Role of rifampin against Propionibacterium acnes biofilm in vitro and in an experimental foreign-body infection model.

Propionibacterium acnes is an important cause of orthopedic-implant-associated infections, for which the optimal treatment has not yet been determined. We investigated the activity of rifampin, alone and in combination, against planktonic and biofilm P. acnes in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration (MBC) were 0.007 and 4 μg/ml for rifampin, 1 and 4 μg/ml for daptomycin, 1 and 8 μg/ml for vancomycin, 1 and 2 μg/ml for levofloxacin, 0.03 and 16 μg/ml for penicillin G, 0.125 and 512 μg/ml for clindamycin, and 0.25 and 32 μg/ml for ceftriaxone. The P. acnes minimal biofilm eradication concentration (MBEC) was 16 μg/ml for rifampin; 32 μg/ml for penicillin G; 64 μg/ml for daptomycin and ceftriaxone; and ≥128 μg/ml for levofloxacin, vancomycin, and clindamycin. In the animal model, implants were infected by injection of 10⁹ CFU P. acnes in cages. Antimicrobial activity on P. acnes was investigated in the cage fluid (planktonic form) and on explanted cages (biofilm form). The cure rates were 4% for daptomycin, 17% for vancomycin, 0% for levofloxacin, and 36% for rifampin. Rifampin cured 63% of the infected cages in combination with daptomycin, 46% with vancomycin, and 25% with levofloxacin. While all tested antimicrobials showed good activity against planktonic P. acnes, for eradication of biofilms, rifampin was needed. In combination with rifampin, daptomycin showed higher cure rates than with vancomycin in this foreign-body infection model.

Activities of fosfomycin, tigecycline, colistin, and gentamicin against extended-spectrum-β-lactamase-producing Escherichia coli in a foreign-body infection model.

Limited antimicrobial agents are available for the treatment of implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli. We compared the activities of fosfomycin, tigecycline, colistin, and gentamicin (alone and in combination) against a CTX-M15-producing strain of Escherichia coli (Bj HDE-1) in vitro and in a foreign-body infection model. The MIC and the minimal bactericidal concentration in logarithmic phase (MBC(log)) and stationary phase (MBC(stat)) were 0.12, 0.12, and 8 μg/ml for fosfomycin, 0.25, 32, and 32 μg/ml for tigecycline, 0.25, 0.5, and 2 μg/ml for colistin, and 2, 8, and 16 μg/ml for gentamicin, respectively. In time-kill studies, colistin showed concentration-dependent activity, but regrowth occurred after 24 h. Fosfomycin demonstrated rapid bactericidal activity at the MIC, and no regrowth occurred. Synergistic activity between fosfomycin and colistin in vitro was observed, with no detectable bacterial counts after 6 h. In animal studies, fosfomycin reduced planktonic counts by 4 log(10) CFU/ml, whereas in combination with colistin, tigecycline, or gentamicin, it reduced counts by >6 log(10) CFU/ml. Fosfomycin was the only single agent which was able to eradicate E. coli biofilms (cure rate, 17% of implanted, infected cages). In combination, colistin plus tigecycline (50%) and fosfomycin plus gentamicin (42%) cured significantly more infected cages than colistin plus gentamicin (33%) or fosfomycin plus tigecycline (25%) (P < 0.05). The combination of fosfomycin plus colistin showed the highest cure rate (67%), which was significantly better than that of fosfomycin alone (P < 0.05). In conclusion, the combination of fosfomycin plus colistin is a promising treatment option for implant-associated infections caused by fluoroquinolone-resistant Gram-negative bacilli.

Beta-lactams against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) have developed resistance to virtually all non-experimental antibiotics. They are intrinsically resistant to beta-lactams by virtue of newly acquired low-affinity penicillin-binding protein 2A (PBP2A). Because PBP2A can build the wall when other PBPs are blocked by beta-lactams, designing beta-lactams capable of blocking this additional target should help solve the issue. Older molecules including penicillin G, amoxicillin and ampicillin had relatively good PBP2A affinities, and successfully treated experimental endocarditis caused by MRSA, provided that the bacterial penicillinase could be inhibited. Newer anti-PBP2A beta-lactams with over 10-fold greater PBP2A affinities and low minimal inhibitory concentrations were developed, primarily in the cephem and carbapenem classes. They are also very resistant to penicillinase. Most have demonstrated anti-MRSA activity in animal models of infection, and two--the carbapenem CS-023 and the cephalosporin ceftopibrole medocaril--have proceeded to Phase II and Phase III clinical evaluation. Thus, clinically useful anti-MRSA beta-lactams are imminent.

