Synthesis, Antimicrobial and Antibiofilm Activities, and Molecular Docking Investigations of 2-(1H-Indol-3-yl)-1H-benzo[d]imidazole Derivatives.

The treatment of many bacterial and fungal infections remains a problem due to increasing antibiotic resistance and biofilm formation by pathogens. In the present article, a methodology for the chemoselective synthesis of 2-(1H-indol-3-yl)-1H-benzo[d]imidazole derivatives is presented. We report on the antimicrobial activity of synthesized 2-(1H-indol-3-yl)-1H-benzo[d]imidazoles with significant activity against Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 43300 (MRSA), Mycobacterium smegmatis (mc(2)155/ATCC 700084), and Candida albicans ATCC 10231. High activity against staphylococci was shown by indolylbenzo[d]imidazoles 3ao and 3aq (minimum inhibitory concentration (MIC) < 1 µg/mL) and 3aa and 3ad (MIC 3.9-7.8 µg/mL). A low MIC was demonstrated by 2-(1H-indol-3-yl)-1-methyl-1H-benzo[d]imidazole (3ag) against M. smegmatis and against C. albicans (3.9 µg/mL and 3.9 µg/mL, respectively). 2-(5-Bromo-1H-indol-3-yl)-6,7-dimethyl-1H-benzo[d]imidazole (3aq) showed a low MIC of 3.9 µg/mL against C. albicans. Compounds 3aa, 3ad, 3ao, and 3aq exhibited excellent antibiofilm activity, inhibiting biofilm formation and killing cells in mature biofilms. Molecular docking analysis identified three potential interaction models for the investigated compounds, implicating (p)ppGpp synthetases/hydrolases, FtsZ proteins, or pyruvate kinases in their antibacterial action mechanism.

The Synthesis and Biological Evaluation of 2-(1H-Indol-3-yl)quinazolin-4(3H)-One Derivatives.

The treatment of many bacterial diseases remains a significant problem due to the increasing antibiotic resistance of their infectious agents. Among others, this is related to Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) and Mycobacterium tuberculosis. In the present article, we report on antibacterial compounds with activity against both S. aureus and MRSA. A straightforward approach to 2-(1H-indol-3-yl)quinazolin-4(3H)-one and their analogues was developed. Their structural and functional relationships were also considered. The antimicrobial activity of the synthesized compounds against Mycobacterium tuberculosis H37Rv, S. aureus ATCC 25923, MRSA ATCC 43300, Candida albicans ATCC 10231, and their role in the inhibition of the biofilm formation of S. aureus were reported. 2-(5-Iodo-1H-indol-3-yl)quinazolin-4(3H)-one (3k) showed a low minimum inhibitory concentration (MIC) of 0.98 µg/mL against MRSA. The synthesized compounds were assessed via molecular docking for their ability to bind long RSH (RelA/SpoT homolog) proteins using mycobacterial and streptococcal (p)ppGpp synthetase structures as models. The cytotoxic activity of some synthesized compounds was studied. Compounds 3c, f, g, k, r, and 3z displayed significant antiproliferative activities against all the cancer cell lines tested. Indolylquinazolinones 3b, 3e, and 3g showed a preferential suppression of the growth of rapidly dividing A549 cells compared to slower growing fibroblasts of non-tumor etiology.

Oxidative Stress Response and E. coli Biofilm Formation under the Effect of Pristine and Modified Carbon Nanotubes.

