The Concept of an Ideal Antibiotic: Implications for Drug Design.

The emergence and spread of antibiotic-resistant pathogens is a major public health issue, which requires global action of an intersectoral nature. Multidrug-resistant (MDR) pathogens-especially "ESKAPE" bacteria-can withstand lethal doses of antibiotics with various chemical structures and mechanisms of action. Pharmaceutical companies are increasingly turning away from participating in the development of new antibiotics, due to the regulatory environment and the financial risks. There is an urgent need for innovation in antibiotic research, as classical discovery platforms (e.g., mining soil Streptomycetes) are no longer viable options. In addition to discovery platforms, a concept of an ideal antibiotic should be postulated, to act as a blueprint for future drugs, and to aid researchers, pharmaceutical companies, and relevant stakeholders in selecting lead compounds. Based on 150 references, the aim of this review is to summarize current advances regarding the challenges of antibiotic drug discovery and the specific attributes of an ideal antibacterial drug (a prodrug or generally reactive compound with no specific target, broad-spectrum antibacterial activity, adequate penetration through the Gram-negative cell wall, activity in biofilms and in hard-to-treat infections, accumulation in macrophages, availability for oral administration, and for use in sensitive patient groups).

Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes.

Natural products from microbes have provided humans with beneficial antibiotics for millennia. However, a decline in the pace of antibiotic discovery exerts pressure on human health as antibiotic resistance spreads, a challenge that may better faced by unveiling chemical diversity produced by microbes. Current microbial genome mining approaches have revitalized research into antibiotics, but the empirical nature of these methods limits the chemical space that is explored.Here, we address the problem of finding novel pathways by incorporating evolutionary principles into genome mining. We recapitulated the evolutionary history of twenty-three enzyme families previously uninvestigated in the context of natural product biosynthesis in Actinobacteria, the most proficient producers of natural products. Our genome evolutionary analyses where based on the assumption that expanded-repurposed enzyme families-from central metabolism, occur frequently and thus have the potential to catalyze new conversions in the context of natural products biosynthesis. Our analyses led to the discovery of biosynthetic gene clusters coding for hidden chemical diversity, as validated by comparing our predictions with those from state-of-the-art genome mining tools; as well as experimentally demonstrating the existence of a biosynthetic pathway for arseno-organic metabolites in Streptomyces coelicolor and Streptomyces lividans, Using a gene knockout and metabolite profile combined strategy.As our approach does not rely solely on sequence similarity searches of previously identified biosynthetic enzymes, these results establish the basis for the development of an evolutionary-driven genome mining tool termed EvoMining that complements current platforms. We anticipate that by doing so real 'chemical dark matter' will be unveiled.

Aqueous stability of alumina and silica perhydrate hydrogels: experiments and computations.

Alumina and silica perhydrate hydrogels were synthesized. Raman spectroscopy and solid (27)Al MAS NMR confirmed alumina perhydrate formation. Thermal and aqueous stability of alumina and silica perhydrates was studied, and they showed exceptionally high stabilities. Alumina perhydrate retained some of the hydrogen peroxide even at 170 °C, higher than any other reported perhydrate, whereas the silica perhydrate lost its hydrogen peroxide content already at 90 °C. The silica perhydrate lost all its peroxide content upon immersion in water, whereas the alumina perhydrate was stable under near-neutral pH conditions. A computational study was conducted in order to glean molecular insight into the observed thermal and aqueous stability of alumina compared to silica perhydrate. Comparison of the hydrogen bond features and the stabilization energies of the hydrate and perhydrate of silica and alumina revealed a higher preference for hydrogen peroxide over water by alumina relative to silica. This is shown to be due to hydrogen peroxide being a better hydrogen donor than water and due to the superior hydrogen accepting propensity of alumina compared to silica.

Sympatric inhibition and niche differentiation suggest alternative coevolutionary trajectories among Streptomycetes.

