Loss Of Synergism Research Articles

Aminophenyl chalcones potentiating antibiotic activity and inhibiting bacterial efflux pump

Chalcones and their derivatives are substances of great interest for medicinal chemistry due to their antibacterial activities. As the bacterial resistance to clinically available antibiotics has become a worldwide public health problem, it is essential to search for compounds capable of reverting the bacterial resistance. As a possibility, the chalcone class could be an interesting answer to this problem. The chalcones (2E)-1-(4′-aminophenyl)-3-(phenyl)‑prop-2-en-1-one (APCHAL), and (2E)-1-(4′-aminophenyl)-3-(4-chlorophenyl)‑prop-2-en-1-one (ACLOPHENYL) were synthesized by the Claisen-Schmidt condensation and characterized by 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and mass spectrometry (MS), In addition, microbiological tests were performed to investigate the antibacterial activity, modulatory potential, and efflux pump inhibition against Staphylococcus aureus (S. aureus) multi-resistant strains. Regarding the S. aureus Gram-positive model, the APCHAL presented synergism with gentamicin and antagonism with penicillin. APCHAL reduced the Minimum inhibitory concentration (MIC) of gentamicin by almost 70%. When comparing the effects of the antibiotic modifying activity of ACLOPHENYL and APCHAL, a loss of synergism is noted with gentamicin due to the addition of a chlorine to the substance structure. For Escherichia coli (E. coli) a total lack of effect, synergistic or antagonistic, was observed between ACLOPHENYL and the antibiotics. In the evaluation of inhibition of the efflux pump, both chalcones presented a synergistic effect with norfloxacin and ciprofloxacin against S. aureus, although the effect is much less pronounced with ACLOPHENYL. The effect of APCHAL is particularly notable against the K2068 (MepA overexpresser) strain, with synergistic effects with both ciprofloxacin and ethidium bromide. The docking results also show that both compounds bind to roughly the same region of the binding site of 1199B (NorA overexpresser), and that this region overlaps with the preferred binding region of norfloxacin. The APCHAL chalcone may contribute to the prevention or treatment of infectious diseases caused by multidrug-resistant S. aureus.

Interactions of resveratrol with other phenolics and activity against food-borne pathogens.

The aim of this study was to investigate the antibacterial activity of individual phenolics and their binary mixtures with resveratrol against selected food‐borne pathogens. The antibacterial activity was quantified using the broth microdilution method by the determination of minimal inhibitory concentrations (MICs). Interactions between compounds in the binary phenolic mixtures were determined by calculating the fractional inhibitory concentration index (FICI). The influence of the number of OH groups in the phenols’ structure on their antibacterial activity was assessed by principal component analysis (PCA). The most effective compounds were flavone luteolin and flavonol rutin, while the weakest antimicrobial activity was observed for phenolic acid and flavan‐3‐ols (catechin and epicatechin). The synergistic effect (FICI ≤0.5) of equimolar mixture of resveratrol with kaempferol was confirmed against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, while the mixture of rutin with resveratrol proved synergistic only against S. aureus. The increasing concentrations of resveratrol in the mixtures with kaempferol and rutin resulted in a loss of synergism which indicates that only selected phenolic mixtures, with optimal concentrations of their individual components, result in synergistic antibacterial activity. We did not find an association between total number of OH groups and antibacterial activity of either individual phenolics or their mixtures.

The cAMP Pathway Amplifies Early MyD88-Dependent and Type I Interferon-Independent LPS-Induced Interleukin-10 Expression in Mouse Macrophages

Interleukin-10 (IL-10) is a key anti-inflammatory cytokine, secreted by macrophages and other immune cells to attenuate inflammation. Autocrine type I interferons (IFNs) largely mediate the delayed expression of IL-10 by LPS-stimulated macrophages. We have previously shown that IL-10 is synergistically expressed in macrophages following a costimulus of a TLR agonist and cAMP. We now show that the cAMP pathway directly upregulates IL-10 transcription and plays an important permissive and synergistic role in early, but not late, LPS-stimulated IL-10 mRNA and protein expression in mouse macrophages and in a mouse septic shock model. Our results suggest that the loss of synergism is not due to desensitization of the cAMP inducing signal, and it is not mediated by a positive crosstalk between the cAMP and type I IFN pathways. First, cAMP elevation in LPS-treated cells decreased the secretion of type I IFN. Second, autocrine/paracrine type I IFNs induce IL-10 promoter reporter activity only additively, but not synergistically, with the cAMP pathway. IL-10 promoter reporter activity was synergistically induced by cAMP elevation in macrophages stimulated by an agonist of either TLR4, TLR2/6, or TLR7, receptors which signal via MyD88, but not by an agonist of TLR3 which signals independently of MyD88. Moreover, MyD88 knockout largely reduced the synergistic IL-10 expression, indicating that MyD88 is required for the synergism displayed by LPS with cAMP. This report delineates the temporal regulation of early cAMP-accelerated vs. late type I IFN-dependent IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.

