Critical view on antimicrobial, antibiofilm and cytotoxic activities of quinazolin-4(3H)-one derived schiff bases and their Cu(II) complexes.

A series of nine 2,3-disubstituted-quinazolin-4(3H)-one derived Schiff bases and their three Cu(II) complexes was prepared and tested for their antimicrobial activities against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the substances were tested in vitro against Mycobacterium tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 and M. smegmatis ATCC 700084. While anti-enterococcal and antimycobacterial activities were insignificant, 3-[(E)-(2-hydroxy-5-nitrobenzylidene)amino]-2-(2-hydroxy-5-nitrophenyl)-2,3-dihydroquinazolin-4(1H)-one (SB3) and its Cu(II) complex (SB3-Cu) demonstrated bacteriostatic antistaphylococcal activity. In addition, both compounds, as well as the other two prepared complexes, showed antibiofilm activity, which resulted in a reduction of biofilm formation and eradication of mature S. aureus biofilm by 80% even at concentrations lower than the values of their minimum inhibitory concentrations. In addition, the compounds were tested for their cytotoxic effect on the human monocytic leukemia cell line THP-1. The antileukemic efficiency was improved by the preparation of Cu(II) complexes from the corresponding non-chelated Schiff base ligands.

Towards Anticancer and Antibacterial Agents: Design and Synthesis of 1,2,3-Triazol-quinobenzothiazine Derivatives.

In this paper, we describe a new method for synthesizing hybrid combinations of 1,2,3-triazoles with a tetracyclic quinobenzothiazinium system. The developed approach allowed for the production of a series of new azaphenothiazine derivatives with the 1,2,3-triazole system in different positions of the benzene ring. In practice, the methodology consists of the reaction of triazole aniline derivatives with thioquinanthrenediinium bis-chloride. The structure of the products was determined by 1H-NMR, 13C-NMR spectroscopy, and HR-MS spectrometry, respectively. Moreover, the spatial structure of the molecule and the arrangement of molecules in the crystal (unit cell) were determined by X-ray crystallography. The anticancer activity profiles of the synthesized compounds were tested in vitro against human cancer cells of the A549, SNB-19, and T47D lines and the normal NHDF cell line. Additional tests of antibacterial activity against methicillin-sensitive and methicillin-resistant staphylococci, vancomycin-sensitive and vancomycin-resistant enterococci, and two mycobacterial strains were also performed. In fact, the dependence of anticancer and antibacterial activity on the substituent type and its position in the quinobenzothiazinium system was observed. Furthermore, the distance-guided property evaluation was performed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) on the pool of the calculated descriptors. Finally, the theoretically approximated partition coefficients (clogP) were (inter-)correlated with each other and cross-compared with the empirically specified logPTLC parameters.

Research Note: A mixture of Bacteroides spp. and other probiotic intestinal anaerobes reduces colonization by pathogenic E. coli strain O78:H4-ST117 in newly hatched chickens.

An experimental group of one-day-old chicken from a commercial hatchery was given a defined mixture of 7 gut anaerobes. The next day the chicks were inoculated by an APEC strain O78:H4-ST117 resistant to ciprofloxacin, alongside with the control group and monitored for 4 wk after the inoculation for the presence of the colonizing strains and ciprofloxacin-resistant E. coli. Significant reduction of colonization rates in the first 2 wk was recorded in the experimental group for the numbers of ciprofloxacin-resistant E. coli. The results show that colonization of chicken by defined anaerobic mixtures may provide a decisive protection during the critical period of the chicken intestinal microflora development.

Design, Synthesis and Antimicrobial Properties of New Tetracyclic Quinobenzothiazine Derivatives.