Soil bacteria produce a diverse array of antibiotics, yet our understanding of the specific roles of antibiotics in the ecological and evolutionary dynamics of microbial interactions in natural habitats remains limited. Here, we show a significant role for antibiotics in mediating antagonistic interactions and nutrient competition among locally coexisting Streptomycete populations from soil. We found that antibiotic inhibition is significantly more intense among sympatric than allopatric Streptomycete populations, indicating local selection for inhibitory phenotypes. For sympatric but not allopatric populations, antibiotic inhibition is significantly positively correlated with niche overlap, indicating that inhibition is targeted toward bacteria that pose the greatest competitive threat. Our results support the hypothesis that antibiotics serve as weapons in mediating local microbial interactions in soil and suggest that coevolutionary niche displacement may reduce the likelihood of an antibiotic arms race. Further insight into the diverse roles of antibiotics in microbial ecology and evolution has significant implications for understanding the persistence of antibiotic inhibitory and resistance phenotypes in environmental microbes, optimizing antibiotic drug discovery and developing strategies for managing microbial coevolutionary dynamics to enhance inhibitory phenotypes.

Isolation and characterization of four gram-positive nickel-tolerant microorganisms from contaminated sediments.

Microbial communities from riparian sediments contaminated with high levels of Ni and U were examined for metal-tolerant microorganisms. Isolation of four aerobic Ni-tolerant, Gram-positive heterotrophic bacteria indicated selection pressure from Ni. These isolates were identified as Arthrobacter oxydans NR-1, Streptomyces galbus NR-2, Streptomyces aureofaciens NR-3, and Kitasatospora cystarginea NR-4 based on partial 16S rDNA sequences. A functional gene microarray containing gene probes for functions associated with biogeochemical cycling, metal homeostasis, and organic contaminant degradation showed little overlap among the four isolates. Fifteen of the genes were detected in all four isolates with only two of these related to metal resistance, specifically to tellurium. Each of the four isolates also displayed resistance to at least one of six antibiotics tested, with resistance to kanamycin, gentamycin, and ciprofloxacin observed in at least two of the isolates. Further characterization of S. aureofaciens NR-3 and K. cystarginea NR-4 demonstrated that both isolates expressed Ni tolerance constitutively. In addition, both were able to grow in higher concentrations of Ni at pH 6 as compared with pH 7 (42.6 and 8.5 mM Ni at pH 6 and 7, respectively). Tolerance to Cd, Co, and Zn was also examined in these two isolates; a similar pH-dependent metal tolerance was observed when grown with Co and Zn. Neither isolate was tolerant to Cd. These findings suggest that Ni is exerting a selection pressure at this site for metal-resistant actinomycetes.

Lack of correspondence between genetic and phenotypic groups amongst soil-borne streptomycetes.

Correspondence between two distinct genetic traits, 16S rRNA gene sequences and repetitive element-sequence-based BOX-PCR DNA fingerprints, and antibiotic inhibition and resistance phenotypes was explored for a spatially explicit sample of Streptomyces from a prairie soil. There was no correspondence between 16S rRNA gene sequence groups and antibiotic phenotypes. However, 16S rRNA gene sequence groups differed significantly in mean inhibition zone sizes. Specific antibiotic phenotypes may reflect local selection pressures, as suggested by the significant differences in mean inhibition zone sizes against specific test isolates by Streptomyces from the same 16S rRNA gene sequence group but from different locations in soil. Significant correlations between antibiotic phenotypes and BOX-PCR fingerprints were found, but were small (r=0.19-0.22). Although genetic characterizations alone were not predictive of specific antibiotic phenotypes, 16S rRNA gene sequence analyses may identify isolates that are most or least likely to possess substantial inhibitory potential, providing insight into the broad ecological strategy for individual isolates.

Effects of living cyanobacteria, cyanobacterial extracts and pure microcystins on growth and ultrastructure of microalgae and bacteria.

