Network pharmacology and experimental verification of the mechanism of licochalcone A against Staphylococcus aureus pneumonia.

Staphylococcus aureus strains cause the majority of pneumonia cases and are resistant to various antibiotics. Given this background, it is very important to discover novel host-targeted therapies. Licochalcone A (LAA), a natural plant product, has various biological activities, but its primary targets in S. aureus pneumonia remain unclear. Therefore, the purpose of this study was to identify its molecular target against S. aureus pneumonia. Network pharmacology analysis, histological assessment, enzyme-linked immunosorbent assays, and Western blotting were used to confirm the pharmacological effects. Network pharmacology revealed 33 potential targets of LAA and S. aureus pneumonia. Enrichment analysis revealed that these potential genes were enriched in the Toll-like receptor and NOD-like receptor signaling pathways. The results were further verified by experiments in which LAA alleviated histopathological changes, inflammatory infiltrating cells and inflammatory cytokines (TNF, IL-6, and IL-1ß) in the serum and bronchoalveolar lavage fluid in vivo. Moreover, LAA treatment effectively reduced the expression levels of NF-κB, p-JNK, p-p38, NLRP3, ASC, caspase 1, IL-1ß, and IL-18 in lung tissue. The in vitro experimental results were consistent with the in vivo results. Thus, our findings demonstrated that LAA exerts anti-infective effects on S. aureus-induced lung injury via suppression of the Toll-like receptor and NOD-like receptor signaling pathways, which provides a theoretical basis for understanding the function of LAA against S. aureus pneumonia and implies its potential clinical application.

Triclosan Reprograms Immunometabolism and Activates the Inflammasome in Human Macrophages.

To gather enough energy to respond to harmful stimuli, most immune cells quickly shift their metabolic profile. This process of immunometabolism plays a critical role in the regulation of immune cell function. Triclosan, a synthetic antibacterial component present in a wide range of consumer items, has been shown to cause immunotoxicity in a number of organisms. However, it is unclear whether and how triclosan impacts immunometabolism. Here, human macrophages were used as model cells to explore the modulatory effect of triclosan on immunometabolism. Untargeted metabolomics using integrated liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) revealed that triclosan changed the global metabolic profile of macrophages. Furthermore, Seahorse energy analysis and 13C isotope-based metabolic flux analysis revealed that triclosan decreased mitochondrial respiratory activity and promoted a metabolic transition from oxidative phosphorylation to glycolysis. Triclosan also polarizes macrophages to the proinflammatory M1 phenotype and activates the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing receptor 3 (NLRP3) inflammasome, which is consistent with triclosan-induced metabolic phenotypic modifications. Collectively, these findings showed that triclosan exposure at micromolar concentrations caused metabolic reprogramming in macrophages, which triggered an inflammatory response. These findings are important for understanding the immunotoxicity caused by triclosan, which is necessary for determining the risk posed by triclosan in the environment.

Roles of the Notch signaling pathway and microglia in autism.

The Notch signaling pathway is mainly involved in the regulation of neural stem cell proliferation, survival and differentiation during the development of the central nervous system. As a neurodevelopmental disorder, autism is associated with an abnormal increase in the number of microglia in several brain regions. These findings suggest that the pathogenesis of autism may be related to the Notch signaling pathway and microglia. In this review, we discuss how Notch pathway activity leads to behavioral abnormalities such as learning and memory impairment by influencing neuronal biological activities. An increase in microglial protein synthesis and abnormal autophagy can affect synaptic development and lead to behavioral abnormalities, and all of these changes can lead to autism. Furthermore, the Notch signaling pathway regulates the activation and differentiation of microglia and promotes inflammatory responses, leading to the occurrence of autism. When excessive reactive oxygen species (ROS) secreted by microglia cannot be cleared by autophagy in a timely manner, Notch signaling pathway activity is affected, possibly further increasing susceptibility to autism. This review reveals the mechanism underlying the role of the Notch signaling pathway, microglia and their interaction in the pathogenesis of autism and provides a theoretical reference for targeted clinical therapies for autism.

A novel NIR fluorescence probe with cysteine-activated structure for specific detection of cysteine and its application in vitro and in vivo.