A new method for modifying the structure of tetracyclic quinobenzothiazinium derivatives has been developed, allowing introduction of various substituents at different positions of the benzene ring. The method consists of reacting appropriate aniline derivatives with 5,12-(dimethyl)thioquinantrenediinium bis-chloride. A series of new quinobenzothiazine derivatives was obtained with propyl, allyl, propargyl and benzyl substituents in 9, 10 and 11 positions, respectively. The structure of the obtained compounds was analyzed by 1H and 13C NMR (HSQC, HMBC) and X-ray analysis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, and representatives of multidrug-resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). In addition, all the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. 9-Benzyloxy-5-methyl-12H-quino [3,4-b][1,4]benzothiazinium chloride (6j), 9-propoxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6a) and 9-allyloxy-5-methyl-12H-quino[3,4-b][1,4]benzothiazinium chloride (6d) demonstrated high activity against the entire tested microbial spectrum. The activities of the compounds were comparable with oxacillin, tetracycline and ciprofloxacinagainst staphylococcal strains and with rifampicin against both mycobacterial strains. Compound 6j had a significant effect on the inhibition of bacterial respiration as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity, but also bactericidal activity. Preliminary in vitro cytotoxicity screening of the compounds performed using normal human dermal fibroblasts (NHDF) proved that the tested compounds showed an insignificant cytotoxic effect on human cells (IC50 > 37 µM), making these compounds interesting for further investigation. Moreover, the intermolecular similarity of novel compounds was analyzed in the multidimensional space (mDS) of the structure/property-related in silico descriptors by means of principal component analysis (PCA) and hierarchical clustering analysis (HCA), respectively. The distance-oriented structure/property distribution was related with the experimental lipophilic data.

Trifluoromethylcinnamanilide Michael Acceptors for Treatment of Resistant Bacterial Infections.

A series of thirty-two anilides of 3-(trifluoromethyl)cinnamic acid (series 1) and 4-(trifluoromethyl)cinnamic acid (series 2) was prepared by microwave-assisted synthesis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. (2E)-3-[3-(Trifluoromethyl)phenyl]-N-[4-(trifluoromethyl)phenyl]prop-2-enamide (1j), (2E)-N-(3,5-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]prop-2-enamide (1o) and (2E)-N-[3-(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)-phenyl]prop-2-enamide (2i), (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)phenyl]-prop-2-enamide (2p) showed antistaphylococcal (MICs/MBCs 0.15-5.57 µM) as well as anti-enterococcal (MICs/MBCs 2.34-44.5 µM) activity. The growth of M. marinum was strongly inhibited by compounds 1j and 2p in a MIC range from 0.29 to 2.34 µM, while all the agents of series 1 showed activity against M. smegnatis (MICs ranged from 9.36 to 51.7 µM). The performed docking study demonstrated the ability of the compounds to bind to the active site of the mycobacterial enzyme InhA. The compounds had a significant effect on the inhibition of bacterial respiration, as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity but also bactericidal activity. Preliminary in vitro cytotoxicity screening was assessed using the human monocytic leukemia cell line THP-1 and, except for compound 2p, all effective agents did show insignificant cytotoxic effect. Compound 2p is an interesting anti-invasive agent with dual (cytotoxic and antibacterial) activity, while compounds 1j and 1o are the most interesting purely antibacterial compounds within the prepared molecules.

Study of Biological Activities and ADMET-Related Properties of Salicylanilide-Based Peptidomimetics.

A series of eleven benzylated intermediates and eleven target compounds derived from salicylanilide were tested against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference strains and against three clinical isolates of methicillin-resistant S. aureus (MRSA) and three isolates of vancomycin-resistant E. faecalis. In addition, the compounds were evaluated against Mycobacterium tuberculosis H37Ra and M. smegmatis ATCC 700084. The in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line THP-1. The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. The benzylated intermediates were found to be completely biologically inactive. Of the final eleven compounds, according to the number of amide groups in the molecule, eight are diamides, and three are triamides that were inactive. 5-Chloro-2-hydroxy-N-[(2S)- 4-(methylsulfanyl)-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (3e) and 5-chloro-2-hydroxy-N-[(2S)-(4-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino)pentan-2-yl)benzamide (3f) showed the broadest spectrum of activity against all tested species/isolates comparable to the used standards (ampicillin and isoniazid). Six diamides showed high antistaphylococcal activity with MICs ranging from 0.070 to 8.95 µM. Three diamides showed anti-enterococcal activity with MICs ranging from 4.66 to 35.8 µM, and the activities of 3f and 3e against M. tuberculosis and M. smegmatis were MICs of 18.7 and 35.8 µM, respectively. All the active compounds were microbicidal. It was observed that the connecting linker between the chlorsalicylic and 4-CF3-anilide cores must be substituted with a bulky and/or lipophilic chain such as isopropyl, isobutyl, or thiabutyl chain. Anticancer activity on THP-1 cells IC50 ranged from 1.4 to >10 µM and increased with increasing lipophilicity.

