Ester-prodrugs of ethambutol control its antibacterial activity and provide rapid screening for mycobacterial hydrolase activity

M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems

Actinomycetes from less explored ecosystems were screened for antibacterial and antimycobacterial activity. Crude bioactive compounds were produced by growing these strains by shake flask fermentation using soybean meal medium. Culture supernatant and mycelia were extracted with ethyl acetate and methanol, respectively. Antibacterial activity of crude extracts was tested by disc diffusion method against gram positive and gram negative bacteria. Actinomycete strains D10, D5, NEK5, ANS2, M104 and R2 showed prominent activity. Culture filtrates and crude extracts were tested against standard strain Mycobacterium tuberculosis H₃₇Rv and drug sensitive and drug resistant clinical isolates of M. tuberculosis by luciferase reporter phage (LRP) assay. Considerable variation was observed in antimycobacterial activity between actinomycete culture filtrates and solvent extracts. Actinomycete strains viz., D10, D5 (desert), CSA14 (forest), CA33 (alkaline soil), NEK5 (Neem plant), MSU, ANS2, R2 and M104 (marine) screened in the present study were found to be highly potent showing good antibacterial and antimycobacterial activity. Five of them such as A3, CSA1, EE9, ANS5 and R9 were exclusively active against M. tuberculosis. Secretary products of actinomycetes of rare ecosystems are meant to antagonize organisms in their respective environments. These are likely to be novel antimycobacterial compounds as they unknown to human pathogens.     

Rapid screening of lactic acid bacteria for restriction endonuclease activity

Summary A rapid microscale heparin Sepharose CL-6B affinity gel procedure was developed for detecting restriction endonuclease (RE-Nase) activity in a variety of lactic acid bacteria. RE-Nase-containing extracts free of DNA, RNA and nonspecific nuclease activity can be produced for forty or more strains daily and only 10–12 ml of each log phase culture was required for screening. RE-Nase activity was detected in several streptococci and lactobacilli. With appropriate modifications, this procedure should allow rapid detection of RE-Nase activity in other bacterial species.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

牙鲆肠道内产蛋白酶菌的分离与筛选

利用酪蛋白培养基和脱脂乳培养基,从健康牙鲆肠道筛选出25株产蛋白酶的益生菌,占健康牙鲆肠道菌群的17.7%,并利用福林-酚试剂法测定其酶活力.结果表明,25株菌具有产蛋白酶酶活力,其中E14、F1、G7和Y2-2株产酶活力特别强,有望成为益生菌在水产养殖上应用.     

Screening antibacterial activity of entomopathogenic bacteria isolated from pests of hazelnut

Thirty-seven entomopathogenic bacteria isolated from six common pests of hazelnut in the Black Sea Region of Turkey have been screened for their potential of antibacterial substance production against indicator bacteria by the agar spot assay and well diffusion assay. Results indicated that 13.5% of entomopathogenic bacteria, Pseudomonas fluorescens (Pf-Xd1), Bacillus polymyxa (Bp-Ar2), Bacillus thuringiensis (Bt-Bn1), Serratia marcescens (Sm-Mm3) and Pseudomonas flourescens (Pf-Aa4) isolated from pests of Xyleborus dispar, Anoplus roboris, Balaninus nucum, Melolontha melolontha and Agelastica alni, respectively, showed significant levels of inhibitor activities against indicator bacteria. Well diffusion assay showed that supernatants of Bp-Ar2, Bt-Bn1 and Sm-Mm3 have antibacterial activity, whereas Pf-Xd1 and Pf-Aa4 did not show any activity. Furthermore, the antibacterial activity of the substance produced by the Bp-Ar2 has a narrow spectrum, whereas those of Bt-Bn1 and Sm-Mm3 exhibit broad spectrum. The production of these antibacterial substances were similarly determined at early logarithmic phase in the growth cycle of three bacteria and continued until the beginning of the stationary phase as primer metabolite. In addition, optimal pH (at 7–9 forBt-Bn1 and 5–9 forSm-Mm3), medium (Muller Hinton broth forBt-Bn1 and Luria Bertani broth forSm-Mm3), temperature (25°C for Bt-Bn1 and Sm-Mm3) and production time (24h forBt-Bn1 and 72h forSm-Mm3) of these substances were determined. Our results demonstrate that entomopathogenic bacteria are a potential source of antibacterial substances.     

The synthesis and screening of 1,4,5,8-naphthalenetetracarboxylic diimide-peptide conjugates with antibacterial activity

We have employed an initial combinatorial approach followed by systematic lead optimization to investigate a series of novel molecules that exhibit antimicrobial activity against Gram-negative and Gram-positive bacteria. The new molecules contain various sequences of amino acids, generally L-lysine and glycine, attached to the 1,4,5,8-naphthalenetetracarboxylic diimide aromatic unit. Systematic structure-activity studies found that increasing positive charge enhanced activity and molecules containing one naphthalenetetracarboxylic diimide unit as well as at least seven lysine residues were optimum for antimicrobial activity. The naphthalenetetracarboxylic diimide derivatives were found to be inactive against mammalian cell lines, making them excellent antimicrobial candidates. Our results indicate that combining positive charge with aromatic and/or hydrophobic elements may be an interesting new approach to antimicrobial agents and adds an important new dimension to the field of cationic peptides.     