Cysteine (Cys) as a vital antioxidant molecule and an effective biomarker for illness, plays an essential role in physiological functions and pathological processes. Extensive work has been done to explore the physiological functions of Cys and develop probes for detection of biothiols. However, the challenge to differentiate Cys from glutathione and homocystine remains. In this work, we constructed a novel near-infrared (NIR) probe, termed TMN-Cys, using TMN-NH2 and thionoesters. The probe could selectively detect Cys over homocysteine and glutathione in solution. It displayed a large Stokes shift (210 nm) upon treatment with Cys, and its detection limit was as low as 79 nM. Moreover, this probe showed low toxicity and was successfully employed in monitoring endogenous Cys in living cells and mice.

Metabolomics Study Reveals Inhibition and Metabolic Dysregulation in Staphylococcus aureus Planktonic Cells and Biofilms Induced by Carnosol.

Staphylococcus aureus (S. aureus) is a global health threat accompanied by increasing in drug resistance. To combat this challenge, there is an urgent need to find alternative antimicrobial agents against S. aureus. This study investigated the antimicrobial efficacy of carnosol against S. aureus using an in vitro model. The effects of carnosol were determined based on the antimicrobial effects or formation and disruption of biofilms. Finally, metabolomics of S. aureus grown as planktonic cells and biofilms with carnosol treatment were analyzed using gas chromatography-mass spectrometry. The minimum inhibitory concentrations (MICs) of carnosol were 32 to 256 µg/mL against the sixteen tested S. aureus strains. Among the biofilms, we observed a reduction in bacterial motility of the S. aureus, biofilm development and preformed biofilm after carnosol treatment. Moreover, the significantly altered metabolic pathways upon carnosol treatment in S. aureus planktonic cells and biofilms were highly associated with the perturbation of glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism, arginine and proline metabolism, alanine, aspartate and glutamate metabolism, arginine biosynthesis, and aminoacyl-tRNA biosynthesis. In addition, glutathione metabolism, D-glutamine and D-glutamate metabolism were significantly changed in the biofilms. This study establishes the antibacterial and antibiofilm properties of carnosol, and will provide an alternative strategy for overcoming the drug resistance of S. aureus.

Aloe-emodin induces autophagy and apoptotic cell death in non-small cell lung cancer cells via Akt/mTOR and MAPK signaling.

Lung cancer has a relatively poor prognosis, and the clinical efficacy of targeted drugs remains unsatisfactory. Therefore, the search for safe and efficient novel antitumor drugs has become an urgent problem in the treatment of lung cancer. Aloe-emodin (AE), a medicinal herb, has been demonstrated to exhibit many pharmacological effects on tumor cells, such as lung cancer cells. However, the anticancer properties of AE have not been fully exploited by modern medicine, as their mechanisms of action are not yet known. In this study, the bioassay results demonstrated that AE reduced the viability of the non-small cell lung cancer cell line A549 and NCI-H1299 in a dose- and time-dependent manner. Moreover, AE induced caspase-dependent apoptosis and autophagy. AE induced autophagy through activation of MAPK signaling and inhibition of the Akt/mTOR pathway. We also found that AE-induced autophagy was attenuated by the reactive oxygen species scavenger N-acetylcysteine, indicating that reactive oxygen species played a key role in AE-mediated autophagy in A549 and NCI-H1299 cells. Furthermore, AE induced reactive oxygen species-dependent autophagy in A549 and NCI-H1299 cells, which triggered apoptosis. Additionally, AE showed synergistic cytotoxic effects with the antitumor drug gemcitabine in A549 and NCI-H1299 cells. In brief, these results showed that AE might be useful for developing a therapeutic candidate for lung cancer complications.

Fosfomycin Protects Mice From Staphylococcus aureus Pneumonia Caused by α-Hemolysin in Extracellular Vesicles by Inhibiting MAPK-Regulated NLRP3 Inflammasomes.

