Various methodological approaches in controlled screening of antibiotic producers among the group of Coryneform bacteria]

A methodical system for directed screening of cultures producing broad-spectrum antibiotics among soil saprophytic coryneform bacteria was developed. To isolate such cultures, it was recommended to use the glucose-yeast medium supplemented with malt extract (No. 18/3) and soybean-glucose medium with sodium sulfate and cobalt chloride (No. 20/3). The preliminary alkaline treatment of the soil substrates and the use of acidic soil samples were found to favour isolation of the Mycobacterium type cultures. It was recommended to use gram-negative tests microbes with relatively low antibiotic resistance for screening cultures producing broad spectrum antibiotics. Various agarized and liquid fermentation media were compared in regard to detection of antibiotic activity in the soil coryneform bacteria. The corn medium supplemented with protein-vitamin concentrate, glucose, lactose and starch (No. 116) proved to be the most efficient.     

Actinomadura species as antibiotic producers

Screening of antibiotic-producing cultures among Actinomadura showed that definite species mainly produced antibiotics of the same groups. Thus, carminomycins were produced by all the 4 studied strains of A. carminata, maduramycins were produced by 3 strains of A. rubra, prodigiozines were produced by 3 strains of A. madurae and luzopeptines were produced by 6 strains of A. recticatena. Supposedly, new antibiotics with original spectral characteristics were isolated from 2 strains of A. fulvescens. There was a clear-cut relation of the number of the active strains and their antibiotic productivity in definite media to their species. The liquid nutrient media, such as yeast-sucrose, soya-glucose and soya-glucose with cobalt chloride proved to be the most efficient in the primary screening of antibiotic-producing cultures.     

Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

Controlled search for the producers of ionophore antibiotics among Streptomycetes

A procedure was developed for directed screening of cultures producing ionophore antibiotics among streptomycetes. The procedure is based on measuring the membrane potential generated in the presence of the Men+/nH+ = -expchanger-protonophore couple. It provided isolation of cultures producing ionophore antibiotics at the fermentation broth stage. It was possible to use the procedure in screening both electrogenic and nonelectrogenic ionophores and to rapidly differentiate them. 5 cultures producing ionophore antibiotics were detected with this procedure; 3 of them carry out nonelectrogenic transport of the cations. The cation transport in the other two cultures was electrogenic. Cation selectivity of the antibiotics produced by the cultures was determined with the procedure. An antibiotic identical to indanomycin was isolated from the culture fluid of Streptomyces chromogenes.     

Isolation of antibiotic-producing Actinomycetes from soil samples exposed to UV light

The use of nonroutine means in isolation of microorganisms from natural substrates extended the possibilities of detecting new cultures which often appear to be producers of previously unknown antibiotics. A new procedure for isolating actinomyces of definite groups was developed. It implies preliminary exposure of soil suspensions to UV light. With the use of the procedure, 2539 strains of actinomycetes belonging to different genera were isolated. There was a marked decrease after the irradiation in isolation of cultures belonging to Streptomyces, a genus most widely distributed in nature and studied in detail while isolation of cultures belonging to other genera, promising as sources of novel antibiotics, increased. Micromonospora, Amycolatopsis and Nocardia proved to be the most stable to the effect of UV light. With the use of the procedure it is possible to increase 2-3-fold isolation of cultures belonging to Micromonospora, a genus known as a producer of many antibiotics including those used clinically.     

A system for screening natural immunosuppressors

Criteria for directed screening of antibiotics with immunosuppressive action were defined. The first stage included screening of cultures producing antiaspergillous antibiotics. At the second stage, the antibiotics whose antifungal activity decreases in the presence of insulin (at the background of calcium salts) and erythromycin and increases in the presence of verapamil were selected. The screening of antibiotic-producing cultures among 123 strains of mycelial fungi and 181 strains of actinomycetes resulted in isolation of 3 fungal cultures and 2 actinomycetes which produced antibiotics corresponding to cyclosporine A as evidenced by thin-layer and high performance liquid chromatographies.     

Formation of broad-spectrum antibiotics by cultures of the genus Micromonospora]

Data on the study of antibiotic production by the representatives of Micromonospora and the use of ion exchange resins for intensification of screening antibiotic-producing organisms among Micromonospora are presented. It was found that out of 172 strains of Micromonospora tested 92 (53.5 per cent) cultures produced antibiotics, 18 of which were active against gramnegative bacteria. The use of carboxylic ion exchange resins at early microbiological stages of the screening provided an increase in the frequency of finding broad spectrum antibiotics from 10.4 to 19.7 per cent.     

Taxonomic characteristics of soil Mycobacteria and their antibiotic properties]

Morphological, cultural and chemotaxonomic properties of 12 gram-positive soil cultures isolated were studied by using a test system developed for screening the organisms producing broad-spectrum antibiotics among Nocardiaforms (Coryneforms). The cultures were found to belong to Actinomycetales, Nocardioforms, Mycobacteriaceae and Mycobacterium. The saprophytic rapidly growing soil mycobacteria showed antibiotic activity against a large number of gram-positive and gram-negative test microbes including those belonging to Pseudomonas and Proteus resistant to the majority of the antibiotics currently used in medicine.     