The discovery of biaryl acids and amides exhibiting antibacterial activity against Gram-positive bacteria

Solid-phase synthetic methods for biaryl-based compounds were developed resulting in the construction of two 1000-member libraries. Numerous compounds were identified by high-throughput screening using whole cell screens to exhibit anti-microbial activity against Gram-positive bacteria. A series of biaryl compounds containing natural and unnatural amino acids were made to explore the SAR of the amino acid functionality.     

The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C.

Brochocin-C is a two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.     

Isolation and characterization of some moderately halophilic bacteria with lipase activity

Lipases are an important class of enzymes which catalyze the hydrolysis of long chain triglycerides and constitute the most prominent group of biocatalysts for biotechnological applications. There are a number of lipases, produced by some halophilic microorganisms. In this study, some lipase producing bacteria from the Maharla salt lake located in south of Iran were isolated. All isolates were screened for true lipase activity on plates containing olive oil. The lipase activity was measured using titrimetric methods. Among thirty three isolates, thirteen strains demonstrating orange zone around colonies under UV light, were selected for identification using the molecular methods and some morphological characteristics. The bacterium Bacillus vallismortis BCCS 007 with 3.41 ± 0.14 U/mL lipase activity was selected as the highest lipase producing isolate. This is the first report of isolation and molecular identification of lipase producing bacteria from the Maharla lake.     

Differential assay for high-throughput screening of antibacterial compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.     

Isolation and identification of a bacteriocin with antibacterial and antibiofilm activity from Citrobacter freundii

Multi- and pan-antibiotic-resistant bacteria area major health challenge in hospital settings. Furthermore,when susceptible bacteria establish surface-attached biofilm populations, they become recalcitrant to antimicrobial therapy. Therefore, there is a need for novel antimicrobials that are effective against multi-drug-resistant and surface-attached bacteria. A screen to identify prokaryote-derived antimicrobials from a panel of over 100 bacterial strains was performed. One compound isolated from Citrobacter freundii exhibited antimicrobial activity against a wide range of Gram-negative bacteria and was effective against biofilms. Random transposon mutagenesis was performed to find mutants unable to produce the antimicrobial compound.Transposons mapped to a bacteriocin gene located on a small plasmid capable of replication in Escherichia coli. The plasmid was sequenced and found to be highly similar to a previously described colicinogenic plasmid.Expression of the predicted bacteriocin immunity gene conferred bacteriocin immunity to E. coli. The predicted bacteriocin gene, colA-43864, expressed in E. coli was sufficient to generate anti-microbial activity, and purified recombinant ColA-43864 was highly effective in killing E. coli, Citrobacter species, and Klebsiella pneumoniae cells in a planktonic and biofilm state. This study suggests that bacteriocins can be an effective way to control surface-attached pathogenic bacteria.     

合成聚β-羟基丁酸(PHB)鞘细菌的分离、鉴定与筛选

通过富集培养和划线分离等手段从活性污泥中分离筛选到一株合成PHB鞘细菌FQ4 0 ,其生物学特征为 :杆菌 ,大小 (0 .6～ 1.5 ) μm× (1.5～ 5 .5 ) μm ;革兰氏染色阴性 ;具衣鞘 ,丝状体很长 ,偶见假分枝 ;亚端生鞭毛 ;胞内具PHB颗粒 ;菌落为光滑型和丝状型两种。经鉴定该菌株为鞘细菌类球衣菌属中的浮游球衣菌 (Sphaerotilusnatans)。对其合成PHB研究表明 :细胞内可积累大量的PHB颗粒 ,含量可达细胞干重的 30 .5 %。     

Isolation,enzyme-bound structure and antibacterial activity of platencin A1 from Streptomyces platensis

Natural products continue to serve as one of the best sources for discovery of antibacterial agents as exemplified by the recent discoveries of platensimycin and platencin. Chemical modifications as well as discovery of congeners are the main sources for gaining knowledge of structure–activity relationship of natural products. Screening for congeners in the extracts of the fermentation broths of Streptomyces platensis led to the isolation of platencin A₁, a hydroxy congener of platencin. The hydroxylation of the tricyclic enone moiety negatively affected the antibacterial activity and appears to be consistent with the hydrophobic binding pocket of the FabF. Isolation, structure, enzyme-bound structure and activity of platencin A₁ and two other congeners have been described.     

Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food

100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.     

An array of target-specific screening strains for antibacterial discovery

As the global threat of drug- and antibiotic-resistant bacteria continues to rise, new strategies are required to advance the drug discovery process. This work describes the construction of an array of Escherichia coli strains for use in whole-cell screens to identify new antimicrobial compounds. We used the recombination systems from bacteriophages lambda and P1 to engineer each strain in the array for low-level expression of a single, essential gene product, thus making each strain hypersusceptible to specific inhibitors of that gene target. Screening of nine strains from the array in parallel against a large chemical library permitted identification of new inhibitors of bacterial growth. As an example of the target specificity of the approach, compounds identified in the whole-cell screen for MurA inhibitors were also found to block the biochemical function of the target when tested in vitro.