α-Hemolysin (Hla) is a significant virulence factor in Staphylococcus aureus (S. aureus)-caused infectious diseases such as pneumonia. Thus, to prevent the production of Hla when treating S. aureus infection, it is necessary to choose an antibiotic with good antibacterial activity and effect. In our study, we observed that Fosfomycin (FOM) at a sub-inhibitory concentration inhibited expression of Hla. Molecular dynamics demonstrated that FOM bound to the binding sites LYS 154 and ASP 108 of Hla, potentially inhibiting Hla. Furthermore, we verified that staphylococcal membrane-derived vesicles (SMVs) contain Hla and that FOM treatment significantly reduced the production of SMVs and Hla. Based on our pharmacological inhibition analysis, ERK and p38 activated NLRP3 inflammasomes. Moreover, FOM inhibited expression of MAPKs and NLRP3 inflammasome-related proteins in S. aureus as well as SMV-infected human macrophages (MΦ) and alveolar epithelial cells. In vivo, SMVs isolated from S. aureus DU1090 (an isogenic Hla deletion mutant) or the strain itself caused weaker inflammation than that of its parent strain 8325-4. FOM also significantly reduced the phosphorylation levels of ERK and P38 and expression of NLRP3 inflammasome-related proteins. In addition, FOM decreased MPO activity, pulmonary vascular permeability and edema formation in the lungs of mice with S. aureus-caused pneumonia. Taken together, these data indicate that FOM exerts protective effects against S. aureus infection in vitro and in vivo by inhibiting Hla in SMVs and blocking ERK/P38-mediated NLRP3 inflammasome activation by Hla.

Aflatoxin B1 Induces Reactive Oxygen Species-Mediated Autophagy and Extracellular Trap Formation in Macrophages.

Aflatoxins are a group of highly toxic mycotoxins with high carcinogenicity that are commonly found in foods. Aflatoxin B1 (AFB1) is the most toxic member of the aflatoxin family. A recent study reported that AFB1 can induce autophagy, but whether AFB1 can induce extracellular traps (ETs) and the relationships among innate immune responses, reactive oxygen species (ROS), and autophagy and the ETs induced by AFB1 remain unknown. Here, we demonstrated that AFB1 induced a complete autophagic process in macrophages (MΦ) (THP-1 cells and RAW264.7 cells). In addition, AFB1 induced the generation of MΦ ETs (METs) in a dose-dependent manner. In particular, the formation of METs significantly reduced the AFB1 content. Further analysis using specific inhibitors showed that the inhibition of either autophagy or ROS prevented MET formation caused by AFB1, indicating that autophagy and ROS were required for AFB1-induced MET formation. The inhibition of ROS prevented autophagy, indicating that ROS generation occurred upstream of AFB1-induced autophagy. Taken together, these data suggest that AFB1 induces ROS-mediated autophagy and ETs formation and an M1 phenotype in MΦ.

Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells.

A recent study reported that Acinetobacter baumannii could induce autophagy, but the recognition and clearance mechanism of intracytosolic A. baumannii in the autophagic process and the molecular mechanism of autophagy induced by the pathogen remains unknown. In this study, we first demonstrated that invading A. baumannii induced a complete, ubiquitin-mediated autophagic response that is dependent upon septins SEPT2 and SEPT9 in mammalian cells. We also demonstrated that autophagy induced by A. baumannii was Beclin-1 dependent via the AMPK/ERK/mammalian target of rapamycin pathway. Of interest, we found that the isochorismatase mutant strain had significantly decreased siderophore-mediated ferric iron acquisition ability and had a reduced the ability to induce autophagy. We verified that isochorismatase was required for the recognition of intracytosolic A. baumannii mediated by septin cages, ubiquitinated proteins, and ubiquitin-binding adaptor proteins p62 and NDP52 in autophagic response. We also confirmed that isochorismatase was required for the clearance of invading A. baumannii by autophagy in vitro and in the mouse model of infection. Together, these findings provide insight into the distinctive recognition and clearance of intracytosolic A. baumannii by autophagy in host cells, and that isochorismatase plays a critical role in the A. baumannii-induced autophagic process.-Wang, Y., Zhang, K., Shi, X., Wang, C., Wang, F., Fan, J., Shen, F., Xu, J., Bao, W., Liu, M., Yu, L. Critical role of bacterial isochorismatase in the autophagic process induced by Acinetobacter baumannii in mammalian cells.

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species.

