Hydroquinones Including Tetrachlorohydroquinone Inhibit Candida albicans Biofilm Formation by Repressing Hyphae-Related Genes.

Candida albicans is an opportunistic pathogenic fungus responsible for candidiasis. The pathogen readily forms antifungal agent-resistant biofilms on implanted medical devices or human tissue. Morphologic transition from yeast to filamentous cells and subsequent biofilm formation is a key virulence factor and a prerequisite for biofilm development by C. albicans. We investigated the antibiofilm and antifungal activities of 18 hydroquinones against fluconazole-resistant C. albicans. Tetrachlorohydroquinone (TCHQ) at subinhibitory concentrations (2 to 10 µg/mL) significantly inhibited C. albicans biofilm formation with an MIC of 50 µg/mL, whereas the backbone hydroquinone did not (MIC > 400 µg/mL), and it markedly inhibited cell aggregation and hyphal formation. Transcriptomic analyses showed that TCHQ downregulated the expressions of several hyphae-forming and biofilm-related genes (ALS3, ECE1, HWP1, RBT5, and UME6) but upregulated hyphae- and biofilm-inhibitory genes (IFD6 and YWP1). Furthermore, it prevented C. albicans biofilm development on porcine skin and at concentrations of 20 to 50 µg/mL was nontoxic to the nematode Caenorhabditis elegans and did not adversely affect Brassica rapa seed germination and growth. This study indicates that hydroquinones, particularly TCHQ, diminish the virulence, biofilm formation, and animal tissue adhesion of C. albicans, which suggests hydroquinones should be considered potential candidate antifungal agents against drug-resistant C. albicans strains. IMPORTANCE Persistence in chronic infections by Candida albicans is due to its ability of biofilm formation that endures conventional antifungals and host immune systems. Hence, the inhibition of biofilm formation and virulence characteristics is another mean of addressing infections. This study is a distinctive one since 18 hydroquinone analogues were screened and TCHQ efficiently inhibited the biofilm formation by C. albicans with significantly changed expressional profile of hyphae-forming and biofilm-related genes. The antibiofilm efficacy was confirmed using a porcine skin model and chemical toxicity was investigated using plant seed germination and nematode models. Our findings reveal that TCHQ can efficiently control the C. albicans biofilms and virulence characteristics.

Appraisal of Cinnamaldehyde Analogs as Dual-Acting Antibiofilm and Anthelmintic Agents.

Cinnamaldehyde has a broad range of biological activities, which include antibiofilm and anthelmintic activities. The ever-growing problem of drug resistance and limited treatment options have created an urgent demand for natural molecules with antibiofilm and anthelmintic properties. Hence, we hypothesized that molecules with a scaffold structurally similar to that of cinnamaldehyde might act as dual inhibitors against fungal biofilms and helminths. In this regard, eleven cinnamaldehyde analogs were tested to determine their effects on fungal Candida albicans biofilm and nematode Caenorhabditis elegans. α-Methyl and trans-4-methyl cinnamaldehydes efficiently inhibited C. albicans biofilm formation (>90% inhibition at 50 µg/mL) with minimum inhibitory concentrations (MICs) of ≥ 200 µg/mL and 4-bromo and 4-chloro cinnamaldehydes exhibited anthelmintic property at 20 µg/mL against C. elegans. α-Methyl and trans-4-methyl cinnamaldehydes inhibited hyphal growth and cell aggregation. Scanning electron microscopy was employed to determine the surface architecture of C. albicans biofilm and cuticle of C. elegans, and confocal laser scanning microscopy was used to determine biofilm characteristics. The perturbation in gene expression of C. albicans was investigated using qRT-PCR analysis and α-methyl and trans-4-methyl cinnamaldehydes exhibited down-regulation of ECE1, IFD6, RBT5, UCF1, and UME6 and up-regulation of CHT4 and YWP1. Additionally, molecular interaction of these two molecules with UCF1 and YWP1 were revealed by molecular docking simulation. Our observations collectively suggest α-methyl and trans-4-methyl cinnamaldehydes are potent biofilm inhibitors and that 4-bromo and 4-chloro cinnamaldehydes are anthelmintic agents. Efforts are required to determine the range of potential therapeutic applications of cinnamaldehyde analogs.