In this study, we demonstrate the inhibitory effect of both cyanobacterial extracts and pure microcystins on the growth of microalgae and bacteria. This inhibitory effect was more persistent in pure microcystins than in the extracts, which lost their properties eight days after exposure. In addition, the effects on bacteria were longerlasting than those on microalgae. The microalgae exposed to both extracts and cultures of microcystin producing species showed morphological and ultrastructural alterations, even in cases where there was no clear effect on growth. The implications for colonisation and benthic communities structure and development are discussed in the context of biomonitoring.

Widespread activation of antibiotic biosynthesis by S-adenosylmethionine in streptomycetes.

The effect of S-adenosylmethionine (SAM) on the production of various antibiotics was investigated to determine whether SAM-dependent methylation is required in biosynthetic pathways of antibiotics. Pristinamycin II(B) and granaticin do not require SAM-dependent methylation in their biosynthesis pathways, and production of these two antibiotics was increased about 2-fold when a low concentration (50 and 10 microM, respectively) of SAM was treated; in contrast, oleandomycin and avermectin B1a require SAM as a methyl donor in their biosynthesis, and production of these two antibiotics was increased 5-fold and 6-fold, depending on the SAM concentration within a certain range. We also found that the transcription of a pathway-specific regulator, gra-ORF9, was activated by exogenous SAM treatment. Production of oleandomycin and avermectin B1a was decreased by using a methyltransferase inhibitor, sinefungin, but the production levels of these antibiotics were restored to the control level by simultaneously adding SAM and sinefungin. Interestingly, we have found a similar stimulatory effect of S-adenosylhomocysteine (SAH), the methylation product of SAM, on antibiotic production in the four strains. Our results clearly demonstrate the widespread activation of antibiotic production using SAM in streptomycetes.

Genetic and phenotypic traits of streptomycetes used to characterize antibiotic activities of field-collected microbes.

Although antibiotic production may contribute significantly to microbial fitness, there is limited information on the ecology of antibiotic-producing microbial populations in soil. Indeed, quantitative information on the variation in frequency and intensity of specific antibiotic inhibitory and resistance abilities within soil microbial communities is lacking. Among the streptomycetes, antibiotic production is highly variable and resistance to antibiotics is highly specific to individual microbial strains. The objective of this work was to genetically and phenotypically characterize a reference collection of streptomycetes for use in distinguishing inhibition and resistance phenotypes of field-collected microbes. Specifically, we examined inhibition and resistance abilities of all isolates in all possible pairwise combinations, genetic relatedness using BOX-PCR and 16S rDNA sequence analyses, nutrient utilization profiles, and antibiotic induction among all possible three-way combinations of isolates. Each streptomycete isolate possessed a unique set of phenotypic and genetic characteristics. However, there was little correspondence between phenotypic and genetic traits. This collection of reference isolates provides the potential for distinguishing 1024 inhibition and resistance phenotypes in field-collected microbes. Relationships between the genetic and phenotypic characteristics examined may provide preliminary insight into the distinct strategies that microbes use in optimizing their fitness in natural environments.

[Search for A factor-dependent variants in actinomycete population]. / Poisk A-factorzavisimykh variantov v populiatsiiakh aktinomitsetov.

A study of 28 nocardia-like, asporogenous, and oligosporous spontaneous morphological variants belonging to 23 species of streptomycetes revealed five strains producing regulators of the A-factor group. Streptomyces griseus 1439, which forms aerial mycelium and spores only in the presence of exogenous A-factor was used as the test strain. Among the 28 spontaneous variants, three new A-factor-dependent strains were revealed, which represented the species Streptomyces griseus, S. citreofluorescens, and S. viridovulgaris subsp. albomarinus. These weakly differentiated variants id not produce A-factor and behaved as its recipients, responding by changes in their morphological characteristics at a concentration of this regulator in the medium of 0.01 microgram/ml and higher. The original collection strains in whose populations the variants were selected produced substances of the A-factor group. The A-factor-dependent variants differed in the level of the regulator required for maximal expression of the morphological characteristics were shown: it was necessary to introduce the A-factor at a concentration of 1 microgram/ml for S. citreofluorescens and S. viridovulgaris subsp. albomarinus and at 10 micrograms/ml for S. griseus.