Isothermal microcalorimetry for antifungal susceptibility testing of Mucorales, Fusarium spp., and Scedosporium spp.

We evaluated isothermal microcalorimetry for real-time susceptibility testing of non-Aspergillus molds. MIC and minimal effective concentration (MEC) values of Mucorales (n = 4), Fusarium spp. (n = 4), and Scedosporium spp. (n = 4) were determined by microbroth dilution according to the Clinical Laboratory Standard Institute M38-A2 guidelines. Heat production of molds was measured at 37 °C in Sabouraud dextrose broth inoculated with 2.5 × 10(4) spores/mL in the presence of amphotericin B, voriconazole, posaconazole, caspofungin, and anidulafungin. As determined by microcalorimetry, amphotericin B was the most active agent against Mucorales (MHIC 0.06-0.125 μg/mL) and Fusarium spp. (MHIC 1-4 μg/mL), whereas voriconazole was the most active agent against Scedosporium spp. (MHIC 0.25 to 8 μg/mL). The percentage of agreement (within one 2-fold dilution) between the MHIC and MIC (or MEC) was 67%, 92%, 75%, and 83% for amphotericin B, voriconazole, posaconazole, and caspofungin, respectively. Microcalorimetry provides additional information on timing of antifungal activity, enabling further investigation of drug-mold and drug-drug interaction, and optimization of antifungal treatment.

Comprehensive approach for the detection of antifungal compounds using a susceptible strain of Candida albicans and confirmation of in vivo activity with the Galleria mellonella model.

An efficient screening strategy for the identification of potentially interesting low-abundance antifungal natural products in crude extracts that combines both a sensitive bioautography assay and high performance liquid chromatography (HPLC) microfractionation was developed. This method relies on high performance thin layer chromatography (HPTLC) bioautography with a hypersusceptible engineered strain of Candida albicans (DSY2621) for bioactivity detection, followed by the evaluation of wild type strains in standard microdilution antifungal assays. Active extracts were microfractionated by HPLC in 96-well plates, and the fractions were subsequently submitted to the bioassay. This procedure enabled precise localisation of the antifungal compounds directly in the HPLC chromatograms of the crude extracts. HPLC-PDA-mass spectrometry (MS) data obtained in parallel to the HPLC antifungal profiles provided a first chemical screening about the bioactive constituents. Transposition of the HPLC analytical conditions to medium-pressure liquid chromatography (MPLC) allowed the efficient isolation of the active constituents in mg amounts for structure confirmation and more extensive characterisation of their biological activities. The antifungal properties of the isolated natural products were evaluated by their minimum inhibitory concentration (MIC) in a dilution assay against both wild type and engineered strains of C. albicans. The biological activity of the most promising agents was further evaluated in vitro by electron microscopy and in vivo in a Galleria mellonella model of C. albicans infection. The overall procedure represents a rational and comprehensive means of evaluating antifungal activity from various perspectives for the selection of initial hits that can be explored in more in-depth mode-of-action studies. This strategy is illustrated by the identification and bioactivity evaluation of a series of antifungal compounds from the methanolic extract of a Rubiaceae plant, Morinda tomentosa, which was used as a model in these studies.

Susceptibility of clinical isolates of Leishmania aethiopica to miltefosine, paromomycin, amphotericin B and sodium stibogluconate using amastigote-macrophage in vitro model.

Cutaneous Leishmaniasis (CL) caused by Leishmania aethiopica is a public health and social problem with a sequel of severe and mutilating skin lesions. It is manifested in three forms: localized CL (LCL), mucosal CL (MCL) and diffuse CL (DCL). Unresponsiveness to sodium stibogluconate (Sb(V)) is common in Ethiopian CL patients. Using the amastigote-macrophage in vitro model the susceptibility of 24 clinical isolates of L. aethiopica derived from untreated patients was investigated. Eight strains of LCL, 9 of MCL, and 7 of DCL patients together with a reference strain (MHOM/ET/82/117/82) were tested against four antileishmanial drugs: amphotericin B, miltefosine, Sb(V) and paromomycin. In the same order of drugs, IC(50) (μg/ml±SD) values for the 24 strains tested were 0.16±0.18, 5.88±4.79, 10.23±8.12, and 13.63±18.74. The susceptibility threshold of isolates originating from the 3 categories of patients to all 4 drugs was not different (p>0.05). Maximal efficacy was superior for miltefosine across all the strains. Further susceptibility test could validate miltefosine as a potential alternative drug in cases of sodium stibogluconate treatment failure in CL patients.

