CFU Data Research Articles

Mycobacterium abscessus pulmonary infection and associated respiratory function in cystic fibrosis-like βENaC mice

IntroductionChronic pulmonary infection with Mycobacterium abscessus (M. abscessus) is a significant cause of morbidity and mortality in people with cystic fibrosis (CF). Developing an animal model of M. abscessus pulmonary infection, especially under CF conditions, is essential to understanding clinical pulmonary M. abscessus infection. βENaC transgenic mice are known to develop spontaneous CF-like disease characterized by airway mucus obstruction and inflammation. The aim of this study was to evaluate the suitability of βENaC mice as a preclinical model and characterize their respiratory function during M. abscessus lung infection.MethodsMice received an intrapulmonary aerosol of M. abscessus using a high-pressure syringe device (Penn-Century) for subsequent characterization of disease progression and respiratory function. Whole body unrestrained plethysmography (WBP) data was collected to monitor lung function and endpoints determined organ bacterial burden and associated pathology.ResultsEndpoint CFU data in the lung and spleen showed that there was no significant difference in bacterial clearance between βENaC and WT mice. WBP data showed an impairment in overall respiratory function during and after M. abscessus infection in both strains of mice. Interestingly, even in wildtype control mice, lung dysfunction persisted after bacterial clearance.DiscussionEven with CF-like features, the βENaC transgenic mice cleared M. abscessus at a similar rate than WT mice, however, the associated respiratory monitoring revealed that there are long-term implications of M. abscessus lung exposure. The clear decline in respiratory function, even after M. abscessus clearance, suggests that WBP coupled animal modeling provides important insight that is relevant to disease burden and treatment efficacy. The M. abscessus clearance in the βENaC mice may help improve the fields understanding of CF-modulated immune deficiencies in M. abscessus pulmonary infection.

EVALUATION OF ANTIBACTERIAL POTENTIAL OF LICORICE AND XYLITOL CONTAINING SUCROSE FREE HARD CANDIES IN HIGH CARIES RISK PATIENTS

Objectives: To evaluate the effi cacy of sucrose-free hard candies containing xylitol and licorice for preventing dental caries in high-risk individuals. Materials and Methods: Total 80 participants were enrolled based on inclusion criteria. Participants were divided at random into four groups of twenty. Group A consumed stevia-sweetened hard candies including licorice extract, Group B consumed stevia-sweetened hard candies containing xylitol extract, Group C consumed stevia-sweetened hard candies, and Group D consumed sucrose-sweetened hard candies. For the fi rst ten days, participants were asked to eat 2 candies each day; saliva samples were collected at Day 0, after fourteen days following the baseline saliva collection, and after six months after the intervention. Each participant was asked to spit into a sterile plastic container until a suffi cient quantity (5ml) of un-stimulated saliva was collected. It took place between 9 and 10 a.m. For at least an hour before the saliva sample was taken, the individual was requested to abstain from all forms of oral care. The statistical analysis was done by SPSS version 20. Results: Eighty participants finish the experiment and follow the instructions. CFU and pH data for streptococcus mutans in saliva were done as experimental analysis. The mean percentage change in CFU of Streptococcus mutans (%) varied signifi cantly across hard candies over the course of each time period (baseline to fourteen days, P<0.001; fourteen days to six months, P<0.001; and baseline to six months, P<0.001). There was a statistically signifi cant difference in mean salivary pH across various assessment periods within Base line to fourteen days (P<0.001) and Fourteen days to six months (P=0.019) and Base line to six months (P<0.001). The connection between bacterial count and salivary pH was found to be -0.142 (P=0.212) using Pearson's correlation coefficient. Conclusion: Licorice or xylitol extract are used to make sugar-free hard candies that are antibacterial, although the effect wears off with time. Hard candies without sugar have been proven to decrease cariogenic bacteria, while having an essentially neutral effect on pH.

Biofilm formation on metal alloys and coatings, zirconia, and hydroxyapatite as implant materials in vivo.