Assessment of Antibiofilm Potencies of Nervonic and Oleic Acid against Acinetobacter baumannii Using In Vitro and Computational Approaches.

Acinetobacter baumannii is a nosocomial pathogen, and its biofilms are tolerant to desiccation, nutrient starvation, and antimicrobial treatment on biotic and abiotic surfaces, tissues, and medical devices. Biofilm formation by A. baumannii is triggered by a quorum sensing cascade, and we hypothesized that fatty acids might inhibit its biofilm formation by interfering with quorum sensing. Initially, we investigated the antibiofilm activities of 24 fatty acids against A. baumannii ATCC 17978 and two clinical isolates. Among these fatty acids, two unsaturated fatty acids, nervonic and oleic acid, at 20 µg/mL significantly inhibited A. baumannii biofilm formation without affecting its planktonic cell growth (MICs were >500 µg/mL) and markedly decreased the motility of A. baumannii but had no toxic effect on the nematode Caenorhabditis elegans. Interestingly, molecular dynamic simulations showed that both fatty acids bind to the quorum sensing acyl homoserine lactone synthase (AbaI), and decent conformational stabilities of interactions between the fatty acids and AbaI were exhibited. Our results demonstrate that nervonic and oleic acid inhibit biofilm formation by A. baumannii strains and may be used as lead molecules for the control of persistent A. baumannii infections.

Antibiofilm and antifungal activities of medium-chain fatty acids against Candida albicans via mimicking of the quorum-sensing molecule farnesol.

Candida biofilms are tolerant to conventional antifungal therapeutics and the host immune system. The transition of yeast cells to hyphae is considered a key step in C. albicans biofilm development, and this transition is inhibited by the quorum-sensing molecule farnesol. We hypothesized that fatty acids mimicking farnesol might influence hyphal and biofilm formation by C. albicans. Among 31 saturated and unsaturated fatty acids, six medium-chain saturated fatty acids, that is, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid and lauric acid, effectively inhibited C. albicans biofilm formation by more than 75% at 2 µg ml-1 with MICs in the range 100-200 µg ml-1 . These six fatty acids at 2 µg ml-1 and farnesol at 100 µg ml-1 inhibited hyphal growth and cell aggregation. The addition of fatty acids to C. albicans cultures decreased the productions of farnesol and sterols. Furthermore, down-regulation of several hyphal and biofilm-related genes caused by heptanoic or nonanoic acid closely resembled the changes caused by farnesol. In addition, nonanoic acid, the most effective compound diminished C. albicans virulence in a Caenorhabditis elegans model. Our results suggest that medium-chain fatty acids inhibit more effectively hyphal growth and biofilm formation than farnesol.

Antimicrobial and antibiofilm activities of prenylated flavanones from Macaranga tanarius.

BACKGROUND: The emergence of antibiotic resistant microorganisms presents a worldwide problem that requires novel antibiotic and non-antibiotic strategies, and biofilm formation is a mechanism of drug resistance utilized by diverse microorganisms. The majority of microorganisms live in biofilms that help their survival against starvation, antimicrobial agents, and immunological defense systems. Therefore, it is important novel compounds be identified that inhibit biofilm formation and cell survival without drug resistance. STUDY DESIGN: In this study, the antimicrobial and antibiofilm activities of five prenylated flavanones (Okinawan propolins) isolated from fruits of Macaranga tanarius (L.) were investigated against 14 microorganisms including 10 pathogens. RESULTS: Of these five propolins, propolin D at 5-10 µg/ml significantly inhibited biofilm formation by three Staphylococcus aureus strains, a Staphylococcus epidermidis strain, and a Candida albicans with MICs from 10 to 50 µg/ml, and in C. albicans, propolin D was found to inhibit biofilm formation by reducing cell aggregation and downregulated the expressions of hypha/biofilm-related genes including ECE1 and HWP1. Interestingly, at sub-MIC concentrations (10-50 µg/ml), propolin D significantly inhibited biofilm formation by enterohemorrhagic E. coli O157:H7, uropathogenic E. coli O6:H1, and Acinetobacter baumannii without affecting planktonic cell growth, but did not inhibit biofilm formation by a commensal E. coli K-12 strain, three probiotic Lactobacillus plantarum strains, or two Pseudomonas aeruginosa strains. And, propolin D reduced fimbriae production by E. coli O157:H7 and repressed gene expression of curli fimbriae genes (csgA and csgB). Also, propolin D was minimally toxic in a Caenorhabditis elegans nematode model. CONCLUSION: These findings show that prenylated flavanones, especially propolin D from Macaranga tanarius (Okinawan propolis), should be considered potential candidates for the development of non-toxic antibacterial and antifungal agents against persistent microorganisms.