Simplified Protocol for the Phenotypic Identification of Carbapenem Resistance Mechanism in Enterobacteriaceae.

Dear Editor, Carbapenems are often one of the last resorts available for the treatment of multi-drug resistant Enterobacteriaceae infections. However, in the recent past, reports were accumulating on the emergence and dissemination of carbapenem resistance [1]. Therefore, carbapenem resistance in Enterobacteriaceae is considered as one of the major public health problems worldwide. According to recent report [2], there are three major mechanisms involved in carbapenemase production, which confers carbapenem resistance among Enterobacteriaceae. They are, production of Klebsiella pneumoniae carbapenemase (blaKPC), production of metallo β-lactamase (MBL) encoded by blaNDM, blaIMP and blaVIM genes, and production of Oxacillinase-48(blaOXA-48) or its derivatives. However, the other mechanisms of carbapenem resistance often observed among Enterobacteriaceae are loss of porin channel along with the co-production of Extended Spectrum β-lactamase (ESBL) or Ambler Cass C (AmpC) β-lactamase [1]. Identification and documentation of carbapenem resistance in Enterobacteriaceae is imperative to contain their spread. Based on the literature review and minor modifications from the previously published protocols [1,3] herewith we present a simplified protocol for the easy detection and differentiation of the mechanism of resistance in Carbapenem Resistant Enterobacteriaceae (CRE) by combined Disk Test (CDT), a phenotypic method. In CDT, the synergism is identified as the significant enlargement (≥5 mm) of the Zone of Inhibition (ZOI) around the antibiotic disk with specific inhibitor, compared to the ZOI around the antibiotic disk without inhibitor. All clinical isolates of Enterobacteriaceae could be initially screened with ertapenem (ETP) disk (10μg), which is reportedly having high sensitivity in detecting carbapenemases among all carbapenems [4]. Then, the clinical isolates showing ZOI <25mm around ETP disk [3] could be further tested with following disks in single 150mm petri plate: ETP (10 μg), ETP+clavulanate (10 + 10μg), ETP+cloxacillin (10 + 750μg), ETP+boronate (10 + 400μg), ETP+EDTA (10 + 585μg), and temocillin (30 μg); optionally aztreonam (30 μg) disk may be included for susceptibility testing. Then the mechanism of carbapenem resistance can be identified based on the synergy observed with specific inhibitor and/or resistance to temocillin as described in [Table/Fig-1]. [Table/Fig-1]: Combined disk tests for the detection of common mechanisms of carbapenem resistance in Enterobacteriaceae (important test for each mechanism is shaded with blue colour) (*ZOI<10mm around the disk) Although the above protocol is highly reliable in detecting most isolates expressing any one of the major carbapenem resistance mechanisms, it may fail in detecting the isolates expressing more than one type of resistance mechanism. That is, production of more than one type of carbapenemases and/or ESBLs or AmpC enzymes with porin loss results in the loss of synergism. Further, this may lead to the false identification of KPC or MBL as OXA-48. Therefore, again we are dependent to molecular tools for the confirmation of OXA-48. However, this protocol would be helpful to reduce the need of molecular diagnosis to selective isolates especially in resource-limited settings. In conclusion, this protocol facilitates timely detection of CRE and identification of the mechanism of carbapenem resistance in Enterobacteriaceae; thus, it could help timely implementation of infection control measures.

