Number Of Colony-forming Units Research Articles

The quality and technological parameters of milk obtained from dairy cows with subclinical mastitis

Mastitis is one of the most common diseases in dairy cattle. It significantly reduces milk quality and yield, thus incurring economic losses for farmers. This study investigates the impact of various bacterial pathogens on the somatic cell count, milk composition, and technological properties of milk samples from 302 clinically healthy Polish Holstein-Friesian cows kept under intensive rearing conditions. Of the 462 milk samples analyzed, 85.06% were contaminated with bacteria. The majority were coagulase-negative staphylococci (52.60%).-Escherichia coli, Streptococcus dysgalactiae, Streptococcus uberis, or Staphylococcus aureus, or Streptococcus agalactiae, a major pathogen, were identified in 16.66% of samples; their presence was associated with higher SCC levels. Additionally, contamination with S. aureus or S. agalactiae had prolonged clotting time, adversely affected curd and whey quality, with curd yield remaining unaltered. Bacterial contamination did not appear to significantly impact the yield of milk or its main components, viz. protein, casein, lactose, fat, total solids, solid nonfat, free fatty acid or citric acid. Although pH, freezing point depression (FDP), and acidity also remained unaffected by bacterial contamination, they were significantly influenced by herd-year-season of calving and herd-year-time of sampling interaction effects. The results indicate that the presence of bacteria causing subclinical mastitis negatively influences milk processing potential. However, fixed linear regression indicated that the number of colony-forming units (cfu/ml) only had a significant influence on FPD and clotting time, and as such, the number of bacteria in a sample did not influence milk yield or quality during subclinical chronic mastitis.

Study on the effect of chlorogenic acid on the antimicrobial effect, physical properties and model accuracy of alginate impression materials.

Dental impressions are essential for accurately capturing the detailed anatomy of teeth and surrounding oral structures. However, these impressions often become contaminated with saliva and blood, making proper disinfection necessary. The application of chemical disinfectants has been associated with negative side effects, leading to suboptimal disinfection practices in clinical settings. The purpose of this study was to evaluate the effectiveness of chlorogenic acid (CA) as a disinfectant for alginate impression materials, the impact of CA disinfection on the physical properties and dimensional accuracy of alginate impressions was also investigated. The physical properties of alginate impression materials, such as elastic recovery, strain-in-compression, initial setting time, and fluidity, were assessed after mixing the alginate impression materials with three different concentrations of CA solution (10 mg/mL, 15 mg/mL, 20 mg/mL). To evaluate the antimicrobial effect of CA, alginate impressions mixed with a 10 mg/mL CA solution and impressions mixed with distilled water (control group) were contaminated with four types of microorganism: Escherichia coli, Staphylococcus aureus, Candida albicans, and Streptococcus pneumoniae. Following a five-minute incubation period, a CA solution at a concentration of either 50mg/mL, 55 mg/mL, or 60 mg/mL was sprayed on the samples for disinfection. Samples were collected at different time intervals (10 min, 20 min, 30min) and cultured to determine the number of colony-forming units (CFU/mL), providing insight into the antimicrobial efficacy of these CA solutions. The dimensional accuracy of alginate impressions was assessed in three groups: one with alginate impressions mixed with distilled water, another with alginate impressions sterilized with available chlorine (2,000 mg/L) mixed with distilled water, and the last group consisting of alginate impressions mixed with 10 mg/mL CA solution and sprayed with 60 mg/mL CA solution. Both the standard model and the plaster model underwent 3D scanning, and the data were processed and compared by software. The root mean square (RMS) was used as a parameter to evaluate the deviation between models. All alginate impression materials mixed with either 10 mg/mL, 15 mg/mL, or 20 mg/mL concentrations of CA solution met the ISO 21563 standard for elastic recovery, strain-in-compression, and fluidity. However, only the material mixed with a concentration of 10 mg/mL CA had an initial setting time within the range specified by the T-6505 Japanese industrial standard. The application of CA solution by mixing or spraying showed significant antimicrobial effects on Staphylococcus aureus, Escherichia coli, Candida albicans, and Streptococcus pneumoniae. There was no significant difference in the dimensional accuracy of the alginate impressions between the group of the CA solution applied, the blank group, or the chlorine intervention group.