Detection of Plasmid-Mediated Resistance to Metronidazole in Clostridioides difficile from River Water.

Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured (6 out of 14 surface water samples yielded 6 isolates, and 2 out of 2 wastewater samples yielded 9 isolates). Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried ermB. Surprisingly, one isolate (RT010, ST15) showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample. IMPORTANCE The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water.

Antistaphylococcal Activities and ADME-Related Properties of Chlorinated Arylcarbamoylnaphthalenylcarbamates.

Pattern 1-hydroxy-N-(2,4,5-trichlorophenyl)-2-naphthamide and the thirteen original carbamates derived from it were prepared and characterized. All the compounds were tested against Staphylococcus aureus ATCC 29213 as a reference and quality control strain and in addition against three clinical isolates of methicillin-resistant S. aureus (MRSA). Moreover, the compounds were evaluated against Enterococcus faecalis ATCC 29212, and preliminary in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line (THP-1). The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. While pattern anilide had no antibacterial activity, the prepared carbamates demonstrated high antistaphylococcal activity comparable to the used standards (ampicillin and ciprofloxacin), which unfortunately were ineffective against E. feacalis. 2-[(2,4,5-Trichlorophenyl)carba- moyl]naphthalen-1-yl ethylcarbamate (2) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl butylcarbamate (4) expressed the nanomolar minimum inhibitory concentrations (MICs 0.018−0.064 µM) against S. aureus and at least two other MRSA isolates. Microbicidal effects based on the minimum bactericidal concentrations (MBCs) against all the tested staphylococci were found for nine carbamates, while 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl heptylcarbamate (7) and 2-[(2,4,5-trichlorophenyl)carbamoyl]naphthalen-1-yl (4-phenylbutyl)carbamate (14) demonstrated MBCs in the range of 0.124−0.461 µM. The selectivity index (SI) for most investigated carbamates was >20 and for some derivatives even >100. The performed tests did not show an effect on the damage to the bacterial membrane, while the compounds were able to inhibit the respiratory chain of S. aureus.

Study of Biological Activities and ADMET-Related Properties of Novel Chlorinated N-arylcinnamamides.

A series of eighteen 4-chlorocinnamanilides and eighteen 3,4-dichlorocinnamanilides were designed, prepared and characterized. All compounds were evaluated for their activity against gram-positive bacteria and against two mycobacterial strains. Viability on both cancer and primary mammalian cell lines was also assessed. The lipophilicity of the compounds was experimentally determined and correlated together with other physicochemical properties of the prepared derivatives with biological activity. 3,4-Dichlorocinnamanilides showed a broader spectrum of action and higher antibacterial efficacy than 4-chlorocinnamanilides; however, all compounds were more effective or comparable to clinically used drugs (ampicillin, isoniazid, rifampicin). Of the thirty-six compounds, six derivatives showed submicromolar activity against Staphylococcus aureus and clinical isolates of methicillin-resistant S. aureus (MRSA). (2E)-N-[3,5-bis(trifluoromethyl)phenyl]- 3-(4-chlorophenyl)prop-2-enamide was the most potent in series 1. (2E)-N-[3,5-bis(Trifluoromethyl)phenyl]-3-(3,4-dichlorophenyl)prop-2-enamide, (2E)-3-(3,4-dichlorophenyl)-N-[3-(trifluoromethyl)phenyl]prop-2-enamide, (2E)-3-(3,4-dichloro- phenyl)-N-[4-(trifluoromethyl)phenyl]prop-2-enamide and (2E)-3-(3,4-dichlorophenyl)- N-[4-(trifluoromethoxy)phenyl]prop-2-enamide were the most active in series 2 and in addition to activity against S. aureus and MRSA were highly active against Enterococcus faecalis and vancomycin-resistant E. faecalis isolates and against fast-growing Mycobacterium smegmatis and against slow-growing M. marinum, M. tuberculosis non-hazardous test models. In addition, the last three compounds of the above-mentioned showed insignificant cytotoxicity to primary porcine monocyte-derived macrophages.