The successful treatment of bacterial infections is the achievement of a synergy between the host's immune defences and antibiotics. Here, we examined whether fosfomycin (FOM) could improve the bactericidal effect of phagocytes, and investigated the potential mechanisms. FOM enhanced the phagocytosis and extra- or intracellular killing of S. aureus by phagocytes. And FOM enhanced the extracellular killing of S. aureus in macrophage (MФ) and in neutrophils mediated by extracellular traps (ETs). ET production was related to NADPH oxidase-dependent reactive oxygen species (ROS). Additionally, FOM increased the intracellular killing of S. aureus in phagocytes, which was mediated by ROS through the oxidative burst process. Our results also showed that FOM alone induced S. aureus producing hydroxyl radicals in order to kill the bacterial cells in vitro. In a mouse peritonitis model, FOM treatment increased the bactericidal extra- and intracellular activity in vivo, and FOM strengthened ROS and ET production from peritoneal lavage fluid ex vivo. An IVIS imaging system assay further verified the observed in vivo bactericidal effect of the FOM treatment. This work may provide a deeper understanding of the role of the host's immune defences and antibiotic interactions in microbial infections.

Vinegar Treatment Prevents the Development of Murine Experimental Colitis via Inhibition of Inflammation and Apoptosis.

This study investigated the preventive effects of vinegar and acetic acid (the active component of vinegar) on ulcerative colitis (UC) in mice. Vinegar (5% v/v) or acetic acid (0.3% w/v) treatment significantly reduced the disease activity index and histopathological scores, attenuated body weight loss, and shortened the colon length in a murine experimental colitis model induced by dextran sulfate sodium (DSS). Further mechanistic analysis showed that vinegar inhibited inflammation through suppressing Th1 and Th17 responses, the NLRP3 inflammasome, and MAPK signaling activation. Vinegar also inhibited endoplasmic reticulum (ER) stress-mediated apoptosis in the colitis mouse model. Surprisingly, pretreatment with vinegar for 28 days before DSS induction increased levels of the commensal lactic acid-producing or acetic acid-producing bacteria, including Lactobacillus, Bifidobacteria, and Enterococcus faecalis, whereas decreased Escherichia coli levels were found in the feces of mice. These results suggest that vinegar supplementation might provide a new dietary strategy for the prevention of UC.

Phenotype and expression profile analysis of Staphylococcus aureus biofilms and planktonic cells in response to licochalcone A.

Staphylococcus aureus is one of the most important pathogens in humans and animals. The formation of biofilm by S. aureus is considered an important mechanism of antimicrobial resistance. Therefore, finding effective drugs against the biofilm produced by S. aureus has been a high priority. Licochalcone A (LAA), a natural plant product, was reported to have antibacterial activities and showed good activity against all 21 tested strains of S. aureus biofilm and planktonic cells. To detect the possible molecular mechanism of LAA against S. aureus biofilm or planktonic cells, Affymetrix GeneChips were used to determine the global comparative transcription of S. aureus biofilm and planktonic cells triggered by treatment with sub-bactericidal and sub-inhibitory concentrations of LAA, respectively. LAA significantly altered (greater than a 2- or less than -2-fold change) the expression of 693 genes in planktonic cells and 817 genes in biofilm. The levels of genes encoding autolysis-associated proteins, cell wall proteins, pathogenic factors, protein synthesis genes, and enzymes involved in capsule synthesis were significantly altered in LAA-treated S. aureus. Furthermore, some differences observed in the microarray analysis were verified by real-time RT-PCR. To our knowledge, this is the first observation of phenotype and expression profiles of S. aureus biofilm and planktonic cells in response to LAA treatment.

Antimicrobial activity of the imipenem/rifampicin combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures.

To investigate the antimicrobial activity of imipenem and rifampicin alone and in combination against clinical isolates of Acinetobacter baumannii grown in planktonic and biofilm cultures. Minimum inhibitory concentrations were determined for each isolate grown in suspension and in biofilm using a microbroth dilution method. Chequerboard assays and the agar disk diffusion assay were used to determine synergistic, indifferent or antagonistic interactions between imipenem and rifampicin. We used the tissue culture plate method for A. baumannii biofilm formation to measure the percentage of biofilm inhibition and the amount of extracellular DNA after the treatment. To understand the synergistic mechanisms, we conducted hydroxyl radical formation assays. The results were verified by confocal laser scanning microscopy. Imipenem and rifampicin showed effective antimicrobial activity against suspensions and biofilm cultures of A. baumannii, respectively. Synergistic antimicrobial effects between imipenem and rifampicin were observed in 13 and 17 of the 20 clinical isolates when in suspension and in biofilms, respectively. Imipenem and rifampicin alone and in combination generated hydroxyl radicals, which are highly reactive oxygen forms and the major components of bactericidal agents. Furthermore, treatment with imipenem and rifampicin individually or in combination has obvious antibiofilm effects. The synergistic activity of imipenem and rifampicin against clinical isolates of A. baumannii (in suspension and in biofilms) was observed in vitro. Therefore, we conclude that imipenem combined with rifampicin has the potential to be used as a combinatorial therapy for the treatment of infectious diseases caused by A. baumannii.