Composition of implant material and its surface structure is decisive for oral biofilm accumulation. This study investigated biofilm formation on eight different materials. Eighteen healthy subjects wore intraoral splints fitted with two sets of eight materials for 24 h: zirconia [ZrO2 ]; silver-gold-palladium [AgAuPd]; titanium zirconium [TiZr]; Pagalinor [PA]; hydroxyapatite [HA]; silver-platinum [AgPt]; titanium aluminum niobium [TAN]; titanium grade4 [TiGr4]. Total biomass was stained by safranin to assess plaque accumulation while conventional culturing (CFU) was conducted to investigate viable parts of the biofilm. Cell viability of human gingival fibroblasts (HGF-1) was assessed in vitro. Statistical evaluation was performed with linear mixed-effects models to compare materials (geometric mean ratios, 95% CI), with the level of significance set at ɑ = .05. Less biofilm mass and CFU were found on noble metal alloys (AgPt, AgAuPd, and PA). Compared to AgPt, PA had 2.7-times higher biofilm mass value, AgAuPd was 3.9-times, TiGr4 was 4.1-times, TiZr was 5.9-times, TAN was 7.7-times, HA was 7.8-times, and ZrO2 was 9.1-times higher (each p < .001). Similarly, CFU data were significantly lower on AgPt, AgAuPd had 4.1-times higher CFU values, PA was 8.9-times, TiGr4 was 11.2-times, HA was 12.5-times, TiZr was 13.3-times, TAN was 16.9-times, and ZrO2 was 18.5-times higher (each p < .001). HGF-1 viability varied between 47 ± 24.5% (HA) and 94.4 ± 24.6% (PA). Noble alloys are considered as beneficial materials for the transmucosal part of oral implants, as less biofilm mass, lower bacterial counts, and greater cell viability were detected than on titanium- or zirconia-based materials.

Zirconia gradation and thermal expansion compatibility between infiltration glass and antimicrobial glass

This study evaluated an infiltration glass over zirconia in order to form a graded layer with thermal expansion coefficient compatibility and antimicrobial capacity. 210 discs of 3Y-TZP were polished and divided into groups: YZ sintered (Sintered), YZ glazed (Glaze), YZ + Ag 5% (Ag 5%), YZ + Ag 4% (Ag 4%), YZ glass infiltrated (INF.), YZ glass infiltrated + Ag 5% (INF. + Ag 5%) and YZ glass infiltrated + Ag 4% (INF. + Ag 4%). Analyzes of biaxial flexural strength, X-ray diffraction (XRD), EDS, translucency, scanning electron microscopy (SEM) and fractographic were performed. The biofilm was quantified by counting the colony forming units (CFU/mL) of S. mutans. Strength values (MPa), translucency and CFU data were analyzed by one-way ANOVA and Tukey's test (α = 0.05). Weibull analysis was performed to calculate the material's modulus (m) and characteristic strength (95% CI). Infiltrated zirconia glass (INF.: 1149.7; INF. + Ag 4%: 928.2; INF. + Ag 5%: 791.5), sintered (819.0) or glazed (790.8) showed higher flexural strength (MPa) than silver-doped soda-lime glass zirconia without infiltration glass (Ag 5%: 612.1; Ag 4%: 519.2). Diffractograms showed combeite crystals in INF. + Ag 4%/5%. EDS analysis of Ag 5% group showed the higher quantity of silver when compared to INF. + Ag 5% group. Silver-doped soda-lime glass did not affect the translucency, while infiltration glass decreased the values. SEM showed irregularities on the surface of the silver-doped soda-lime glass groups and a smooth surface on the glass and glaze groups. Fracture origin of all groups was in the glaze layer and silver-doped soda-lime glass, while it was inside the zirconia in the INF. group. Lowest colony growth was observed in the Ag 5%. Glass-infiltrated zirconia showed greater strength, while Ag 5% presented greater antimicrobial capacity. The application of silver-doped soda-lime glass did not influence zirconia translucency, while glass infiltration decreased it.

Strategies to improve the survival of probiotic Lacticaseibacillus rhamnosus R0011 during the production and storage of granola bars.

The goal of this study was to evaluate the effectiveness of two approaches to protect the viability of probiotic cells during granola bar manufacturing and storage: microencapsulation (ME) and inclusion in chocolate chips. In the process used, hot honey (138 °C) was blended with cereal ingredients, resulting in an initial blend temperature of 52 °C. Chocolate chips carrying probiotics were added; however, when the blend was cooled to 42 °C. The viability of Lacticaseibacillus rhamnosus R0011 probiotic was assessed by flow cytometry (FC) and plating (CFU). There was an uneven distribution of inoculated probiotic bacteria throughout the cereal bars, resulting in variability in the CFU data. By providing total and viable counts, FC assessed the correct number of inoculated cells in the sample, which enabled the accurate calculation of survival levels. Spray coating with ME increased survival during manufacturing, but ME in alginate particles was detrimental. Including the cultures in chocolate improved the stability of the probiotics during storage at 25 °C, but only in the first 4 weeks. FC analyses showed that viability losses during bar manufacturing could be linked to damage to the cell membrane, but less so during storage.

Levofloxacin pharmacodynamics against Stenotrophomonasmaltophilia in a neutropenic murine thigh infection model: implications for susceptibility breakpoint revision.