Preliminary in vivo investigation of the mesenchymal stromal cell secretome as a novel treatment for methicillin-resistant Staphylococcus aureus in equine skin wounds.

We aimed to study the antimicrobial and pro-healing potential of equine mesenchymal stromal cell secreted products (i.e. secretome), collected as conditioned media (mesenchymal stromal cell-conditioned media, MSC CM), in a novel in vivo model of methicillin-resistant Staphylococcus aureus (MRSA)-inoculated equine thorax wounds. Prospective in vivo study. Two Thoroughbred geldings. Six full-thickness cutaneous wounds were created bilaterally on the dorsal thorax of two horses (n = 12 wounds/horse). Wounds on the left thoraces were inoculated with MRSA on day 0. All wounds were then treated with either mupirocin ointment, MSC CM, or vehicle control (n = 4 wounds per group) once daily for 3 days. Photographs were taken to quantify wound scores and sizes, as well as samples to determine bacterial colony forming units (CFUs), at days 0, 1, 2, 3, 7, 14, 21, and 28. The wound edge was biopsied on days 0, 7, and 28, and scored histologically. Inoculated wounds had more bacterial CFUs at day 1 (p < .0001) and were larger in size at day 28 (p = .0009) than noninoculated wounds. Mupirocin-treated wounds were smaller than MSC CM and vehicle control-treated wounds at day 28 (p = .003). Mesenchymal stromal cell-conditioned media did not affect CFU numbers in inoculated and noninoculated wounds. Moreover, MSC CM did not affect the parameters of wound size or gross or microscopic wound scores over time. Mesenchymal stromal cell-conditioned media did not exhibit antimicrobial or pro-healing properties in the current study; however, the in vivo model of inoculated equine thorax wounds requires further optimization. This pilot study contributes to a growing understanding of the equine MSC secretome as an antimicrobial and pro-healing therapeutic for equine wounds.

Antimicrobial efficacy of nanoparticle calcium hydroxide and silver nanoparticles on Enterococcus faecalis: An in vitro study

Abstract Background: In root canal disinfection, intracanal medicaments play a vital role. Typical intracanal medicament, calcium hydroxide (CH), has the lowest efficacy against the biofilm of Enterococcus faecalis. Nanoparticles and their combinations may increase antimicrobial activity, making them better than conventional CH. Aim: The aim is to determine the efficacy of nanoparticle CH (NPCH) and silver nanoparticles (AgNP) against E. faecalis. Materials and Methods: A total of 60 premolars were chosen. The samples were cleaned and decoronated. Cleaning and shaping were done. The apices were tightly sealed with modeling wax before being injected with 5 μl of E. faecalis (ATCC 29212) bacterial suspension, and then left to incubate. Samples were categorized into CH (G-I), NPCH (G-II), AgNP (G-III), and a combination of NPCH + AgNP (G-IV) according to the intracanal medication used. Eppendorf tubes were used to transfer dentin chips after they had been incubated for 7 days. The process of obtaining bacterial colony-forming units (CFUs) involves inoculating sediments onto blood agar. Results: The findings are statistically significant, number of CFUs seen with the Combination of NPCH + AgNP is lower than the other experimental groups, the group treated with CH had the largest number of CFUs. Conclusions: The highest antibacterial effect was seen in combination of NPCH + AgNP.

Nicotine promotes pathogenic bacterial growth and biofilm formation in peri-implant.