Synergistic Activity of Econazole-Nitrate and Chelerythrine against Clinical Isolates of Candida albicans.

The aim of this investigation was to assess the in-vitro interaction of two antifungal agents, econazole-nitrate and chelerythrine, against ten fluconazole-resistant clinical isolates and one ATCC type strain 10231 of Candida albicans. The checkerboard microdilution method was performed according to the recommendations of the National Committee for Clinical Laboratory Standards, and the results were determined by visual examination. The interaction intensity was tested in all isolates using the fractional inhibitory concentration index (FICI). These experiments showed synergism between econazole-nitrate and chelerythrine in antifungal activity against C. albicans, and no antagonistic activity was observed in any of the strains tested. Moreover, time-kill curves were performed with selected strains to confirm the positive interactions. The similarity between the results of the FICI values and the time-kill curves revealed that chelerythrine greatly enhances the antifungal effects of econazole-nitrate against isolates of C. albicans. This synergistic effect may markedly reduce the dose of econazole-nitrate required to treat candidiasis, thereby decreasing the econazole-nitrate toxic side effects. This novel synergism might provide a potential combination treatment against fungal infections.

Genome-wide expression profiling of the response to linezolid in Mycobacterium tuberculosis.

Tuberculosis (TB) is still one of the most common causes of death in the world. The emergence of multidrug-resistant and extensively drug-resistant (XDR-TB) Mycobacterium tuberculosis (M. tuberculosis) strains has increased the importance of searching for alternative targets to develop new antimycobacterial drugs. Linezolid, the first of oxazolidinones, is active in vitro against M. tuberculosis, but the response mechanisms of M. tuberculosis to linezolid are still poorly understood. To reveal the possible mechanism of action of linezolid against M. tuberculosis, commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes triggered by treatment with subinhibitory concentrations of linezolid. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. A total of 729 genes were found to be differentially regulated by linezolid. Among these, 318 genes were upregulated, and 411 genes were downregulated. A number of important genes were significantly regulated that are involved in various pathways, such as protein synthesis, sulfite metabolism, and genes involved in the cell envelope and virulence. This genome-wide transcriptomics approach produced the first insights into the response of M. tuberculosis to a linezolid challenge.

Efficacy of trans-cinnamaldehyde against Psoroptes cuniculi in vitro.

The acaricidal activity of trans-cinnamaldehyde was evaluated in vitro on Psoroptes cuniculi. In this study, different concentrations of trans-cinnamaldehyde were tested, and the observed mites mortality was compared with that observed in untreated and treated (Acacerulen R®) controls. The morphological changes in P. cuniculi treated with trans-cinnamaldehyde were examined with light microscopy. By the analysis of variance one-way test, up to 8 µg/ml of trans-cinnamaldehyde gave highly significant (P < 0.01) percentages of mite mortality compared with the untreated controls, but only up to 256 µg/ml, it showed the same efficacy of Acacerulen R®. At the same time, a bioassay was conducted by exposing mites to varying doses of trans-cinnamaldehyde in vitro cultures. The resulting data were analyzed by using a time-dose-mortality modeling technique, yielding the parameters for time and dose effects of P. cuniculi. The ß value was 2.01, indicating that trans-cinnamaldehyde had a good activity to kill P. cuniculi adults. Based on the time-dose-mortality relationships fitted and the virulence indices estimated, trans-cinnamaldehyde is a promising microbial agent for mites control.

In-vitro Antimycoplasmal Activity of Triclosan in Combination with Fluoroquinolones against Five mycoplasma Species.