The article investigates the expression of oxyR and soxS oxidative stress genes in E. coli under the effect of pristine multi-walled carbon nanotubes (MWCNTs) and pristine single-walled carbon nanotubes (SWCNTs), MWCNTs and SWCNTs functionalized with carboxyl groups (MWCNTs-COOH and SWCNTs-COOH, respectively), SWCNTs functionalized with amino groups (SWCNTs-NH2) and SWCNTs functionalized with octadecylamine (SWCNTs-ODA). Significant differences were found in the expression of the soxS gene, while no changes were observed in the expression level of the oxyR gene. The pro-oxidant effect of SWCNTs, SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA is presented, and the contrary antioxidant effect of pristine MWCNTs and MWCNTs-COOH in the presence of methyl viologen hydrate (paraquat) is shown. The article shows that SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA added to the medium generate the production of reactive oxygen species (ROS) in bacterial cells. SWCNTs-COOH intensified the E. coli biofilm formation, and the biofilm biomass exceeded the control by 25 times. Additionally, it is shown that the rpoS expression increased in response to MWCNTs-COOH and SWCNTs-COOH, and the effect of SWCNTs-COOH was more significant. SWCNTs-COOH and SWCNTs-NH2 initiated an increase in ATP concentration in the planktonic cells and a decrease in the biofilm cells. The atomic force microscopy (AFM) method showed that the volume of E. coli planktonic cells after the exposure to carbon nanotubes (CNTs) decreased compared to that without exposure, mainly due to a decrease in cell height. The absence of a strong damaging effect of functionalized SWCNTs on E. coli K12 cells, both in suspension and in biofilms, is shown. Contact with functionalized SWCNTs initiated the aggregation of the polymeric substances of the biofilms; however, the cells did not lyse. Among the studied CNTs, SWCNTs-COOH caused an increased expression of the soxS and rpoS, the formation of ROS, and stimulation of the biofilm formation.

Cadaverine biosynthesis contributes to decreased Escherichia coli susceptibility to antibiotics.

Some bacterial stress responses are involved in survival under antibiotic treatment and contribute to less susceptible microbial forms selection. Here, we tested the role of cadaverine, one of the biogenic polyamines considered as universal adaptogens, in the processes. The expression of ldcC and cadA genes, encoding cadaverine-producing lysine decarboxylase, increased in Escherichia coli cells exposed to ß-lactams and fluoroquinolones but not aminoglycosides. The transcriptional regulators RpoS and SoxS controlled the expression of ldcC and cadA, respectively, in response to antibiotics. Exogenous cadaverine had little effect on E. coli antibiotic susceptibility, whereas non-antibiotic-induced endogenous cadaverine contributed to its tolerance to ß-lactams, fluoroquinolones, and aminoglycosides. Antibiotic-induced cadaverine synthesis promoted bacterial survival under fluoroquinolone exposure, as well as could contribute to low-resistant bacterial forms development. Selection under the fluoroquinolone levofloxacin exposure toward bacteria with an increased ability to synthesize cadaverine and negative correlation between LdcC activity and fluoroquinolone susceptibility in the selected forms were demonstrated. The same correlation in a special group of low-level resistant clinical E. coli isolates was revealed. So, cadaverine biosynthesis appeared to be a significant player in decreased E. coli antibiotic susceptibility development.

A synthetic diterpene analogue inhibits mycobacterial persistence and biofilm formation by targeting (p)ppGpp synthetases.

Bacterial persistence coupled with biofilm formation is directly associated with failure of antibiotic treatment of tuberculosis. We have now identified 4-(4,7-DiMethyl-1,2,3,4-tetrahydroNaphthalene-1-yl)Pentanoic acid (DMNP), a synthetic diterpene analogue, as a lead compound that was capable of suppressing persistence and eradicating biofilms in Mycobacterium smegmatis. By using two reciprocal experimental approaches - ΔrelMsm and ΔrelZ gene knockout mutations versus relMsm and relZ overexpression technique - we showed that both RelMsm and RelZ (p)ppGpp synthetases are plausible candidates for serving as targets for DMNP. In vitro, DMNP inhibited (p)ppGpp-synthesizing activity of purified RelMsm in a concentration-dependent manner. These findings, supplemented by molecular docking simulation, suggest that DMNP targets the structural sites shared by RelMsm, RelZ, and presumably by a few others as yet unidentified (p)ppGpp producers, thereby inhibiting persister cell formation and eradicating biofilms. Therefore, DMNP may serve as a promising lead for development of antimycobacterial drugs.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

Escherichia coli Isolated from Cases of Colibacillosis in Russian Poultry Farms (Perm Krai): Sensitivity to Antibiotics and Bacteriocins.