Levofloxacin displays in vitro activity against Stenotrophomonas maltophilia (STM); however, current susceptibility breakpoints are supported by limited data. We employed the murine neutropenic thigh infection model to assess levofloxacin pharmacodynamics against STM. Twenty-six clinical STM were studied using the neutropenic murine thigh infection model. Human simulated regimens (HSR) of levofloxacin 750 mg q24h were administered over 24 h. Efficacy was measured as the change in log10 cfu/thigh at 24 h compared with 0 h. Composite cfu data were fitted to an Emax model to determine the fAUC/MIC needed for stasis and 1 log10 reduction at 24 h. Monte Carlo simulation was performed to determine PTA. Levofloxacin MICs ranged from 0.5-8 mg/L. Mean bacterial burden at 0 h was 6.21 ± 0.20 log10 cfu/thigh. In the 24 h controls, bacterial growth was 1.64 ± 0.66 log10 cfu/thigh. In isolates with levofloxacin MICs ≤1, 2 and ≥4 mg/L, changes in bacterial density following levofloxacin HSR were -1.66 ± 0.89, 0.13 ± 0.97 and 1.54 ± 0.43 log10 cfu/thigh, respectively. The Emax model demonstrated strong agreement between fAUC/MIC and change in bacterial density (R2 = 0.82). The fAUC/MIC exposure needed for stasis and 1 log10 reduction was 39.9 and 54.9, respectively. PTAs for the 1 log10 reduction threshold were 95.8, 72.2, and 26.6% at MICs of 0.5, 1 and 2 mg/L, respectively. These are the first data to describe fAUC/MIC thresholds predictive of cfu reductions for levofloxacin against STM. Due to poor in vivo efficacy and PTA at MICs ≥2 mg/L, reassessment of the current susceptibility breakpoint is warranted.

Direct-Contact Low-Frequency Ultrasound and Pulse Lavage Eradicates Biofilms on Implant Materials In Vitro.

Pulse lavage (PL) debridement and ultrasound are both known to be the treatment of biofilm-related periprosthetic joint infection (PJI). However, the efficacy of these in combination is unknown in eradicating biofilm from the orthopaedic metal implant surface. This study was conducted to understand the efficacy of PL and ultrasound in combination in eradicating bacterial biofilms on titanium alloy in vitro. Biofilms of Staphylococcus aureus strains were grown on titanium alloy coupons for 24 h. Then, the coupons were taken to each treatment group: (i) debrided with PL, (ii) exposed to ultrasound, or (iii) exposed to both. An untreated biofilm was set as a control group. Viable plate count and confocal microscopy using live/dead staining was used to measure the amount of biofilm. Viable plate count showed an approximate two-log reduction in CFU/cm2 in PL alone, from an initial cell count on the mental surface of approximately 109 CFU/cm2. The ultrasound caused an approximate seven-log reduction, and the combination group eradicated viable biofilm bacteria completely. Confocal imaging corroborated the CFU data. Our results indicate that PL and ultrasound both are remarkably in eradicating biofilm, and the combination of PL and ultrasound is more effective than alone in reducing biofilm.

Aerosol and bioaerosol particle size and dynamics from defective sanitary plumbing systems.

Aerosols are readily transported on airstreams through building sanitary plumbing and sewer systems, and those containing microbial pathogens (known as bioaerosols) are recognized as contributors to infection spread within buildings. When a defect occurs in the sanitary plumbing system that affects the system integrity, a cross-transmission route is created that can enable the emission of bioaerosols from the system into the building. These emission occurrences are characterized as short-burst events (typically <1min in duration) which make them difficult to detect and predict. The characterization of these emission events is the focus of this research. Two methods were used to characterize bioaerosol emission events in a full-scale test rig: (a) an Aerodynamic Particle Sizer (APS) for particle size distribution and concentrations; and (b) a slit-to-agar sampler to enumerate the ingress of a viable tracer microorganism (Pseudomonas putida). The APS data confirmed that most particles (>99.5%) were <5μm and were therefore considered aerosols. Particles generated within the sanitary plumbing system as a result of a toilet flush leads to emissions into the building during system defect conditions with an equivalence of someone talking loudly for over 6 and a half minutes. There were no particles detected of a size >11μm anywhere in the system. Particle count was influenced by toilet flush volume, but it was not possible to determine if there was any direct influence from airflow rate since both particle and biological data showed no correlation with upward airflow rates and velocities. Typical emissions resulting from a 6L toilet flush were in the range of 280-400 particles per second at a concentration of typically 9-12 number per cm3 and a total particle count in the region of 3000 to 4000 particles, whereas the peak emissions from a 1.2L toilet flush were 60-80 particles per second at a concentration of 2.4-3 number per cm3 and a total particle count in the region of 886 to 1045 particles. The reduction in particles is in direct proportion to the reduction in toilet flush volume. The slit-to-agar sampler was able to provide viable time course CFU data and confirmed the origin of the particles to be the tracer microorganism flushed into the system. The time course data also have characteristics consistent with the unsteady nature of a toilet flush.

