulinal toxin by the enzyme-linked immunosorbent assay and conventional mouse toxicity test. The amount of toxin associated with 48-h colonies of stock strains was comparable to that of 96-h broth culture. The quantity of toxin present in a single colony or combination of two was shown to be sufficient for toxin detection by the enzyme-linked immunosorbent assay. Of 42 additional stock strains tested in this manner, 41 (97.5%) were identified as toxigenic C. botulinum type A or B. The remaining one strain also proved to be toxigenic when it was tested as a concentrated cell suspension. This procedure should prove useful for large-scale serological screening of food and clinical specimens.

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type A and type B toxins in stool samples of infants with botulism.

An enzyme-linked immunosorbent assay (ELISA) for Clostridium botulinum type A and type B toxins was assessed for diagnostic accuracy in cases of infant botulism. This test was positive in all 22 cases confirmed by the conventional tests, which included the mouse lethality assay and stool culture. Stool specimens from five cases were positive by culture, but the mouse lethality bioassay was either negative or toxicity was judged nonspecific since it could not be neutralized by specific antitoxin. The positive ELISA results in these specimens suggested that this assay may be more reliable, in some cases, than the mouse bioassay. Of the 21 fecal specimens from suspected foodborne cases, 2 contained botulinal toxin demonstrable by the mouse assay and the ELISA. With regard to specificity, 35 fecal specimens from infants and 19 from suspected foodborne cases which were negative in the bioassay for botulinal toxins A and B were also negative in the ELISA. Only two fecal specimens with negative bioassay gave positive ELISA readings, providing a specificity rate of 96%. These results suggest that the ELISA may serve as a useful screening test to detect C. botulinum toxin in clinical specimens.

Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals.

Immunological tolerance is a state of unresponsiveness to foreign substances (antigens) which can develop in human and animal species as the result of continued exposure to antigens early in life. We utilized this principle for the preparation of antibodies against Clostridium botulinum type A toxin. By selective suppression of the immunological response of rabbits to unwanted antigens and subsequent immunization with a toxoid, we were able to produce a specific type A antitoxin without the need to purify the toxin. Despite cross-reactivity with C. botulinum type B, our type A antitoxin was otherwise specific since it did not react with culture filtrates of nontoxigenic variants of type B, any other C. botulinum type (C, D, E, F, and G), nor with 18 other Clostridium species, including Clostridium sporogenes. Using this antitoxin, we developed a sensitive enzyme-linked immunosorbent assay for detection of C. botulinum type A toxin.

Relative efficacy and critical interval of antimicrobial agents in experimental infections involving bacteroides fragilis.

Activity of seven antimicrobial agents was examined using a mouse model of a subcutaneous infection that involved Bacteroides fragilis. Untreated mice had encapsulated abscesses with approximately 10(10) bacteria. Pharmacokinetic studies showed that all drugs tested penetrated into abscesses to provide mean peak levels that were 17% to 53% of mean peak serum levels. In vivo efficacy v 15 strains was measured by the reduction in counts of viable organisms when treatment was initiated one hour after challenge. This showed that the most active agents, in order of activity, were metronidazole hydrochloride, clindamycin phosphate, moxalactam disodium, and cefoxitin sodium. A delay in treatment of eight to 120 hours after challenge showed a noticeable reduction in activity, except with metronidazole. It is presumed that bacteria within an abscess are in a stationary growth phase, and this has an important influence on in vivo efficacy.

Efficacy of moxalactam in an animal model of subcutaneous abscesses: penetration into infected sites and in vivo activity against Bacteroides fragilis.

Moxalactam was compared with alternative antimicrobial agents for in vivo activity against Bacteroides fragilis. Mice were challenged with one of 15 strains and evaluated by quantitative bacteriologic analysis of abscess contents after five days of treatment. Optimal results were achieved with clindamycin, moxalactam, and cefoxitin. The mean decrease in bacteria with these three drugs was significantly greater than with chloramphenicol, cefotaxime, carbenicillin, cephalothin, ampicillin, and cefoperazone. The penetration of antimicrobial agents into subcutaneous abscesses was examined. The mean peak level in sterile abscesses of moxalactam was 27% of the mean peak serum level, a result indicating relatively good penetration compared with other beta-lactam antimicrobial agents. Stability of moxalactam to the beta-lactamase of B. fragilis was demonstrated in vivo by comparing levels of biologically active drug in sterile and infected abscesses and in vitro by comparing levels of antibiotic incubated in sterile exudate and exudate infected with B. fragilis. Moxalactam compares favorably with clindamycin and cefoxitin in activity against B. fragilis.

Serum antibody response to Clostridium botulinum toxin in infant botulism.

A serum antibody response has not been previously demonstrated after infection with Clostridium botulinum. We developed an enzyme immunoassay for measuring serum antibody to C. botulinum toxins A, B, and E. This assay system detected a specific immunoglobulin G and immunoglobulin M antibody response to C. botulinum toxin in two patients with infant botulism.

Selective medium for isolation of Clostridium botulinum from human feces.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.