[A new regulatory function of the A-factor--stimulation of the streptomyces spore germination]. / Obnaruzhenie novoi reguliatornoi funktsii A-faktora--stimuliatsii prorastaniia spor streptomitsetov.

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs, S. coelicolor A3(2), which forms six AcL-factors (A-factor analogues), and S. avermitilis JCM5070, which fails to form regulators of this group. The count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition A-factor to the medium in an amount 10 micrograms/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 microgram/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.

Effect of dissolved oxygen levels on biosynthesis of aureofungin by Streptoverticillium cinnamoneum var. terricola.

Biosynthesis of aureofungin by Streptoverticillium cinnamoneum var, terricola was found to be an oxygen dependent reaction. An accelerated rate of aureofungin production, along with a better yield coefficient were obtained under conditions of enhanced aeration during fermentation. A higher oxygen transfer rate was found to stimulate aureofungin A, and suppresses aureofungin B formation.

[Screening of beta-lactamase inhibitors among representatives of the genus Micromonospora and the genus Streptoverticillium]. / Poisk ingibitorov beta-laktamaz sredi predstavitelei roda Micromonospora i roda Streptoverticillium.

A total of 123 cultures of the genus Micromonospora and 89 cultures of the genus Streptoverticillium were studied for their capacity to produce beta-lactamase inhibitors. It was shown that streptoverticilla were highly resistant to beta-lactam antibiotics. No organisms producing beta-lactamase inhibitors were detected among the cultures of this genus. As for Micromonospora, 30 cultures (24.4 per cent) of this genus produced inhibitors. They were most frequently isolated from orange and brown cultures of Micromonospora. The inhibitors had no antibacterial activity, inhibited beta-lactamases of gram-negative bacteria and did not inhibit exogenic beta-lactamases of staphylococci. Their inhibitory activity was evident only with the use of isolated enzymes. With the use of intact growing cultures of gram-negative bacteria producing the same enzymes (Pseudomonas, Klebsiella, Enterobacter) no inhibitory activity was observed. Though the inhibitors had some features in common, they did not seem to be similar, since they had different spectra with respect to their effect on various beta-lactamases of gram-negative bacteria.

[Auxotrophic mutants of the producer of the antibiotic mycoheptin Streptoverticillium mycoheptinicum]. / Auksotrofnye mutanty produtsenta antibiotika mikogeptina Streptoverticillium mycoheptinicum.

The lethal and mutagenic effects of N-nitrozo-N-methyl biuret (NMB), N-nitrozo-N-methyl urea (NMU) and UV light on Streptoverticillium mycoheptinicum, strains O883 and 852, were studied. The concentrations of NMB were 0.005, 0.1 and 0.25 per cent, the exposure time was 2, 4 and 6 hours. The concentration of NMU was 1 per cent and the exposure time was 1, 2, 3, 4 and 5 hours. The dose of UV light was 2000 erg/mm2. When the spores of Streptoverticillium mycoheptinicum, strain O883, were treated with NMB, the frequency of auxotrophic mutants increased from 0.63 to 3.4 per cent with an increase of the mutagen concentration from 0.05 to 0.25 per cent and the exposure time from 2 to 6 hours. More than 80 auxotrophic mutants were selected. When Streptoverticillium mycoheptinicum, strain 852, was treated with NMU, the frequency of auxotrophic mutants ranged from 0.5 to 2.4 per cent. Fifty-seven auxotrophic mutants were selected. The majority of the auxotrophic mutants selected with the use of NMB and NMU was unstable. Exposure of Streptoverticillium mycoheptinicum, strains 852, 10/69 Met and 54/100 Lys to UV light resulted in formation of groups of polyauxotrophic mutants.