Targets and assays for discovering novel antibacterial agents

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.     

Elimination of mycoplasmas from cell cultures by a novel soft agar technique

Summary Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12–21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1–3 days. In separate studies, it was shown that certainMycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas. These studies were supported in part by grant 15748 from the National Institute of Allergy and Infectious Diseases and the W. W. Smith Charitable Trust.     

Early identification of streptothricin group antibiotics]

Methodical approaches to detecting cultures producing streptothricins at the early stages of screening new antibiotics were developed. The approaches are based on chromatographic and electrophoretic mobility of streptothricins and the products of their hydrolysis in the extracts from agar cultures of actinomycetes. Application of the method for screening new antibiotics is illustrated with an example. Nine strains of actinomyces with broad antibacterial spectra isolated from soil samples were studied and 6 of them belonging to 4 species were shown to produce streptothricins in agar cultures. The new streptothricin-producing culture S. roseolilacinus was isolated.     

Robust and Systematic Drug Screening Method Using Chemical Arrays and the Protein Library: Identification of Novel Inhibitors of Carbonic Anhydrase II

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.     

Robust and systematic drug screening method using chemical arrays and the protein library: identification of novel inhibitors of carbonic anhydrase II

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.     

Comparative antibiotic eradication of mycoplasma infections from continuous cell lines

Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method.     

The role of combichem in antibiotic discovery

Combinatorial chemistry (combichem) has had a significant impact on the discovery of new antibiotics. Most of the successes have come from the use of small libraries to explore a specific pharmacophore. However, large diverse libraries are more appropriate when identifying hits by screening specific bacterial or fungal targets. Combichem has been used to optimize new azole and oxazolidinone leads. An entirely new class of antibiotics, inhibitors of bacterial peptidyl-deformylase, has been discovered by combining mechanism-based drug design and combichem. These compounds are active in vivo. The impact of combichem on discovery projects that aim to develop new antibiotics for the treatment of infectious diseases is discussed.     

Irregular nuclear localization and anucleate cell production in Escherichia coli induced by a Ca2+ chelator, EGTA

A screening system for specific inhibitors of chromosome partitioning in Escherichia coli was constructed using the blue assay method developed for detection of anucleate cell production. Effects of known chemical compounds and antibiotics were examined in the system. It was found that a calcium-chelating reagent, EGTA, induced blue zones around the paper disks containing EGTA at concentrations which did not induce growth inhibition zones. Induction of anucleate cell production by EGTA was confirmed by fluorescence microscopy after DAPI staining. Even in the nucleated cells, irregular intracellular localization of nucleoids was frequently observed. The effect of EGTA was reversed by addition of Ca(2+). These results suggest the possible role of calcium ion in the process of chromosome partitioning in E. coli.     

Use of the method of enriching of soil samples with calcium carbonate for isolation of Actinomyces

Possible application of a modified procedure for treatment of soil samples with calcium carbonate under humid conditions to isolation of actinomycetes was studied. The procedure proved to be rather efficient in regard to increasing the number of the isolates including representatives of rare genera as compared with the routine methods. The number of microorganisms isolated from the soil samples treated with calcium carbonate under humid conditions was 2 orders higher than that from untreated soil samples. 1033 actinomycete cultures were isolated from the soil samples treated in accordance with the above procedure and only 597 cultures were isolated from the untreated soil samples. The antibiotic activity of the isolates was studied and their taxonomic position at the genus level was determined. The preliminary treatment of soil samples equally stimulated development of actinomycetes belonging to different genera. It is advisable that the described procedure for isolation of actinomycetes from soil samples in screening of cultures producing new antibiotics be used in combination with other selective methods of isolation.     

Detection of novel secondary metabolites.

The study of antibiotics and other fermentation products has shown that a seemingly unlimited number of compounds with diverse structures are produced by microorganisms. The continued high rate of discovery of new chemical entities, in the light of the abundance of microbial products already described, is due to creative screening procedures that incorporate such features as the emphasis on unusual microorgnaisms, their special propagation and fermentation requirements, supersensitive and highly selective assays, genetic engineering both for the biosynthesis of new compounds and in the development of screening systems, early in vivo evaluation, improved isolation techniques, modern procedures for structure determination, computer-assisted identification, and an efficient multidisciplinary approach. This review focuses on the genesis and development of the gamut of methodologies that have led to the successful detection of the wide variety of novel secondary metabolites that include antibacterial, antigungal, antiviral and antitumour antibiotics, enzyme inhibitors, pharmacologically and immunologically active agents, products useful in agriculture and animal husbandry, microbial regulators, and other compounds for which no bioactive role has yet been found.