Inhibition of Biofilm Formation by Candida albicans and Polymicrobial Microorganisms by Nepodin via Hyphal-Growth Suppression.

Candida albicans is an opportunistic pathogenic yeast and is responsible for candidiasis. It readily colonizes host tissues and implant devices, and forms biofilms, which play an important role in pathogenesis and drug resistance. In this study, the antibiofilm, antihyphal, and antivirulence activities of nepodin, isolated from Rumex japonicus roots, were investigated against a fluconazole-resistant C. albicans strain and against polymicrobial-microorganism-biofilm formation. Nepodin effectively inhibited C. albicans biofilm formation without affecting its planktonic cell growth. Also, Rumex-root extract and nepodin both inhibited hyphal growth and cell aggregation of C. albicans. Interestingly, nepodin also showed antibiofilm activities against Candida glabrata, Candida parapsilosis, Staphylococcus aureus, and Acinetobacter baumannii strains and against dual biofilms of C. albicans and S. aureus or A. baumannii but not against Pseudomonas aeruginosa. Transcriptomic analysis performed by RNA-seq and qRT-PCR showed nepodin repressed the expression of several hypha- and biofilm-related genes (ECE1, HGT10, HWP1, and UME6) and increased the expression of several transport genes (CDR4, CDR11, and TPO2), which supported phenotypic changes. Moreover, nepodin reduced C. albicans virulence in a nematode-infection model and exhibited minimal cytotoxicity against the nematode and an animal cell line. These results demonstrate that nepodin and Rumex-root extract might be useful for controlling C. albicans infections and multispecies biofilms.

Subclinical Mastitis in a European Multicenter Cohort: Prevalence, Impact on Human Milk (HM) Composition, and Association with Infant HM Intake and Growth.

Background: Subclinical mastitis (SCM) is an inflammatory condition of the mammary gland. We examined the effects of SCM on human milk (HM) composition, infant growth, and HM intake in a mother-infant cohort from seven European countries. Methods: HM samples were obtained from 305 mothers at 2, 17, 30, 60, 90, and 120 days postpartum. SCM status was assessed using HM Sodium (Na): Potassium (K) ratio >0.6. Levels of different macro- and micronutrients were analyzed in HM. Results: SCM prevalence in the first month of lactation was 35.4%. Mean gestational age at delivery was lower and birth by C-section higher in SCM mothers (p ≤ 0.001). HM concentrations of lactose, DHA, linolenic acid, calcium, and phosphorous (p < 0.05 for all) was lower, while total protein, alpha-lactalbumin, lactoferrin, albumin, arachidonic acid to DHA ratio, n-6 to n-3 ratio and minerals (iron, selenium, manganese, zinc, and copper) were higher (p < 0.001 for all) in mothers with SCM. There were no differences in infant growth and HM intake between non-SCM and SCM groups. Conclusion: We document, for the first time, in a large European standardized and longitudinal study, a high prevalence of SCM in early lactation and demonstrate that SCM is associated with significant changes in the macro- and micronutrient composition of HM. Future studies exploring the relation of SCM with breastfeeding behaviors and developmental outcomes are warranted.

Knowledge and attitudes of primary care physicians in the management of patients at risk for cardiovascular events.