Escherichia coli strains isolated from case of colibacillosis in Russian poultry farms in the region of Perm Krai were analyzed for their sensitivity to main antibiotics and bacteriocins. Sensitivity profiles for 9 antibiotics and 20 bacteriocins were determined with the disc diffusion method and the overlay test, respectively. Further, with the PCR the presence of several bla and integron 1 genes was revealed and the phylogenetic group for each strain determined. Among the 28 studied E. coli strains 85.7% were resistant to at least three antibiotics, 53.6% to five or more drugs, and 10.7% to eight antibiotics. PCR revealed that the blaTEM gene was harbored by 71.4% of strains and the blaCTX-M gene by 53.6% of strains. The class 1 integrons were found in 28.6% of strains. All of the studied strains were insensitive to ten or more bacteriocins. More than 90% of the studied strains were insensitive to pore-forming colicins of group A and B colicins, while 60.7% were insensitive to colicins with DNase and RNase activity. All of the analyzed strains were insensitive to at least two of the tested microcins. Neither the antibiotic resistance profile nor the bacteriocin resistance profile correlated with phylogenetic group of the strains. Thus, the studied strains were shown to possess high levels of multiple resistance to antibiotics and insensitivity to bacteriocins.

Interactions of Pseudomonas aeruginosa in predominant biofilm or planktonic forms of existence in mixed culture with Escherichia coli in vitro.

Pseudomonas aeruginosa and Escherichia coli are known to be involved in mixed communities in diverse niches. In this study we examined the influence of the predominant form of cell existence of and the exometabolite production by P. aeruginosa strains on interspecies interactions, in vitro. Bacterial numbers of P. aeruginosa and E. coli in mixed plankton cultures and biofilms compared with their numbers in single plankton cultures and biofilms changed in a different way, but were in accordance with the form of P. aeruginosa cell existence. The mass of a mixed-species biofilm was greater than the mass of a single-species biofilm. Among the mixed biofilms, the one with the "planktonic" P. aeruginosa strain had the least biomass. The total pyocyanin and pyoverdin levels were found to be lower in all mixed plankton cultures. Despite this, clinical P. aeruginosa strains irrespective of the predominant form of existence ("biofilm" or "planktonic") had a higher total concentration of exometabolites than did the reference strain in 12-24 h mixed cultures. The metabolism of E. coli, according to its bioluminescence, was reduced in mixed cultures, and the decrease was by 20- to 100-fold greater with the clinical Pseudomonas strains than the reference Pseudomonas strain. Thus, both the predominant form of existence of and the exometabolite production by distinct P. aeruginosa strains should be considered to fully understand the interspecies relationship and bacteria survival in natural communities.

Polyamines reduce oxidative stress in Escherichia coli cells exposed to bactericidal antibiotics.

Bactericidal antibiotics (fluoroquinolones, aminoglycosides and cephalosporins) at their sublethal concentrations were able to produce hydroxyl radicals, hydrogen peroxide and superoxide anions (ROS) in Escherichia coli cells, which resulted in damage to proteins and DNA. The cells responded to oxidative stress by a 2-3-fold increase in cell polyamines (putrescine, spermidine) produced as a consequence of upregulation of ornithine decarboxylase (ODC). Relief of oxidative stress by cessation of culture aeration or addition of antioxidants substantially diminished or even completely abolished polyamine accumulation observed in response to antibiotics. Alternatively, inhibition of polyamine synthesis resulted in enhancement of oxidative stress in antibiotic-processed cells. When added to antibiotic-inhibited culture, polyamines reduced intracellular ROS production and thereby prevented damage to proteins and DNA. These effects eventually resulted in a substantial increase in cell viability, growth recovery and antibiotic resistance that were more strongly expressed in polyamine-deficient mutants.