Statistical evaluation of a newly modified Robbins device using a bioluminescent pseudomonad to quantify adhesion to plastic

A modified Robbins device (MRD) has frequently been used as a model system to study adhesion and biofilm formation. This study investigates the reproducibility of attachment and whether a statistically significant gradient of adhesion exists along the 25 sampling ports of a MRD. A simple, quantitative, non‐destructive, bioluminescence assay was developed in order to measure attachment of bioluminescent P. veronii BL146bio cells to plastic discs of Thermanox? in newly modified Robbins devices (nMRD). No statistically significant difference in mean bioluminescence values occurred between pairs of nMRDs run in parallel, but there was a significant difference in bioluminescence values between different batches of bacteria (p < 0.05). Generalised Linear Modelling showed that the position of the sample disc influenced the numbers attaching. In 50% of devices a significant positive gradient of attachment occurred and bioluminescence values varied from disc 1 to disc 25 by 29.6–58.0%. In the other 50% of nMRDs there was a smaller, non‐significant gradient. A disc sampling regime was devised to take this gradient into account and used to prove a positive correlation between bioluminescence and numbers of viable P. veronii BL146bio cells during a 6h biofilm accumulation period.     

Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Use of a serum-based antifungal susceptibility assay to predict the in vivo efficacy of novel echinocandin compounds

In vitro susceptibility assays of antifungal activity do not always accurately predict in vivo efficacy. As well as having a clear clinical importance, the ability to predict efficacy is also essential for effective screening of novel drug compounds. Initial screening of novel compounds must often be based on in vitro data. The present report describes the use of serum-MIC, an in vitro test of antifungal susceptibility, to accurately predict in vivo efficacy of echinocandin drugs in a mouse model of disseminated candidiasis. The basis of the serum-MIC method was to measure the inhibitory activity of a test compound against Candida albicans hyphal growth in the presence of pooled mouse serum. For 13 previously uncharacterized echinocandin compounds, as well as for the known echinocandin drugs, micafungin and caspofungin, serum-MIC determinations were shown to give better correlation to efficacy in the animal model than conventional, CLSI standard, in vitro antifungal susceptibility tests. The most accurate prediction of efficacy was obtained when the serum-MIC was adjusted in relation to the serum concentration at 30 min post-treatment. Furthermore, when the efficacy of micafungin was determined by measuring C. albicans kidney burden in the mouse model of infection, the adjusted serum-MIC consistently reflected the effective serum concentrations. Our data indicate that determination of serum-MIC values will facilitate prediction of the in vivo potency of new antifungal compounds such as novel echinocandins.     

In vitro efficacy of the lipopeptide biosurfactant surfactin-C15 and its complexes with divalent counterions to inhibit Candida albicans biofilm and hyphal formation

Abstract

Surfactin is a type of cyclic lipopeptide biosurfactant implicated in a wide range of applications. Although its antimicrobial activity has been characterized, its effect on Candida albicans physiology remains to be elucidated. The present study evaluated the influence of surfactin-C₁₅ (SF) and its complexes with divalent counterions on C. albicans biofilm formation and preformed biofilms. The SF and metal(II)-SF complexes inhibited biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. The same concentrations of the compounds studied dislodged preexisting biofilms grown on polystyrene plates. Moreover, SF and its metal(II) complexes reduced the mRNA expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1 and SAP4 without exhibiting significant growth inhibition. Further research showed that the compounds tested reduced cellular surface hydrophobicity (CSH). These results suggest that SF and metal(II)-SF complexes could be used as anti-biofilm agents against C. albicans hypha-related infections in clinical practice.     