Paraphocaeicola brunensis gen. nov., sp. nov., Carrying Two Variants of nimB Resistance Gene from Bacteroides fragilis, and Caecibacteroides pullorum gen. nov., sp. nov., Two Novel Genera Isolated from Chicken Caeca.

Three difficult-to-cultivate, strictly anaerobic strains, AN20T, AN421T, and AN502, were analyzed within a project studying possible probiotics for newly hatched chickens. Phylogenetic analyses showed that strains AN20T, AN421T, and AN502 formed two well-separated phylogenetic lineages in all phylogenetic and phylogenomic trees comprising members of the family Bacteroidaceae. Comparison to reference genomes of type species Bacteroides fragilis NCTC 9343T, Phocaeicola abscessus CCUG 55929T, and Capsularis zoogleoformans ATCC 33285T showed low relatedness based on the calculated genome-to-genome distance and orthologous average nucleotide identity. Analysis of fatty acid profiles showed iso-C15:0, anteiso-C15:0, C16:0, C18:1ω9c, and iso-C17:0 3OH as the major fatty acids for all three strains and additionally C16:0 3OH for AN421T and AN502. A specific combination of respiratory quinones different from related taxa was found in analyzed strains, MK-5 plus MK-11 in strain AN20T and MK-5 plus MK-10 in strains AN421T and AN502. Strains AN421T and AN502 harbor complete CRISPR loci with CRISPR array, type II-C, accompanied by a set of cas genes (cas9, cas1, and cas2) in close proximity. Interestingly, strain AN20T was found to harbor two copies of nimB gene with >95% similarity to nimB of B. fragilis, suggesting a horizontal gene transfer between these taxa. In summary, three isolates characterized in this study represent two novel species, which we proposed to be classified in two novel genera of the family Bacteroidaceae, for which the names Paraphocaeicola brunensis sp. nov. (AN20T = CCM 9041T = DSM 111154T) and Caecibacteroides pullorum sp. nov. (AN421T= CCM 9040T = DSM 111155T) are proposed. IMPORTANCE This study represents follow-up research on three difficult-to-cultivate anaerobic isolates originally isolated within a project focused on strains that are able to stably colonize newly hatched chickens, thus representing possible probiotics. This project is exceptional in that it successfully isolates several miscellaneous strains that required modified and richly supplemented anaerobic media, as information on many gut-colonizing bacteria is based predominantly on metagenomic studies. Superior colonization of newly hatched chickens by Bacteroides spp., Phocaeicola spp., or related taxa can be considered of importance for development of future probiotics. Although different experiments can also be performed with provisionally characterized isolates, precise taxonomical definition is necessary for subsequent broad communication. The aim of this study is therefore to thoroughly characterize these isolates that represent novel genera and precisely determine their taxonomic position among related taxa to facilitate further research and communication involving these strains.

Towards Property Profiling: SYNTHESIS and SAR Probing of New Tetracyclic Diazaphenothiazine Analogues.