BACKGROUND: Adherence to clinical practice guidelines for management of cardiovascular disease (CVD) is suboptimal. The purposes of this study were to identify practice patterns and barriers among U.S. general internists and family physicians in regard to cardiovascular risk management, and examine the association between physician characteristics and cardiovascular risk management. METHODS: A case vignette survey focused on cardiovascular disease risk management was distributed to a random sample of 12,000 U.S. family physicians and general internists between November and December 2006. RESULTS: Responses from a total of 888 practicing primary care physicians who see 60 patients per week were used for analysis. In an asymptomatic patient at low risk for cardiovascular event, 28% of family physicians and 37% of general internists made guideline-based preventive choices for no antiplatelet therapy (p < .01). In a patient at high risk for cardiovascular event, 59% of family physicians and 56% of general internists identified the guideline-based goal for serum fasting LDL level (< 100 mg/dl). Guideline adherence was inversely related to years in practice and volume of patients seen. Cost of medications (87.7%), adherence to medications (74.1%), adequate time for counseling (55.7%), patient education tools (47.1%), knowledge and skills to recommend dietary changes (47.8%) and facilitate patient adherence (52.0%) were cited as significant barriers to CVD risk management. CONCLUSION: Despite the benefits demonstrated for managing cardiovascular risks, gaps remain in primary care practitioners' management of risks according to guideline recommendations. Innovative educational approaches that address barriers may facilitate the implementation of guideline-based recommendations in CVD risk management.

Age-related determinants of outcome after acute myocardial infarction: a dobutamine-atropine stress echocardiographic study.

OBJECTIVES: To investigate the cause of worse survival in older patients after myocardial infarction (MI). DESIGN: Prospective 18-month and longer follow-up study of a cohort of 167 patients (mean age +/- standard deviation 58 +/- 12, including 71 aged >or=65) with acute MI for cardiac events, defined as cardiac death, recurrent MI, or resuscitated ventricular tachycardia or fibrillation (VT/VF). SETTING: Milwaukee County Medical Complex and the Zablocki Veterans Affairs Medical Center, Milwaukee, WI. PARTICIPANTS: One hundred sixty-seven patients who underwent dobutamine-atropine stress echocardiography (DASE) in the first week (2-7 days) after acute MI and were medically managed. MEASUREMENTS: Comparison of event rates in older (>or=65 years) and younger (<65 years) patients and of clinical, resting echocardiographic, DASE, and angiographic findings in patients with and without events. Coronary angiography was performed in 141. RESULTS: Older and younger patients tolerated DASE well. During follow-up, there were 29 cardiac events (cardiac death in 17, nonfatal MI in 10, and VT/VF in 2). Events were more common in older patients (26% vs 12%, P <.05), especially death (19% vs 5%, P <.05). Scar size in the infarct zone by DASE was larger (4.0 +/- 2.8 vs 3.0 +/- 2.7 segments, P <.05) and remote wall motion abnormalities more common (47 vs 29%, P <.05) in older patients. Univariate determinants of outcome (P <.05) in older patients were diabetes mellitus; remote wall motion abnormalities; angiographic multivessel disease; scar size; ejection fraction; and resting, low-, and peak-dose wall motion score. Univariate determinants in younger patients were similar, but diabetes mellitus was not. Multivariate analysis revealed that remote wall motion abnormalities and scar size by DASE were independently predictive of outcome in older and younger patients and diabetes mellitus only in older patients. Low- and peak-dose DASE data enhanced (P <.01) the prediction of outcome in all patients with acute MI relative to clinical data and resting echocardiography. CONCLUSION: DASE was more predictive of outcome than clinical data and resting echocardiography in both age groups. Scar size and remote wall motion abnormalities were the primary determinants of outcome irrespective of age. The worse prognosis of older patients correlated with diabetes mellitus, greater scar size, and higher incidence of remote inducible ischemia.

Diagnostic and Prognostic Use of Stress Echocardiography and Radionuclide Scintigraphy.

Stress echocardiography and radionuclide scintigraphy are effective diagnostic and prognostic techniques in patients with known or suspected coronary artery disease (CAD), myocardial infarction (MI), chronic left ventricular dysfunction (LVD), and those undergoing noncardiac surgery. Both are sensitive and specific for the detection and extent of CAD. Negative tests confer a high negative predictive value for cardiac events irrespective of clinical risk. Positive studies confer a high positive predictive value for ischemic events in patients with intermediate to high clinical risk. Both provide incremental diagnostic and prognostic information relative to clinical, resting echocardiographic, and angiographic data. Meta-analysis studies have shown that the diagnostic and prognostic information provided by stress echocardiography is comparable with radionuclide scintigraphic stress tests. Stress echocardiography may be more specific for the detection and extent of CAD, whereas radionuclide scintigraphy may be more sensitive for single-vessel disease. Sensitivities are similar for the detection and extent of disease in patients with multivessel CAD.