Distribution of Xanthomonas hortorum pv. carotae Populations in Naturally Infested Carrot Seed Lots.

Bacterial blight of carrot (Daucus carota subsp. sativus), caused by the plant-pathogenic bacterium Xanthomonas hortorum pv. carotae, is a common seedborne disease of carrot wherever the crop is grown. Carrot seed lots were evaluated to determine the variability and distribution of populations of X. hortorum pv. carotae among individual carrot seeds. Twenty-four carrot seed lots harvested between 2014 and 2016 were subjected to a bulk seed wash dilution-plate assay to obtain mean X. hortorum pv. carotae levels. Mean infestation levels resulting from the bulk seed wash assays among the 24 seed lots ranged from 1.2 × 107 and 9.6 × 108 CFU/g seed and averaged 3.6 × 108 CFU/g seed. Individual seeds from the same 24 lots were also tested with a scaled-down wash assay of individual seeds. Among the 1,380 seeds that were individually assayed, 475 X. hortorum pv. carotae-positive seeds were detected (34.4%). Rates of X. hortorum pv. carotae detection on individual seed in seed lots ranged from 0% (not detected) to 97.9%, and the mean and median X. hortorum pv. carotae population on an individual seed was 8.3 × 104 and 6.3 × 101 CFU/seed, respectively. Among individual seeds, X. hortorum pv. carotae populations ranged from 2 (the limit of detection of the assay) to 3.6 × 107 CFU/seed. CFU data for 23 of the 24 seed lots were nonnormal and the Log-Logistic (3P) distribution best described populations of X. hortorum pv. carotae recovered from individual carrot seeds. The influence and impact of nonnormal distributions of X. hortorum pv. carotae in commercial carrot seed lots on seed health tests, seedborne transmission, and bacterial blight epidemiology requires further study.

A model-based analysis identifies differences in phenotypic resistance between in vitro and in vivo: implications for translational medicine within tuberculosis

Proper characterization of drug effects on Mycobacterium tuberculosis relies on the characterization of phenotypically resistant bacteria to correctly establish exposure–response relationships. The aim of this work was to evaluate the potential difference in phenotypic resistance in in vitro compared to murine in vivo models using CFU data alone or CFU together with most probable number (MPN) data following resuscitation with culture supernatant. Predictions of in vitro and in vivo phenotypic resistance i.e. persisters, using the Multistate Tuberculosis Pharmacometric (MTP) model framework was evaluated based on bacterial cultures grown with and without drug exposure using CFU alone or CFU plus MPN data. Phenotypic resistance and total bacterial number in in vitro natural growth observations, i.e. without drug, was well predicted by the MTP model using only CFU data. Capturing the murine in vivo total bacterial number and persisters during natural growth did however require re-estimation of model parameter using both the CFU and MPN observations implying that the ratio of persisters to total bacterial burden is different in vitro compared to murine in vivo. The evaluation of the in vitro rifampicin drug effect revealed that higher resolution in the persister drug effect was seen using CFU and MPN compared to CFU alone although drug effects on the other bacterial populations were well predicted using only CFU data. The ratio of persistent bacteria to total bacteria was predicted to be different between in vitro and murine in vivo. This difference could have implications for subsequent translational efforts in tuberculosis drug development.

Genomic instability of TnSMU2 contributes to Streptococcus mutans biofilm development and competence in a cidB mutant.

Streptococcus mutans is a key pathogenic bacterium in the oral cavity and a primary contributor to dental caries. The S. mutans Cid/Lrg system likely contributes to tolerating stresses encountered in this environment as cid and/or lrg mutants exhibit altered oxidative stress sensitivity, genetic competence, and biofilm phenotypes. It was recently noted that the cidB mutant had two stable colony morphologies: a “rough” phenotype (similar to wild type) and a “smooth” phenotype. In our previously published work, the cidB rough mutant exhibited increased sensitivity to oxidative stress, and RNAseq identified widespread transcriptomic changes in central carbon metabolism and oxidative stress response genes. In this current report, we conducted Illumina‐based genome resequencing of wild type, cidB rough, and cidB smooth mutants and compared their resistance to oxidative and acid stress, biofilm formation, and competence phenotypes. Both cidB mutants exhibited comparable aerobic growth inhibition on agar plates, during planktonic growth, and in the presence of 1 mM hydrogen peroxide. The cidB smooth mutant displayed a significant competence defect in BHI, which was rescuable by synthetic CSP. Both cidB mutants also displayed reduced XIP‐mediated competence, although this reduction was more pronounced in the cidB smooth mutant. Anaerobic biofilms of the cidB smooth mutant displayed increased propidium iodide staining, but corresponding biofilm CFU data suggest this phenotype is due to cell damage and not increased cell death. The cidB rough anaerobic biofilms showed altered structure relative to wild type (reduced biomass and average thickness) which correlated with decreased CFU counts. Sequencing data revealed that the cidB smooth mutant has a unique “loss of read coverage” of ~78 kb of DNA, corresponding to the genomic island TnSMU2 and genes flanking its 3′ end. It is therefore likely that the unique biofilm and competence phenotypes of the cidB smooth mutant are related to its genomic changes in this region.