A series of new tertiary phenothiazine derivatives containing a quinoline and a pyridine fragment was synthesized by the reaction of 1-methyl-3-benzoylthio-4-butylthioquinolinium chloride with 3-aminopyridine derivatives bearing various substituents on the pyridine ring. The direction and mechanism of the cyclization reaction of intermediates with the structure of 1-methyl-4-(3-pyridyl)aminoquinolinium-3-thiolate was related to the substituents in the 2- and 4-pyridine position. The structures of the compounds were analyzed using 1H, 13C NMR (COSY, HSQC, HMBC) and X-ray analysis, respectively. Moreover, the antiproliferative activity against tumor cells (A549, T47D, SNB-19) and a normal cell line (NHDF) was tested. The antibacterial screening of all the compounds was conducted against the reference and quality control strain Staphylococcus aureus ATCC 29213, three clinical isolates of methicillin-resistant S. aureus (MRSA). In silico computation of the intermolecular similarity was performed using principal component analysis (PCA) and hierarchical clustering analysis (HCA) on the pool of structure/property-related descriptors calculated for the novel tetracyclic diazaphenothiazine derivatives. The distance-oriented property evaluation was correlated with the experimental anticancer activities and empirical lipophilicity as well. The quantitative shape-based comparison was conducted using the CoMSA method in order to indicate the potentially valid steric, electronic and lipophilic properties. Finally, the numerical sampling of similarity-related activity landscape (SALI) provided a subtle picture of the SAR trends.

New Unnatural Gallotannins: A Way toward Green Antioxidants, Antimicrobials and Antibiofilm Agents.

Nature has been a source of inspiration for the development of new pharmaceutically active agents. A series of new unnatural gallotannins (GTs), derived from d-lyxose, d-ribose, l-rhamnose, d-mannose, and d-fructose have been designed and synthesized in order to study the protective and antimicrobial effects of synthetic polyphenols that are structurally related to plant-derived products. The structures of the new compounds were confirmed by various spectroscopic methods. Apart from spectral analysis, the antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and iron reducing power (FRAP) assays. Antibacterial activity of compounds was tested in vitro against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 (reference and control strains), three methicillin-resistant isolates of S. aureus, and three isolates of vancomycin-resistant E. faecalis. For screening of antimycobacterial effect, a virulent isolate of Mycobacterium tuberculosis and two non-tuberculous mycobacteria were used. Furthermore, antibiofilm activity of structurally different GTs against S. aureus, and their ability to inhibit sortase A, were inspected. Experimental data revealed that the studied GTs are excellent antioxidants and radical-scavenging agents. The compounds exhibited only a moderate antibacterial effect against Gram-positive pathogens S. aureus and E. faecalis and were practically inactive against mycobacteria. However, they were efficient inhibitors and disruptors of S. aureus biofilms in sub-MIC concentrations, and interacted with the quorum-sensing system in Chromobacteriumviolaceum. Overall, these findings suggest that synthetic GTs could be considered as promising candidates for pharmacological, biomedical, consumer products, and for food industry applications.

Different Bacteroides Species Colonise Human and Chicken Intestinal Tract.

Bacteroidaceae are common gut microbiota members in all warm-blooded animals. However, if Bacteroidaceae are to be used as probiotics, the species selected for different hosts should reflect the natural distribution. In this study, we therefore evaluated host adaptation of bacterial species belonging to the family Bacteroidaceae. B. dorei, B. uniformis, B. xylanisolvens, B. ovatus, B. clarus, B. thetaiotaomicron and B. vulgatus represented human-adapted species while B. gallinaceum, B. caecigallinarum, B. mediterraneensis, B. caecicola, M. massiliensis, B. plebeius and B. coprocola were commonly detected in chicken but not human gut microbiota. There were 29 genes which were present in all human-adapted Bacteroides but absent from the genomes of all chicken isolates, and these included genes required for the pentose cycle and glutamate or histidine metabolism. These genes were expressed during an in vitro competitive assay, in which human-adapted Bacteroides species overgrew the chicken-adapted isolates. Not a single gene specific for the chicken-adapted species was found. Instead, chicken-adapted species exhibited signs of frequent horizontal gene transfer, of KUP, linA and sugE genes in particular. The differences in host adaptation should be considered when the new generation of probiotics for humans or chickens is designed.

Consensus-Based Pharmacophore Mapping for New Set of N-(disubstituted-phenyl)-3-hydroxyl-naphthalene-2-carboxamides.