Interfacial and aggregation properties of some anionic/cationic surfactant binary systems II. Mixed micelle formation and surface tension reduction effectiveness

Mixed micelle formation and surface tension reduction effectiveness (γcmc) were investigated for the following systems: triethanolammonium dodecylpoly(oxyethylene)sulfate (TADPS, containing about two ethylene oxide units)/dodecyltrimethylammonium bromide, TADPS/hexadecyltrimethylammonium bromide and TADPS/hexadecylpyridinium chloride. For all these anionic/cationic systems, the mixed critical micelle concentration (cmc) values reflect a strong synergism in mixed micelle formation, with βM values ranging from −13.8 to −18.3. The mixed micelle composition is mixing-ratio dependent and, for equimolar mixtures, the mixed micelle is richer in the surfactant with the lower cmc. Precipitation is inhibited to a certain extent, thanks to the presence of ethylene oxide groups in the anionic species. The conditions for synergism in γcmc, differently expressed in the literature, can be derived from the surface tension equations established in our previous article. They can be conveniently described by a few characteristic constants: Γi∞ (saturated Gibbs excess), Ki (constant in the Szyszkowski equation), the cmc of the individual surfactants and the interaction parameters, βS and βM, of their mixtures. Excellent agreement between theoretically predicted and experimental results is obtained. With the increase in surfactant chain length, the βM values decrease faster than the βS ones and this can result in the loss of synergism in γcmc.

Imipenem resistance in Bacteroides distasonis mediated by a novel beta-lactamase

Imipenem is a highly active drug against the Bacteroides fragilis group of organisms. On the basis of a nationwide survey of over 500 isolates, it was found that the frequency of imipenem resistance was less than 0.1%. We have a highly resistant Bacteroides distasonis isolate, TAL7860, for which the following MICs (micrograms per milliliter) were determined by agar dilution: cefoxitin, greater than 128; moxalactam, greater than 128; piperacillin, greater than 128; imipenem, 16; and SCH34343, 16. Resistance was shown to involve both a beta-lactamase and an outer membrane permeability barrier. beta-Lactamase kinetics studies with several beta-lactams, including imipenem, revealed similar hydrolytic efficiency in comparison with those found for the B. fragilis strains. An imipenem outer membrane permeability barrier was detected for TAL7860, which was approximately sixfold more effective for B. fragilis TAL3636 and TAL2480. Significant inhibition of nitrocefin destruction was also shown with sulbactam and clavulanic acid at 10 mumol and dithiothreitol at 10 mM. No inhibition was seen with 10 mM EDTA. Differences in physicochemical properties and inhibition studies suggest that this beta-lactamase is different from the imipenem-inactivating metallo-beta-lactamase previously described in B. fragilis. We demonstrated a significant permeability barrier to clavulanic acid and sulbactam, which resulted in loss of synergism between these clinically employed beta-lactamase inhibitors and beta-lactam drugs. The novel beta-lactamase activity in conjunction with a limited permeability in TAL7860 resulted in resistance to all commonly employed beta-lactams, including the newest and most potent beta-lactam drugs.

Properties of a CH3-blocked creatine kinase with altered catalytic activity. Kinetic consequences of the presence of the blocking group.

Steady state kinetic parameters for rabbit muscle creatine kinase (EC 2.7.3.2) and this enzyme stoichiometrically blocked at the iodoacetamide-sensitive cysteinyl residue with a CH3S-group have been measured at 30+/-0.1 degrees, pH 9.00, using Mg(II) as the required metal ion cofactor. The double reciprocal plots for the CH3S-blocked enzyme with MgATP as the variable substrate are biphasic, each curve showing a break at approximately 1.9 mM MgATP, and suggest the possibility of negative cooperativity in metal-nucleotide binding. Furthermore, extrapolated lines at high MgATP concentrations intersect on the abscissa, indicating loss of synergism in binding of substrates. In contrast, observed Michaelis constants for creatine are, within experimental error, the same for both native and blocked enzymes. The maximal velocity of the CH3S-blocked enzyme is found to be 28.1% of the value of the native enzyme. Product inhibition patterns for both native and blocked enzyme are also compared. Again, these patterns indicate that the CH3S-blocking group alters the nucleotide binding site more than the guanidino substrate binding site. Calculations using the methods of Chou and Fasman (1970) Biochemistry 13, 211-222) lead to the prediction that the active cysteinyl residue occurs at the beginning of a beta turn which separates two portions of beta sheet structure of the enzyme, and so may be in a position to mediate conformational changes in the protein.