[Action of acridine dyes on antibiotic, pigment and aerial mycelium formation in streptomycete producers of multicomponent antibiotics]. / Deistvie akridinovykh krasitelei na obrazovanie antibiotika, pigmenta i vozdushnogo mitseliia u streptomitsetov--prudutsentov mnogokomponentnykh antibiotikov.

The ability of 5 streptomycetous species synthesizing multicomponent antibiotic to produce the antibiotic and water-soluble pigment and to form the aerial mycelium in the presence of acridine dyes was studied. It was found that the character of the produced complex changed, when acridine dyes were added to the medium under conditions not affecting the culture growth and the temperature was elevated. Colonies deficient with respect to formation of the aerial mycelium and with changed pigment and antibiotic production were detected in the monospore cultures of the streptomycetes treated with acridine dyes, when the spore survival was equal to 100 percent, the frequency of the colonies being about 40 per cent.

[Postradiation effect of caffeine on the producer of mycoheptin, Streptoverticillium mycoheptinicum]. / Postradiatsionnoe vliianie kofeina na produtsent mikogeptina Streptoverticillium mycoheptinicum.

The modifying effect of caffeine on irradiated spores of Streptoverticillium mycoheptinicum, producing mycoheptin was found. Postradiation treatment of the strain O883: spores with caffeine resulted in decreased survival of the spores proportionally to the radiation dose increase and postradiation caffeine treatment. An increase in the frequency of the morphologically changed colonies, as well as the low and highly active variants with respect to mycoheptin production was observed. The effect may be explained by the fact that caffeine possibly inhibited the reparation process in the irradiated spores of the strain tested. The method of postradiation treatment of the spores of the mycoheptin-producing organism with caffeine which provided selection of highly active variants by the antibiotic production with the use of definite doses may be considered promising in selection of actinomycetes.

[Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]. / Letal'noe i mutagennoe deistvie N-nitrozo-N-metilbiureta na produtsenty levorina, amfoteritsina i mikogeptina.

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.

[Dependence of the lethal and mutagenic effect of mitomycin C and UV rays on the Streptoverticillium mucoheptinicum spore germination process]. / Zavisimost' letal'nogo i mutagennogo éffekta mitomitsina C i UF-luchei ot protsessa prorastaniia spor Streptoverticillium mucoheptinicum.

The lethal and mutagenic effects of the DNA-tropic antibiotic mithomycin C and UV-light on Streptoverticillium mycoheptinicum during the period of the spore replication were studied. The spores were more stable to the effect of the mutagens during DNA replication after 120 minutes of incubation. The mutagenic activity of mithomycin C and UV-light was closely connected with the quantitative changes in the genetic material of the pores. During DNA replication the rate of the morphological mutants was the highest. With respect to production of mycoheptin the spores were most mutable under the effect of mithomycin C during the period of DNA replication, while under the effect of UV-light they were most mutable at the lag-phase.

[Effect of mineral phosphorus on mycoheptin biosynthesis]. / Vliianie mineral'nogo fosfora na biosintez mikogeptina

The effect of mineral phosphorus in doses of 22.8 to 684 gamma/ml in a synthetic medium on biosynthesis of mycoheptin, an antifungal antibiotic was studied. It was found that the maximum productivity of the mycelium was at the phosphorus concentration in the medium equal to 22.8--45.6 gamma/ml and the concentration of 91.2 gamma/ml was optimal for the growth of the antibiotic-prodicing organism. The phosphorus optimal concentrations for the growth and antibiotic synthesis phases did not coincide that provided consideration of phosphorus as a limiting factor useful in control of the antibiotic biosynthesis. The content of pphosphorus in the medium 6 times higher than the optimal concentration did not inhibit the biomass accumulation and had no significant effect on the colony morphology.