A study on the effects of short-, medium- and long-term professional oral care in elderly persons requiring long-term nursing care at a chronic or maintenance stage of illness

Objective: The purpose of the present study is to clarify the frequency of provision of effective professional oral care by dental health workers in order to improve the oral hygiene state of elderly persons requiring long‐term care. Methods: In 105 patients requiring long‐term nursing care, 55 subjects with positive oral Candida albicans were divided into five groups according to the frequency of oral care intervention at intervals of 1, 2, 3, 4 and 6 weeks. The effects of professional oral care on the improvement of the oral hygiene state were investigated taking the frequency and duration of care into consideration. Results: The results showed that oral hygienic condition could be improved by performing professional oral care at intervals of 1 week for 12 consecutive weeks, and the improved condition could be maintained when professional oral care was continued at intervals of 1 week thereafter. Conclusion: It is necessary to continue professional oral care at intervals of 1 week for 12 consecutive weeks and at intervals of 2 weeks for more than 20 weeks. The oral hygiene state could be improved by professional intervention at intervals of three or four weeks in long‐term oral care, but this was limited to subjects whose Functional Independence Measure (FIM) scores were higher than 3.     

A novel in vitro flat‐bed perfusion biofilm model for determining the potential antimicrobial efficacy of topical wound treatments

Aims: To develop an in vitro flat‐bed perfusion biofilm model that could be used to determine the antimicrobial efficacy of topically applied treatments. Methods and Results: Pseudomonas aeruginosa and Staphylococcus aureus biofilms were grown within continuously perfused cellulose matrices. Enumeration of the biofilm density and eluate was performed at various sampling times, enabling determination of the biofilm growth rate. Two antimicrobial wound dressings were applied to the surface of mature biofilms and periodically sampled. To enable real‐time imaging of biofilm growth and potential antimicrobial kinetics, a bioluminescent Ps. aeruginosa biofilm was monitored using low‐light photometry. Target species produced reproducible steady‐state biofilms at a density of c. 10⁷ per biofilm support matrix, after 24‐h perfusion. Test dressings elicited significant antimicrobial effects, producing differing kill kinetic profiles. There was a good correlation between photon and viable count data. Conclusions: The model enables determination of the antimicrobial profile of topically applied treatments against target species biofilms, accurately differentiating bactericidal from bacteriostatic effects. Moreover, these effects could be monitored in real time using bioluminescence. Significance and Impact of the Study: This is the first in vitro biofilm model which can assess the antimicrobial potential of topical therapies in a dynamic growth environment.     

Standardized reactors for the study of medical biofilms: a review of the principles and latest modifications

Biofilms can cause severe problems to human health due to the high tolerance to antimicrobials; consequently, biofilm science and technology constitutes an important research field. Growing a relevant biofilm in the laboratory provides insights into the basic understanding of the biofilm life cycle including responses to antibiotic therapies. Therefore, the selection of an appropriate biofilm reactor is a critical decision, necessary to obtain reproducible and reliable in vitro results. A reactor should be chosen based upon the study goals and a balance between the pros and cons associated with its use and operational conditions that are as similar as possible to the clinical setting. However, standardization in biofilm studies is rare. This review will focus on the four reactors (Calgary biofilm device, Center for Disease Control biofilm reactor, drip flow biofilm reactor, and rotating disk reactor) approved by a standard setting organization (ASTM International) for biofilm experiments and how researchers have modified these standardized reactors and associated protocols to improve the study and understanding of medical biofilms.     

In vivo evaluation of modified gum karaya as a carrier for improving the oral bioavailability of a poorly water-soluble drug,nimodipine

This work examines the influence of modified gum karaya (MGK) on the oral bioavailability of a poorly water-soluble drug, nimodipine (NM), in comparison with that of gum karaya (GK). A cogrinding method was selected to prepare mixtures of NM and GK or MGK in a 1:9 ratio (NM:GK/MGK). Differential scanning calorimetry (DSC), Fourier transmission infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), solubility studies, and in vitro release studies were performed to characterize the properties of the cogrinding mixtures. No drug-carrier interactions were found, as confirmed by DSC and FT-IR studies. The XRD study revealed that the crystallinity of NM was identical in both the cogrinding mixtures and was decreased when compared to that of physical mixtures or pure NM. The in vitro release rate of NM from both cogrinding mixtures was significantly higher than that of physical mixtures or pure NM. The in vivo study revealed that the bioavailability of NM from pure drug was significantly lower when compared to the cogrinding mixtures. The oral bioavailability was found to be NM powder < cogrinding mixtures of NM and GK < cogrinding mixtures of NM and MGK < NM solution. It can be inferred from the above results that MGK, an economical carrier, could be used for the dissolution enhancement of NM.     

Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study

OBJECTIVE: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol. METHODS: (i) Group without lesions. Smears obtained from the lower lip, border of the tongue and floor of the mouth of 31 control individuals (group I), 49 tobacco users (group II) and 27 tobacco/alcohol users (group III) were stained using the Papanicolaou method. The first 100 cells counted on each smear determined the maturation pattern and the keratinization index (KI). Analysis of variance (ANOVA) and the Tukey multiple comparison test were used for statistical analysis, at a 5% significance level. (ii) Group with lesions. Cytopathological and histopathological studies were conducted for 15 patients: eight with leucoplakia without epithelial dysplasia, two with epithelial dysplasia and five with squamous cell carcinoma. RESULTS: (i) Group without lesions. Statistical analysis revealed a smaller number of superficial cells with nuclei in all sites of the group of tobacco/alcohol users (group III) when compared to the control group (group I), and this difference was statistically significant (P<0.005). (ii) Group with lesions. The severity of histopathological findings increased with the increase in the number of cells of the deeper epithelial layers, with a statistically significant difference in the number of intermediate (P=0.013) and parabasal cells (P=0.049), which increased with the severity of the epithelial maturation disorder: leucoplakias with dysplasia had a greater number of intermediate and parabasal cells than leucoplakias without dysplasia; and the number in squamous cell carcinomas was greater than in leucoplakias with dysplasia. CONCLUSION: The maturation pattern of cells in the three anatomic sites showed changes that may be associated with the synergistic effect of tobacco and alcohol. Also, the severity of histopathological findings was associated with the increase in the number of cells in the deeper epithelial layers.     

A biofilm growth protocol and the design of a magnetic field exposure setup to be used in the study of magnetic fields as a means of controlling bacterial biofilms

The use of prosthetic implants is increasing both in the United States and around the world and there is a concomitant rise in cases of biofilm‐based, persistent infections that are quite serious and virtually impervious to antibiotic treatment. The development of alternate therapies that do not involve long term use of high levels of antibiotics or surgical intervention is needed. Based on the success of using electric or magnetic fields to alter certain physiological processes, it is hypothesized that relatively low level magnetic fields, in conjunction with the appropriate antibiotic, may be able to help control and eventually clear bacterial biofilms on a prosthetic. In order to test this hypothesis, it is necessary to first develop a means of growing laboratory grade biofilms on specific materials in a way that is repeatable between experiments and that can be reproduced by other laboratories. Secondly, a means of applying controlled magnetic fields to the surfaces supporting the biofilms at a defined temperature must be developed. This article addresses both of these points. Bioelectromagnetics 31:56–63, 2010. © 2009 Wiley‐Liss, Inc.     

Use of a novel nonantibiotic triple marker gene cassette to monitor high survival of Pseudomonas fluorescens SBW25 on winter wheat in the field