A series of twenty-two novel N-(disubstituted-phenyl)-3-hydroxynaphthalene- 2-carboxamide derivatives was synthesized and characterized as potential antimicrobial agents. N-[3,5-bis(trifluoromethyl)phenyl]- and N-[2-chloro-5-(trifluoromethyl)phenyl]-3-hydroxy- naphthalene-2-carboxamide showed submicromolar (MICs 0.16-0.68 µM) activity against methicillin-resistant Staphylococcus aureus isolates. N-[3,5-bis(trifluoromethyl)phenyl]- and N-[4-bromo-3-(trifluoromethyl)phenyl]-3-hydroxynaphthalene-2-carboxamide revealed activity against M. tuberculosis (both MICs 10 µM) comparable with that of rifampicin. Synergistic activity was observed for the combinations of ciprofloxacin with N-[4-bromo-3-(trifluoromethyl)phenyl]- and N-(4-bromo-3-fluorophenyl)-3-hydroxynaphthalene-2-carboxamides against MRSA SA 630 isolate. The similarity-related property space assessment for the congeneric series of structurally related carboxamide derivatives was performed using the principal component analysis. Interestingly, different distribution of mono-halogenated carboxamide derivatives with the -CF3 substituent is accompanied by the increased activity profile. A symmetric matrix of Tanimoto coefficients indicated the structural dissimilarities of dichloro- and dimetoxy-substituted isomers from the remaining ones. Moreover, the quantitative sampling of similarity-related activity landscape provided a subtle picture of favorable and disallowed structural modifications that are valid for determining activity cliffs. Finally, the advanced method of neural network quantitative SAR was engaged to illustrate the key 3D steric/electronic/lipophilic features of the ligand-site composition by the systematic probing of the functional group.

Insight into antimicrobial activity of substituted phenylcarbamoyloxypiperazinylpropanols.

3-[4-(Substituted)phenyl-/4-(diphenylmethyl)phenylpiperazin-1-yl]-2-hydroxypropyl-1-[(substituted)phenyl]carbamates and their salts with hydrochloric acid were synthesized, characterized, and tested in vitro against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference and quality control strains, against three methicillin-resistant isolates of S. aureus, and three isolates of vancomycin-resistant E. faecalis. All the compounds were evaluated against Mycobacterium tuberculosis H37Ra/ATCC 25177, M. kansasii DSM 44162, and M. smegmatis ATCC 700084. All of the tested compounds demonstrated very good activity against all the tested strains/isolates comparable with or better than that of clinically used drugs (ampicillin, ciprofloxacin, vancomycin, isoniazid). 1-[{(3-Trifluoromethyl)phenyl}carbamoyloxy-2-hydroxypropyl]-4-(3,4-dichlorophenyl)piperazin-1-ium chloride demonstrated the highest potency against all the tested strains/isolates (MICs ranged from 3.78 to 30.2 µM), and 1-[{(3-trifluoromethyl)phenyl}carbamoyloxy-2-hydroxypropyl]-4-(diphenylmethyl)piperazin-1-ium chloride was the most effective against all the screened mycobacterial strains (MICs ranged from 3.64 to 14.5 µM). All the investigated derivatives had strong antibiofilm activity against S. aureus ATCC 29123 and a synergistic or additive effect with gentamicin against isolates of E. faecalis with both intrinsic and acquired resistance to gentamicin. The screening of the cytotoxicity of the compounds was performed using human monocytic leukemia THP-1 cells. The IC50 values of the most effective compounds ranged from ca. 2.8 to 7.3 µM; thus, it can be stated that the antimicrobial effect is closely connected with their cytotoxicity. These observations disqualify these compounds from further development as antimicrobial agents, but they can be considered potential multi-target drugs with a preferred anticancer effect with good water solubility and additional anti-infectious activity.

The Colonisation of Calves in Czech Large-Scale Dairy Farms by Clonally-Related Clostridioides difficile of the Sequence Type 11 Represented by Ribotypes 033 and 126.