Mycoplasmosis caused by mycoplasma has immensely reduced the performance of commercial animal husbandry, along with prevalence and increase of drug resistance in mycoplasma, thus new agents and therapies are urgently needed. Triclosan is a broad spectrum antimicrobial agent with a favorable safety profile. In the present study, we tested the antimycoplasmal activity of triclosan alone, as well as the in-vitro interaction of triclosan and the fluoroquinolones, gatifloxacin (GAT), moxifloxacin (MXF), levofloxacin (LVX), sparfloxacin (SPX), ciprofloxacin (CIP), enrofloxacin (EFX), and norfloxacin (NOR), against five mycoplasma species. This study demonstrated that triclosan had antimycoplasmal activity against both fluoroquinolones-sensitive species and a fluoroquinolones-resistant species isolated from clinic, with minimum inhibitory concentrations (MICs) of 16.0-64.0 µg/mL and 64.0 µg/ mL, respectively. A synergistic antimycoplasmal effect between triclosan and GAT, MFX or EFX against the five mycoplasma species was observed, with modulation factors (MFs) of 4-8, 4-16, 8-32, respectively, and fractional inhibitory concentration indexes (FICIs) of 0.375- 0.500, 0.350-0.500, 0.281-0.375, respectively. The combination of triclosan with LVX, SPX, CIP or NOR displayed either synergistic activity or indifference against the same mycoplasma species with MFs of 2-64, 4-16, 2-16, 2-64, respectively, while FICI values range from 0.516- 0.750, 0.500-0.625, 0.306-0.750, and 0.615-0.750, respectively. No antagonism was observed for any drug combination against any of the species tested. To the best of our knowledge, this is the first report that triclosan has synergistic activity with fluoroquinolones against mycoplasma species.

Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

BACKGROUND: The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL) shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA). MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. CONCLUSIONS/SIGNIFICANCE: MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

Transcriptional and functional analysis shows sodium houttuyfonate-mediated inhibition of autolysis in Staphylococcus aureus.

Sodium houttuyfonate (SH), an addition compound of sodium bisulfite and houttuynin, showed in vitro antibacterial activity against 21 Staphylococcus aureus (S. aureus) strains grown in planktonic cultures. Microarray results showed decreased levels of autolysin atl, sle1, cidA and lytN transcripts in the SH-treated strain as compared to the control strain, consistent with the induction of the autolytic repressors lrgAB and sarA and with the downregulation of the positive regulators agrA and RNAIII. Triton X-100-induced autolysis was significantly decreased by SH in S. aureus ATCC 25923, and quantitative bacteriolytic assays and zymographic analysis demonstrated SH-mediated reduction of extracellular murein hydrolase activity in these cells. Anti-biofilm assay showed that SH is poorly active against S. aureus grown in biofilm cultures, whereas SH diminished the amounts of extracellular DNA (eDNA) of S. aureus in a dose-dependent manner, which suggested that SH may impede biofilm formation by reducing the expression of cidA to inhibit autolysis and eDNA release in the early phase. Some of the microarray results were confirmed by real-time RT-PCR.

In Vitro Synergy of Biochanin A and Ciprofloxacin against Clinical Isolates of Staphylococcus aureus.

Many clinical isolates of Staphylococcus aureus (S. aureus) are resistant to numerous antimicrobials, including the fluoroquinolones (FQs). Flavonoids such as biochanin A (BCA) are compounds that are naturally present in fruits, vegetables, and plant-derived beverages. The goal of this investigation was to study the possible synergy between the antimicrobial agents BCA and ciprofloxacin (CPFX) when used in combination; CPFX was chosen as a representative FQ compound. We used S. aureus strain ATCC 25923 and 11 fluoroquinolone (FQ)-resistant methicillin-resistant S. aureus (MRSA) strains. Results from the drug susceptibility testing and checkerboard assays show that the minimum inhibitory concentration (MIC) of BCA ranged from 64 µg/mL to 512 µg/mL. When BCA was combined with CPFX, the fractional inhibitory concentration index (FICI) data showed that there was synergy in all 12 of the S. aureus strains tested. No antagonistic activity was observed in any of the strains tested. The results of time-kill tests and agar diffusion tests confirm that there was synergy between BCA and CPFX against S. aureus strains. These results suggest that BCA can be combined with FQs to produce a powerful antimicrobial agent.