Pseudomonas fluorescens SBW25 was tagged with a triple marker gene cassette containing gfp, encoding green fluorescent protein; luxAB, encoding luciferase; and telABkilA, encoding tellurite resistance, and the tagged strain was monitored in the first Swedish field release of a genetically modified microorganism (GMM). The cells were inoculated onto winter wheat seeds and the GMM cells (SBW25:tgl) were monitored in the field from September 2005 to May 2006 using plating, luminometry and microscopic analyses. Cell numbers were high on all sampling occasions and metabolically active cells were detected on all plant parts. Field results were similar to those obtained in a parallel phytotron study, although the amount of SBW25:tgl detected on shoots was significantly higher in the phytotron than in the field. After winter, cell counts were 100-fold higher on the roots and root-associated soil compared with prewinter measurements, although the cells had a lower relative metabolic activity. The wheat seeds were naturally infested with Microdochium nivale, but no treatment resulted in reduction of disease symptoms. No SWB25:tgl cells were ever found in bulk soil or uninoculated plants. The Swedish field trial results complement and contrast with prior field studies performed with the same parent organism in the United Kingdom under different soil, plant and climatic conditions.     

A Phase 2a randomized,single-center,double-blind,placebo-controlled study to evaluate the safety and preliminary efficacy of oral iOWH032 against cholera diarrhea in a controlled human infection model

Cholera remains a major cause of infectious diarrhea globally. Despite the increased availability of cholera vaccines, there is still an urgent need for other effective interventions to reduce morbidity and mortality. Furthermore, increased prevalence of antibiotic-resistant Vibrio cholerae threatens the use of many drugs commonly used to treat cholera. We developed iOWH032, a synthetic small molecule inhibitor of the cystic fibrosis transmembrane conductance regulator chloride channel, as an antisecretory, host-directed therapeutic for cholera. In the study reported here, we tested iOWH032 in a Phase 2a cholera controlled human infection model. Forty-seven subjects were experimentally infected with V. cholerae El Tor Inaba strain N16961 in an inpatient setting and randomized to receive 500 mg iOWH032 or placebo by mouth every 8 hours for 3 days to determine the safety and efficacy of the compound as a potential treatment for cholera. We found that iOWH032 was generally safe and achieved a mean (± standard deviation) plasma level of 4,270 ng/mL (±2,170) after 3 days of oral dosing. However, the median (95% confidence interval) diarrheal stool output rate for the iOWH032 group was 25.4 mL/hour (8.9, 58.3), compared to 32.6 mL/hour (15.8, 48.2) for the placebo group, a reduction of 23%, which was not statistically significant. There was also no significant decrease in diarrhea severity and number or frequency of stools associated with iOWH032 treatment. We conclude that iOWH032 does not merit future development for treatment of cholera and offer lessons learned for others developing antisecretory therapeutic candidates that seek to demonstrate proof of principle in a cholera controlled human infection model study.Trial registration: This study is registered with ClinicalTrials.gov as {"type":"clinical-trial","attrs":{"text":"NCT04150250","term_id":"NCT04150250"}}NCT04150250.     

Pivaloylcodeine,a new codeine derivative,for the inhibition of morphine glucuronidation. An in vitro study in the rat

We have previously found that phenanthrenic opioids, including codeine, modulate morphine glucuronidation in the rat. Here codeine and five of its derivatives were compared in their effects on the synthesis of morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) from morphine by rat liver microsomal preparations, and by primary cultures of rat hepatocytes previously incubated for 72 h with either codeine or its derivatives. Acetylcodeine and pivaloylcodeine shared the capability of the parent compound of inhibiting the synthesis of M3G by liver microsomes through a noncompetitive mechanism of action. Their IC₅₀ were 3.25, 2.27, and 4.32 μM, respectively. Dihydrocodeine, acetyldihydrocodeine, and lauroylcodeine were ineffective. In all the experimental circumstances M6G was undetectable in the incubation medium. In primary hepatocyte cultures codeine only inhibited M3G formation, but with a lower efficacy than that observed with microsomes (IC₅₀ 20.91 vs 4.32 μM). Preliminary results show that at micromolar concentrations codeine derivatives exhibit a low rate of affinity for μ opiate receptors. In conclusion, acetyl and pivaloyl derivatives of codeine noncompetitively inhibit liver glucuronidation of morphine interacting with microsomes. This study further strengths the notion that phenanthrenic opioids can modulate morphine glucuronidation independently from their effects on μ opiate receptors.     