To investigate a possible Clostridioides difficile reservoir in the Czech Republic, we performed a study in 297 calves from 29 large-scale dairy farms. After enrichment, faecal samples were inoculated onto selective agar for C. difficile. From the 297 samples, 44 C. difficile isolates were cultured (prevalence of 14.8%, 10 farms). The Holstein breed and use of digestate were associated with C. difficile colonisation (p ˂ 0.05). C. difficile isolates belonged to the ribotype/sequence type: RT033/ST11 (n = 37), RT126/ST11 (n = 6) and RT046/ST35 (n = 1). A multiple-locus variable-number tandem-repeat analysis revealed four clonal complexes of RT033 isolates and one clonal complex of RT126 isolates. All isolates were sensitive to amoxicillin, metronidazole and vancomycin. Forty isolates were resistant to ciprofloxacin, twenty-one to clindamycin, seven to erythromycin, seven to tetracycline and six to moxifloxacin. Moxifloxacin resistant isolates revealed an amino-acid substitution Thr82Ile in the GyrA. In conclusion, the calves of Holstein breed from farms using digestate as a product of bio-gas plants are more likely to be colonised by clonally-related C. difficile of ST 11 represented by ribotypes 033 and 126. The identified resistance to moxifloxacin with a Thr82Ile substitution in the GyrA highlights the need for further monitoring by the "One health approach".

Genomic analysis of Escherichia coli strains isolated from diseased chicken in the Czech Republic.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) can cause various extraintestinal infections in poultry, resulting in massive economic losses in poultry industry. In addition, some avian E. coli strains may have zoonotic potential, making poultry a possible source of infection for humans. Due to its extreme genetic diversity, this pathotype remains poorly defined. This study aimed to investigate the diversity of colibacillosis-associated E. coli isolates from Central European countries with a focus on the Czech Republic. RESULTS: Of 95 clinical isolates subjected to preliminary characterization, 32 were selected for whole-genome sequencing. A multi resistant phenotype was detected in a majority of the sequenced strains with the predominant resistance to ß-lactams and quinolones being associated with TEM-type beta-lactamase genes and chromosomal gyrA mutations respectively. The phylogenetic analysis confirmed a great diversity of isolates, that were derived from nearly all phylogenetic groups, with predominace of B2, B1 and C phylogroups. Clusters of closely related isolates within ST23 (phylogroup C) and ST429 (phylogroup B2) indicated a possible local spread of these clones. Besides, the ST429 cluster carried blaCMY-2, - 59 genes for AmpC beta-lactamase and isolates of both clusters were generally well-equipped with virulence-associated genes, with considerable differences in distribution of certain virulence-associated genes between phylogenetically distant lineages. Other important and potentially zoonotic APEC STs were detected, incl. ST117, ST354 and ST95, showing several molecular features typical for human ExPEC. CONCLUSIONS: The results support the concept of local spread of virulent APEC clones, as well as of zoonotic potential of specific poultry-associated lineages, and highlight the need to investigate the possible source of these pathogenic strains.

Dibasic Derivatives of Phenylcarbamic Acid as Prospective Antibacterial Agents Interacting with Cytoplasmic Membrane.

1-[2-[({[2-/3-(Alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium/azepan- ium oxalates or dichlorides (alkoxy = butoxy to heptyloxy) were recently described as very promising antimycobacterial agents. These compounds were tested in vitro against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 (reference and control strains), three methicillin-resistant isolates of S. aureus, and three isolates of vancomycin-resistant E. faecalis. 1-[3-(Dipropylammonio)-2-({[3-(pentyloxy-/hexyloxy-/heptyloxy)phenyl]carbamoyl}oxy)propyl]pyrrolidinium dichlorides showed high activity against staphylococci and enterococci comparable with or higher than that of used controls (clinically used antibiotics and antiseptics). The screening of the cytotoxicity of the compounds as well as the used controls was performed using human monocytic leukemia cells. IC50 values of the most effective compounds ranged from ca. 3.5 to 6.3 µM, thus, it can be stated that the antimicrobial effect is closely connected with their cytotoxicity. The antibacterial activity is based on the surface activity of the compounds that are influenced by the length of their alkoxy side chain, the size of the azacyclic system, and hydro-lipophilic properties, as proven by in vitro experiments and chemometric principal component analyses. Synergistic studies showed the increased activity of oxacillin, gentamicin, and vancomycin, which could be explained by the direct activity of the compounds against the bacterial cell wall. All these compounds demonstrate excellent antibiofilm activity, when they inhibit and disrupt the biofilm of S. aureus in concentrations close to minimum inhibitory concentrations against planktonic cells. Expected interactions of the compounds with the cytoplasmic membrane are proven by in vitro crystal violet uptake assays.