Can phenotypic data complement our understanding of antimycobacterial effects for drug combinations?

ObjectivesTo demonstrate how phenotypic cell viability data can provide insight into antimycobacterial effects for the isoniazid/rifampicin treatment backbone.MethodsData from a Mycobacterium komossense hollow-fibre infection model comprising a growth control group, rifampicin at three different exposures (Cmax = 0.14, 0.4 and 1.47 mg/L with t½ = 1.57 h and τ = 8 h) and rifampicin plus isoniazid (Cmax rifampicin = 0.4 mg/L and Cmax isoniazid = 1.2 mg/L with t½ = 1.57 h and τ = 8 h) were used for this investigation. A non-linear mixed-effects modelling approach was used to fit conventional cfu data, quantified using solid-agar plating. Phenotypic proportions of respiring (alive), respiring but with damaged cell membrane (injured) and ‘not respiring’ (dead) cells data were quantified using flow cytometry and Sytox Green™ (Sigma–Aldrich, UK) and resazurin sodium salt staining and fitted using a multinomial logistic regression model.ResultsIsoniazid/rifampicin combination therapy displayed a decreasing overall antimicrobial effect with time ( = 438 h) on cfu data, in contrast to rifampicin monotherapy where this trend was absent. In the presence of isoniazid a phenotype associated with cell injury was displayed, whereas with rifampicin monotherapy a pattern of phenotypic cell death was observed. Bacterial killing onset time on cfu data correlated negatively ( = 28.9 h, = 0.132 mg/L) with rifampicin concentration up to 0.165 mg/L and this coincided with a positive relationship between rifampicin concentration and the probability of phenotypic cell death.ConclusionsCell viability data provide structured information on the pharmacodynamic interaction between isoniazid and rifampicin that complements the understanding of the antibacillary effects of this mycobacterial treatment backbone.

Analysis of 154 Cord Blood Unit (CBU) Reports Demonstrates High Variability in Potency Data and Highlights the Need for Standardization of CBU Quality Assessment

Introduction Both total nucleated cell (TNC) & CD34+ cell doses are critical for CB engraftment. However, how CBU potency characteristics are reported is not established. Methods We evaluated the completeness of CBU potency information in NMDP Traxis system & individual Bank Reports at the time of selection/ release. We then compared the reported data to CBU post-thaw values. At our center, CBU underwent albumin-dextran dilution (1:8). Post-thaw flow cytometry for CD45+/CD34+ cell counts & 7-AAD was done within 2 hours. Results Between 1/2016-4/2018, 154 CBU were obtained from domestic (N=88) or international (N=66) Banks. Post-processing/ pre-cryopreservation results: At least 1 result was reported for 147/154 (95%) CBU (Table 1). CD34+ was present in only 75/154 (49%) Reports. In most, the method was not stated (presumed 7-AAD). cell viability (not specifying CD45+ or TNC) was present in 110/154 (71%); 26 of these were by trypan blue or ethidium bromide. Total CFU were reported in 96/154 (62%), mostly domestic CBU & 7 reported only CFU-GM. Of 7/154 (5%) CBU without any viability, 2 were domestic (collected 2006, 2012) & 5 international (collected 2012-2015). Only 6 domestic CBU did not have CFU data (5 collected in 2006). Post-thaw segment results: A minority of Reports (47/154, 31%), all international, included segment CD34+ data. 43/154 (28%) also had segment CFU results but few included pre-cryopreservation values for comparison. Five CBU Reports had only cryovial data. Results after CBU thaw: Median CD34+ was 93% (SD: 7%). Except one very low CD34+ (22%, no pre-freeze value reported), all others were >77%. Post-thaw CD34+ was similar between domestic (mean: 92%; SD: 8.1%) & international CBU (mean: 92%; SD: 3.6%). However, viable CD34+ cell recovery (ratio of viable CD34+ cells post-thaw to those pre-freeze) was significantly lower for international CBU (p 84% & 28/43 (65%) had higher post-thaw CBU CD34+ than the segment (median increase: 10%). Conclusions CBU Reports include highly variable potency data & may be incomplete even when the information is available at the Banks. Efforts to standardize CBU potency testing & reporting are needed. Disparities in CD34+ recovery likely indicate high inter-laboratory variability & possibly differences in flow gating strategies. Also, Banks need to provide interpretation of segment results as reduced may be due to technical challenges, not CBU quality problems. Finally, the clinical significance of this data requires validation.