In pancreatic carcinoma, dual EGFR/HER2 targeting with cetuximab/trastuzumab is more effective than treatment with trastuzumab/erlotinib or lapatinib alone: implication of receptors' down-regulation and dimers' disruption.

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2(low) human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab')(2) fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

Enrichment cultures should be performed in the detection of bacterial oral human pathogens in DUWLs

Water delivered by dental units during routine dental practice is densely contaminated by bacteria. The aim of this study was to determine actual isolation of the microorganisms sprayed from Dental Unit Water Lines (DUWLs) when enrichment cultures are performed and to compare frequencies with those obtained without enrichment cultures. Moreover, the antimicrobial susceptibilities of the microorganisms isolated were also studied. Water samples were collected from one hundred dental equipments in use at Dental Hospital of our University in order to evaluate the presence/absence of microorganisms and to perform their presumptive identification. Aliquots from all of the samples were inoculated in eight different media including both enrichment and selective media. Minimal inhibitory concentrations (MIC) were determined by the broth dilution method. The results herein reported demonstrate that most of the DUWLs were colonized by bacteria from human oral cavity; when enrichment procedures were applied the percentage of DUWLs with detectable human bacteria was one hundred percent. The results showed that in order to evaluate the actual risk of infections spread by DUWLs the inclusion of a step of pre-enrichment should be performed. The need for devices preventing bacterial contamination of DUWLs is a goal to be achieved in the near future that would contribute to maintain safety in dental medical assistance

Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes). Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin). All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

Synthesis and antitubercular activity of isoniazid condensed with carbohydrate derivatives

A series of 13 compounds analogous of isoniazid condensed with carbohydrate was synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H37Rv using Alamar Blue susceptibility test and the activity expressed as the minimum inhibitory concentration (MIC90) in μg/mL. Several compounds exhibited antitubercular activity (0.31-3.12 μg/mL) when compared with first line drugs such as isoniazid (INH) and rifampicin (RIP) and could be a good starting point to develop new compounds against tuberculosis.

Isolamento e seleção de micro-organismos resistentes e capazes de volatilizar mercúrio

Mercury (Hg) occurs in the environment as a natural and anthropogenic element, and through the years the accumulation of mercury has affected the integrity of ecosystems and human health. This study presents a screening of microorganisms resistant to organic and inorganic mercury, the determination of the minimum inhibitory concentration of Hg, the estimation of the mercury volatilization by selected microorganisms and the dynamics of volatilization. Eight Gram-negative bacteria resistant to high concentrations of mercury (60 to 210 mg L-1) were selected, and these isolates showed ability to volatilize the metal. The dynamics of the volatilization of the Proteus mirabilis M50C demonstrated that in only 4 h of incubation it was possible to volatilize 72% of the mercury present in the culture. The results showed promising application for bioremediation strategies.

Atividade anticolinesterásica e perfil químico de uma fração cromatográfica ativa do extrato etanólico das flores Bellis perennis L. (Asteraceae)

This work describes the isolation of an active flavonoid fraction and identification of isorhamnetin 3-O-β-D-(6''-acetyl)-galactopyranoside from flowers of B. perennis, and also the evaluation of anticholinesterase (AChE) activity of ethanolic extract from flowers (EEF) and the active fraction. The chemical structure of the flavonoid was defined on the basis of spectroscopic ¹H NMR, IR and UV data. EEF or flavonoid reduces AChE activity in vivo, while flavonoid also reduces AChE activity in vitro, showing a value of 1.49 mM for 50% inhibitory concentration (IC50), suggesting potential use as an insecticide or in the treatment of neurodegenerative diseases such as Alzheimer's disease.