Evaluation of the clinical efficacy of a mouthwash and oral gel containing the antimicrobial proteins lactoperoxidase, lysozyme and lactoferrin in elderly patients with dry mouth--a pilot study

Objectives: To evaluate the clinical efficacy of a mouthwash and oral gel containing the antimicrobial proteins lactoperoxidase, lactoferrin and lysozyme, in a sample of elderly individuals with dry mouth. Material and methods: Twenty elderly institutionalised subjects with dry mouth and with a certain degree of independence for daily life activities were included in this pilot study. A randomised, double blind and cross‐over design was used. The study variables comprised subjective dry mouth sensation, the severity of discomfort assessed by means of a visual analogical scale (VAS), the Oral Health Impact Profile (OHIP), the presence of signs and symptoms of dry mouth, sialometry and Candida albicans culture. All the variables were recorded before and after each of the two periods of the study. Results: The 20 selected subjects we made up of 16 women and four men, with a mean age of 81.3 years. Improvement was observed on analysing the data between the first and second intervention period in terms of the OHIP values, the presence of dry mouth, and the need to drink fluids to swallow. However, the improvement in certain variables before and after treatment did not take a positive course in all cases, and some subjects even improved with placebo. Conclusions: The evaluated mouthwash and oral gel improved some subjective and clinical aspects in elderly individuals with dry mouth, though a placebo effect cannot be entirely discarded.     

Deadlock, a novel protein of Drosophila, is required for germline maintenance, fusome morphogenesis and axial patterning in oogenesis and associates with centrosomes in the early embryo

The deadlock gene is required for a number of key developmental events in Drosophila oogenesis. Females homozygous for mutations in the deadlock gene lay few eggs and those exhibit severe patterning defects along both the anterior-posterior and dorsal-ventral axis. In this study, we analyzed eggs and ovaries from deadlock mutants and determined that deadlock is required for germline maintenance, stability of mitotic spindles, localization of patterning determinants, oocyte growth and fusome biogenesis in males and females. Deadlock encodes a novel protein which colocalizes with the oocyte nucleus at midstages of oogenesis and with the centrosomes of early embryos. Our genetic and immunohistological experiments point to a role for Deadlock in microtubule function during oogenesis.     

Development and validation of a liquid chromatography-tandem mass spectrometry for the determination of BPR0L075, a novel antimicrotuble agent, in rat plasma: application to a pharmacokinetic study

A rapid and sensitive liquid chromatography-tandem mass spectrometric method (LC-MS/MS) had been developed and validated to determine the concentrations of BPR0L075 in rat plasma. After a simple protein precipitation of plasma samples by acetonitrile, BPR0L075 was analyzed on a C(8) column at a flow rate of 0.5 mL/min. The mobile phase consisted of a mixture of 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile (20:80, v/v). Both BPR0L075 (analyte) and the internal standard (BPR0L092) were determined using electro-spray ionization and the MS data acquisition was via multiple reactions monitoring (MRM) in positive scanning model. The MS/MS ion transitions monitored are m/z 342.2/195.2 and 312.5/165.2 for BPR0L075 and BPR0L092, respectively. The low limit of quantitation was 0.5 ng/mL. Each plasma sample was chromatographed within 5 min. The method was validated with respect to linearity, accuracy, precision, recovery, and stability. A good linear relationship was observed over the concentration range of 0.5-1000 ng/mL (r>0.9994). Absolute recoveries ranged from 63.45 to 68.34% in plasma at the concentrations of 2, 40, 400, and 800 ng/mL. The intra- and inter-day accuracy ranged from 92.04 to 111.80%. Intra- and inter-day relative standard deviations were 1.08-3.29% and 1.96-5.46%, respectively. This developed and validated assay method had been successfully applied to a pharmacokinetic study after intravenous injection of BPR0L075 in rats at a dose of 5mg/kg.