Antibiotic loaded calcium sulfate bead and pulse lavage eradicates biofilms on metal implant materials in vitro.

Pulse lavage (PL) debridement and antibiotic loaded calcium sulfate beads (CS-B) are both used for the treatment of biofilm related periprosthetic joint infection (PJI). However, the efficacy of these alone and in combination for eradicating biofilm from orthopaedic metal implant surfaces is unclear. The purpose of the study was to understand the efficacy of PL and antibiotic loaded CS-B in eradicating bacterial biofilms on 316L stainless steel (SS) alone and in combination in vitro. Biofilms of bioluminescent strains of Pseudomonas aeruginosa Xen41 and a USA300 MRSA Staphylococcus aureus SAP231 were grown on SS coupons for 3 days. The coupons were either, (i) debrided for 3 s with PL, (ii) exposed to tobramycin (TOB) and vancomycin (VAN) loaded CS-B for 24 h, or (iii) exposed to both. An untreated biofilm served as a control. The amount of biofilm was measured by bioluminescence, viable plate count and confocal microscopy using live/dead staining. PL alone reduced the CFU count of both strains of biofilms by approximately 2 orders of magnitude, from an initial cell count on metal surface of approximately 109 CFU/cm2 . The antibiotic loaded CS-B caused an approximate six log reduction and the combination completely eradicated viable biofilm bacteria. Bioluminescence and confocal imaging corroborated the CFU data. While PL and antibiotic loaded CS-B both significantly reduced biofilm, the combination of two was more effective than alone in removing biofilms from SS implant surfaces. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2349-2354, 2018.

In vitro evaluation of microbial adhesion on the different surface roughness of acrylic resin specific for ocular prosthesis.

Objective:The purpose of this study was to evaluate the influence of surface roughness in biofilm formation of four microorganisms (Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans) on acrylic resin surface of ocular prostheses.Materials and Methods:Acrylic resin samples were divided into six groups according to polishing: Group 1200S (1200 grit + silica solution); Group 1200; Group 800; Group 400; Group 120 and Group unpolished. Surface roughness was measured using a profilometer and surface images obtained with atomic force microscopy. Microbial growth was evaluated after 4, 24, and 48 hours of incubation by counting colony-forming units.Statistical Analysis Used:For roughness, it was performed 1-way ANOVA and parametric Tukey test α5% (P ≤ 0.05). For CFU data found, it was applied Kruskal-Wallis and Mann-Whitney tests.Results:Group 120 and 400 presented the highest roughness values. For S. epidermidis and S. aureus, Group 1200S presented the lowest values of microbial growth. For E. faecalis at 4 hour, microbial growth was not observed. C. albicans did not adhere to the acrylic resin. Except for Group 1200S, different surface roughnesses did not statistically interfere with microbial adhesion and growth on acrylic surfaces of ocular prostheses.Conclusions:The roughness did not interfere with the microbial adhesion of the microorganisms evaluated. The use of silica decreases significantly microbial growth.

Improved power for TB Phase IIa trials using a model-based pharmacokinetic-pharmacodynamic approach compared with commonly used analysis methods.

Background: The demand for new anti-TB drugs is high, but development programmes are long and costly. Consequently there is a need for new strategies capable of accelerating this process. Objectives: To explore the power to find statistically significant drug effects using a model-based pharmacokinetic–pharmacodynamic approach in comparison with the methods commonly used for analysing TB Phase IIa trials. Methods: Phase IIa studies of four hypothetical anti-TB drugs (labelled A, B, C and D), each with a different mechanism of action, were simulated using the multistate TB pharmacometric (MTP) model. cfu data were simulated over 14 days for patients taking once-daily monotherapy at four different doses per drug and a reference (10 mg/kg rifampicin). The simulated data were analysed using t-test, ANOVA, mono- and bi-exponential models and a pharmacokinetic–pharmacodynamic model approach (MTP model) to establish their respective power to find a drug effect at the 5% significance level. Results: For the pharmacokinetic–pharmacodynamic model approach, t-test, ANOVA, mono-exponential model and bi-exponential model, the sample sizes needed to achieve 90% power were: 10, 30, 75, 20 and 30 (drug A); 30, 75, 245, 75 and 105 (drug B); 70, >1250, 315, >1250 and >1250 (drug C); and 30, 110, 710, 170 and 185 (drug D), respectively. Conclusions: A model-based design and analysis using a pharmacokinetic–pharmacodynamic approach can reduce the number of patients required to determine a drug effect at least 2-fold compared with current methodologies. This could significantly accelerate early-phase TB drug development.