In vivo sensitivity reduction of Puccinia triticina races, causal agent of wheat leaf rust, to DMI and QoI fungicides

Experiments were carried out to determine in vivo the IC50 and the IC90 for demethylation-inhibitor fungicides (DMIs, triazoles) and quinone outside inhibitors (QoIs, strobilurins) to the five most frequent races of Puccinia triticina in 2007 growing season in Southern Brazil. The tests were done in a greenhouse with wheat seedlings. DMI fungicides were tested at the concentrations, in mg/L, 0.0; 0.02; 0.2; 2.0; 20.0; 100.0 and 200.0, and QoIs at the concentrations 0.0; 0.0001; 0.001; 0.01; 0.1; 1 and 10.0 mg of active ingredient/L water. Fungicides were preventively applied at 24 hours before the inoculation of seedlings with the fungal spores. The effect of treatments was assessed based on the number of uredia/cm². The lowest IC50 (inhibitory concentration) for DMI fungicides determined for MCG-MN, sensitive race, ranged from 0.33 to 0.91 mg/L, while the highest values for MDP-MR, MDT-MR, MDK-MR, MFH-HT races, varied from 9.63 to 85.64 mg/L (suspected insensitivity). QoI fungicide presented an IC50 varying from 0.0018 to 0.14 mg/L. The sensitivity reduction factor for DMIs varied from 8.8 to 238.8, and for QoIs from 0.3 to 1.5 mg/L. Sensitivity reduction was confirmed for the races MDP-MR, MDT-MR, MDK-MR, MFH-HT to DMIs, as well as their sensitivity to QoI fungicides.

In vitro sensitivity of Fusarium graminearum isolates to fungicides

Head blight of wheat is a disease of global importance. In Brazil, it can cause damage of up to 27%. As resistant cultivars are not available yet, short-term disease control relies on the use of fungicides. The first step to reach effective management is to identify potent fungicides. In vitro experiments were conducted to determine the inhibitory concentration 50% (IC50) for mycelial growth or conidial germination, according to the chemical group of fungicides, of five Fusarium graminearum isolates of different origins. The following demethylation inhibitor (DMI) fungicides were tested: epoxiconazole, cyproconazole, metconazole, prochloraz, protioconazole and tebuconazole. In addition, azoxystrobin, kresoxim-methyl, pyraclostrobin and trifloxystrobin were included in the study, representing Quinone outside inhibitor fungicides (QoI), as well as a tubulin synthesis inhibitor, carbendazim and two ready mixtures, trifloxystrobin + tebuconazole or trifloxistrobin + prothioconazole. DMI's showed lower IC50 values compared to the QoI's. For the five tested isolates, in the overall mean, IC50 considering mycelial growth ranged for DMI's from 0.01 mg/L (metconazole, prochloraz and prothioconazole) to 0.12 mg/L (cyproconazole) and considering conidial germination for QoI's from 0.21 mg/L (azoxystrobin) to 1.33 mg/L (trifloxystrobin). The IC50 for carbendazim was 0.07 mg/L. All tested isolates can be considered sensitive to the studied DMI's, although certain differences in sensitivity could be detected between the isolates originating from one same state.

In vitro sensitivity reduction of Fusarium graminearum to DMI and QoI fungicides

In Brazil, Fusarium head blight (FHB) affecting wheat can cause up to 39.8% damage. Resistant cultivars are not available yet; thus, short-term disease control relies on the use of fungicides. The first step to improve control is to monitor fungal populations that are sensitivity to chemicals in order to achieve efficient FHB management. In vitro experiments were conducted to evaluate the inhibitory concentration (IC50) of fungicides for both mycelial growth and conidial germination of ten Fusarium graminearum isolates. The following demethylation inhibitor (DMI) fungicides were tested: metconazole, prothioconazole and tebuconazole. In addition, pyraclostrobin and trifloxystrobin were included, representing QoI fungicides, as well as three co-formulations containing metconazole + pyraclostrobin, prothioconazole + trifloxystrobin, and tebuconazole + trifloxystrobin. For mycelial growth, the overall mean IC50 of isolates was: metconazole 0.07, prothioconazole 0.1, and tebuconazole 0.19 mg/L. For the co-formulations, it was: prothioconazole + trifloxystrobin 0.08, tebuconazole + trifloxystrobin 0.12, and metconazole + pyraclostrobin 0.14 mg/L. Regarding spore germination inhibition, IC50 for prothioconazole + trifloxystrobin was 0.06, for tebuconazole + trifloxystrobin, 0.12 mg/L, for QoI alone pyraclostrobin, was 0.09, and for trifloxystrobin, 0.28 mg/L. There was a sensitivity shift among isolates and the highest fungitoxicity to F. graminearum was confirmed for prothioconazole, metconazole and tebuconazole .