Multiple In vitro biological effects of phenolic compounds from Morus alba root bark.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus alba L. is used in traditional Chinese medicine for the treatment of various diseases, including bacterial infections and inflammation. As a rich source of phenolic compounds, the plant is an object of many phytochemical and pharmacological studies. AIM OF THE STUDY: The aim of the study was to isolate and evaluate possible parallel antiviral, antibacterial, and anti-inflammatory activities of phenolic mulberry compounds. MATERIALS AND METHODS: Extensive chromatographic separation of mulberry root bark extract and in vitro biological screening of 26 constituents identified promising candidates for further pharmacological research. Selected compounds were screened for anti-infective and anti-inflammatory activities. Antiviral activity was determined by the plaque number reduction assay and by the titer reduction assay, antibacterial using broth microdilution method, and anti-inflammatory activity using COX Colorimetric inhibitor screening assay kit. One compound was evaluated in vivo in carrageenan-induced paw-edema in mice. RESULTS: Five prenylated compounds 1, 2, 8, 9, and 11, together with a simple phenolic ester 13, exhibited inhibitory activity against the replication of herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2), with IC50 values ranging from 0.64 to 1.93 µg/mL, and EC50 values 0.93 and 1.61 µg/mL. Molecular docking studies demonstrated the effects of the active compounds by targeting HSV-1 DNA polymerase and HSV-2 protease. In antibacterial assay, compounds 1, 4, 11, and 17 diminished the growth of all of the Gram-positive strains tested, with MIC values of 1-16 µg/mL. The anti-inflammatory ability of several compounds to inhibit cyclooxygenase 2 (COX-2) was tested in vitro, and compound 16 displayed greater activity than the indomethacin, positive control. Mulberrofuran B (11) showed anti-inflammatory activity in vivo against carrageenan-induced paw-edema in mice. CONCLUSIONS: Experimental investigation showed promising antiviral, antibacterial, and/or anti-inflammatory activities of the phenolic mulberry constituents, often with multiple inhibitory effects that might be used as a potential source of new medicine.

Gut Anaerobes Capable of Chicken Caecum Colonisation.

Chicks in commercial production are highly sensitive to enteric infections and their resistance can be increased by administration of complex adult microbiota. However, it is not known which adult microbiota members are capable of colonising the caecum of newly hatched chicks. In this study, we therefore orally inoculated chicks with pure cultures of 76 different bacterial isolates originating from chicken caecum on day 1 of life and determined their ability to colonise seven days later. The caecum of newly hatched chickens could be colonised by bacteria belonging to phyla Bacteroidetes, Proteobacteria, Synergistetes, or Verrucomicrobia, and isolates from class Negativicutes (phylum Firmicutes). On the other hand, we did not record colonisation with isolates from phyla Actinobacteria and Firmicutes (except for Negativicutes), including isolates from families Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, and Lactobacillaceae. Representatives of genera commonly used in probiotics such as Lactobacillus, Enterococcus, or Bacillus therefore did not colonise the chicken intestinal tract after a single dose administration. Following challenge with Salmonella enterica serovar Enteritidis, the best protecting isolates increased the chicken's resistance to S. Enteritidis only tenfold, which, however, means that none of the tested individual bacterial isolates on their own efficiently protected chicks against S. Enteritidis.