The Effects of Galactic Cosmic Radiation Exposure on Hematopoietic Stem Cell Dysfunction and Oncogenesis

Natural sources of radiation in space include galactic cosmic rays (GCR), solar energetic particles (SPE) and trapped energetic particles in a planetary magnetic field. These different sources of space radiation consist of protons of various energies, particle nuclei of high energy and charge (HZE) and neutrons of different energies. These sources are difficult to shield because of their high energies and dense ionization patterns, thus posing significant health risks to astronauts on long term inter-planetary missions. Efforts to protect astronauts from harmful cosmic radiation require a deeper understanding of the effects of GCR on human health. In particular, very little is known about the effects of GCR exposure on the hematopoietic stem cell (HSC) population and whether disruptions in genetic stability in HSCs could result in the development of hematopoietic malignancies in astronauts on deep space missions.The average age of shuttle crew has risen above 46 years, and our work and others have shown that HSCs display diminished function with age. Recent data from our group has demonstrated that middle-aged individuals show frequent defects in DNA mismatch repair (MMR) in HSCs. MMR corrects DNA mismatches generated by DNA polymerase during replication which prevents mutations from becoming permanent in dividing cells. Thus, MMR plays a crucial role in the DNA damage response pathway to prevent short-term mutagenesis and long-term tumorigenesis. Several human MMR proteins have been identified as MutS and MutL homologues consisting of MSH2 and MLH1 heterodimers that functions in DNA mismatch/damage recognition, endonuclease activity and termination of mismatch-provoked excision. Our group has shown that humans accumulate microsatellite instability (MSI) with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age. Therefore, we employed a DNA mismatch repair deficient mouse model (MLH1+/- and MLH1-/-) to study the effects of different radiation sources including 56Fe, 28Si, 4He, 1H and γ-rays on HSCs to examine HSCs of potential astronaut population under GCR conditions. The complete blood count (CBC) data after 5 months and 9 months of whole body irradiation with different ions showed a slight dose-dependent decrease in all blood counts but absence of any significant difference in CBC of MLH1+/+ vs MLH1+/- mice. In addition, CFU and competitive repopulation data demonstrated a radiation quality effects on HSC function, but not an MLH1 effect. These results demonstrate that hematopoietic stem cell function is normal and that a MLH1 defect does not differentiate progenitor and mature effector cells following HZE radiation.To study long term effects of different ions on the potential for disease progression in a MLH1 dependent manner, we performed whole body irradiation with 56Fe, 28Si, 1H and γ-rays on MLH1+/+ and MLH1+/- mice and followed them up to 18 months post exposure. We observed that MLH1+/- mice show dramatic increases in lymphomagenesis 10-12 months after 56Fe irradiation compared to wild type mice, with greater than 60 % of MLH1+/- mice developed lymphomas at doses 10 cGy and 100 cGy compared to less than 10 % of wild type. For comparison, roughly 10 % of MLH1+/- mice developed lymphomas when mice were treated with whole body sparsely ionizing γ-rays at 100 cGy compared to none of the control. Thus the date show that MMR defects in HSCs lead to sensitization to radiation induced hematopoietic malignancy and that radiation quality effects exacerbate the sensitivity. The findings could have profound effects on astronaut screening, as well as lead to important questions regarding safety of ion therapy and development of second malignancies for cancer patients who remain on Earth. DisclosuresNo relevant conflicts of interest to declare.

Bacterial Colonization in the Marginal Region of Ceramic Restorations: Effects of Different Cement Removal Methods and Polishing.

This study evaluated the effects of excess cement removal techniques, with or without subsequent polishing, on biofilm formation and micromorphology in the marginal region of the tooth/restoration. From bovine teeth, 96 dentin blocks (4 × 8 × 2 mm) were produced, molded, and reproduced in type IV gypsum, on which 96 pressed ceramic blocks (Vita PM9, Vita Zahnfabrik; 4 × 8 × 2 mm) were produced via the lost wax technique. The dentin blocks and their respective ceramic blocks were cemented with a self-adhesive resin cement (RelyX U200, 3M ESPE), and cement excess was removed from the margin using four different techniques, followed or not by polishing with silicone rubber tips: MBr, removal with microbrush and photoactivation; MBr-Pol, MBr + polishing; Br, removal with brush and photoactivation; Br-Pol, Br + polishing; Photo-Expl, 5 seconds of initial photoactivation, removal with explorer, and final curing; Photo-Expl-Pol, Photo-Expl + polishing; Photo-SB, 5 seconds of initial photoactivation, removal with scalpel, and final curing; and Photo-SB-Pol, Photo-SB + polishing. After 24 hours, the roughness in the marginal region was analyzed using a profilometer (three measurements on each sample). Micromorphological analyses of the region were performed by stereomicroscope and scanning electron microscopy (SEM). Then the samples were contaminated with sucrose broth standardized suspension with Streptococcus mutans , Staphylococcus aureus , and Candida albicans and incubated for a period of 48 hours. The samples were quantitatively analyzed for bacterial adherence in the marginal region by confocal laser scanning microscopy and counting of colony-forming units (CFUs/mL) and qualitatively analyzed using SEM. Roughness data (Ra) were submitted to two-way analysis of variance, Tukey test at a confidence level of 95%, and Student t-tests. CFU, biomass, and biothickness data were analyzed by Kruskal-Wallis, Mann-Whitney, and Dunn tests. The removing technique statistically influenced Ra (MBr, p=0.0019; Br, p=0.002; Photo-Expl, p=0.0262; Photo-SB, p=0.0196) when comparing the polished and unpolished groups. The MBr and MBr-Pol technique differed significantly for CFU/mL values (p=0.010). There was no significant difference in the amounts of biomass and biothickness comparing polished and unpolished groups and when all groups were compared (p>0.05). Different morphological patterns were observed (more regular surface for polished groups). We conclude that margin polishing after cementation of feldspar/pressed ceramic restorations is decisive for achieving smoother surfaces, as the excess cement around the edges can increase the surface roughness in these areas, influencing bacterial adhesion.

Oral biofilm and caries-infiltrant interactions on enamel

ObjectivesThis study aimed to analyze interactions between oral biofilms and a dental triethylene glycol dimethacrylate (TEGDMA)-based resin infiltration material on enamel. MethodsDemineralized enamel specimens (14days, acidic buffer, pH 5.0) were either infiltrated with a commercial TEGDMA resin and subjected to a three-species biofilm (Streptococcus mutans UAB 159, Streptococcus oralis OMZ 607 and Actinomyces oris OMZ 745) (group 1), applied to the biofilm (group 2), or merely resin infiltrated (group 3). A control group received no treatment (4). Biofilm formation and metabolic activity of biofilms were measured for group (1) and (2) after 24h CFU and a resazurin assay. Resin biodegradation was measured for group (1) and (3) by high performance liquid chromatography (HPLC) coupled with mass spectrometry after 6 and 24h incubation. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) images were taken to study the biofilm and material’s autofluorescence in groups (1–4) after 24h. ResultsSEM and CLSM images showed reduced biofilm formation on resin-infiltrated specimens (group 1) compared to group 2, while no biofilm was detectable on groups 3 and 4. CFU data (log10 CFU per mL) of group 1 showed significantly reduced bacterial numbers (p<0.05) compared to group 2. However, HPLC analysis of TEGDMA leakage after 6h and 24h revealed no differences between group 1 and group 3. ConclusionsThe results of the current study indicate that freshly resin-infiltrated enamel surfaces show a biofilm reducing effect, while monomer leakage was not affected by bacterial presence. Clinical significanceResin infiltrated enamel surfaces are constantly exposed to the oral microflora. Yet, it is not known how biofilms interact with enamel-penetrated resins and if and to which extent accessory alignments in oral hygiene are needed.

Antimicrobial photodynamic effect of phenothiazinic photosensitizers in formulations with ethanol on Pseudomonas aeruginosa biofilms

Background dataMethylene blue (MB) and toluidine blue (TB) are recognized as safe photosensitizers (Ps) for use in humans. The clinical effectiveness of the antimicrobial photodynamic therapy with MB and TB needs to be optimized, and ethanol can increase their antimicrobial effect. Formulations of MB and TB containing ethanol were evaluated for their ability to produce singlet oxygen and their antibacterial effect on Pseudomonas aeruginosa biofilms. MethodsPhotoactivated formulations were prepared by diluting the Ps (250μM) in buffered water (pH 5.6, sodium acetate/acetic acid), 10% ethanol (buffer: ethanol, 90:10), or 20% ethanol (buffer: ethanol, 80:20). Biofilms also were exposed to the buffer, 10% ethanol, or 20% ethanol without photoactivation. Untreated biofilm was considered the control group. The production of singlet oxygen in the formulations was measured based on the photo-oxidation of 1,3-diphenylisobenzofuran. The photo-oxidation and CFU (log10) data were evaluated by two-way ANOVA and post-hoc Tukey’s tests. ResultsIn all the formulations, compared to TB, MB showed higher production of singlet oxygen. In the absence of photoactivation, neither the buffer nor the 10% ethanol solution showed any antimicrobial effect, while the 20% ethanol solution significantly reduced bacterial viability (P=0.009). With photoactivation, only the formulations containing MB and both 10% and 20% ethanol solutions significantly reduced the viability of P. aeruginosa biofilms when compared with the control. ConclusionsMB formulations containing ethanol enhanced the antimicrobial effect of the photodynamic therapy against P. aeruginosa biofilms in vitro.