Introduction. Peri-implantitis is a plaque-associated disease that leads to implant loss and arises from bacterial biofilms on the surface of the implant. Smoking is a risk factor for peri-implantitis and impedes treatment effectiveness. Additionally, aryl hydrocarbon receptor (AHR), IL-6, and IL-22 levels are related to peri-implantitis.Aim. We aimed to investigate the effects of nicotine on inflammatory response, bacterial growth and biofilm formation.Hypothesis/Gap Statement. We hypothesized that nicotine promoted pathogenic bacterial growth and biofilm formation, thereby aggravating inflammation.Methodology. The expression of AHR, IL-6 and IL-22 was measured in peri-implant sulci fluid using quantitative PCR and Western blot analyses. The cementum was incubated with bacterial suspension including Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum and treated with 100, 200, 250 and 300 µg ml-1 nicotine, and then, the absorbance and number of colony-forming units were detected. Biofilm formation was evaluated using the tissue culture plate method and safranin O staining. Carbohydrates and proteins were measured by the phenol-sulfuric acid method and the bicinchoninic acid method, respectively.Results. The results indicated that smoking increased the levels of AHR, IL-6 and IL-22. Functionally, nicotine promoted the growth of P. gingivalis, S. sanguinis and F. nucleatum. Additionally, it promoted the biofilm formation of these bacteria and increased the contents of carbohydrates and proteins.Conclusion. Nicotine promoted bacterial growth and biofilm build-up, suggesting that smoking may aggravate the progression of peri-implantitis.

The Influence of Selected Titanium Alloy Micro-Texture Parameters on Bacterial Adhesion.

The colonization of microbes and the resulting formation of biofilms on dental implants are significant contributors to peri-implantitis and the failure of these implants. The aim of the research was to analyze the impact of density and depth of laser texturing of the Ti-6Al-7Nb alloy surface on the colonization of selected microorganisms and biofilm formation. Standard strains of Gram-negative and Gram-positive bacteria and yeasts from the American Type Culture Collection-ATCC-were used to demonstrate the ability to form single-species biofilms in vitro. The study evaluated three types of titanium samples with different texture density and depth. The colonization and biofilm formation abilities of the tested microorganisms were assessed. The obtained results were subjected to statistical analysis. Among the analyzed strains, L. rhamnosus showed the highest colonization of the tested surfaces. It was found that there is no relationship between the texture parameters and the number of colony-forming units (CFU/mL) for C. albicans, S. mutans, and L. rhamnosus. For the F. nucleatum strain, it was shown that the number of colony-forming bacteria is related to the texture density.

Incorporating an artificially synthesized fluoride complex into urethane-acrylate-based 3D printing resin: Effects on mechanical properties, cytotoxicity, antimicrobial actions, and its long-term fluoride-releasing properties

ObjectivesTo synthesize a 3D printing resin with antibacterial and long-term fluoride-releasing properties. Methods(4,4-Bis-4-[2‑hydroxy-3-(2-methacryloyloxy)propoxy]-phenyl-pentanol-amine)-N,N-diacetic acid zirconium (IV) fluoride complex was synthesized from 4,4-bis-(4-hydroxyphenyl)-pentanoic acid and monitored using proton nuclear magnetic resonance spectroscopy. The synthesized complex was incorporated into a urethane-acrylate-based (UA) resin at 5 wt% and 10 wt% (5F-UA and 10F-UA groups, respectively). The UA resin without the synthesized complex was considered as the control group. All groups were 3D printed using a DLP printer, followed by 10 min of washing and 20 min of curing. Surface characteristics were observed using scanning electron microscopy. The mechanical properties were assessed by measuring its flexural strength and Vickers hardness. The antibacterial property was investigated with direct and indirect contact tests and a WST-8 metabolic activity assay. The suspension was fully mixed and diluted for counting the number of colony-forming units. The cell viability test was performed using a cell proliferation assay. The amount of fluoride released was measured daily for 28 days using ion chromatography. One-way analysis of variance was performed for statistical analyses using SPSS software. ResultsThe amount of fluoride released increased with the concentration of fluoride complex in the resin. The fluoride ions were constantly released at a low concentration from the 3D printed specimens (5F-UA: around 0.13 ppm daily; 10F-UA: around 0.22 ppm daily). The antibacterial efficacy was acceptable in both the 5F-UA and 10F-UA groups, and higher in the latter. No cytotoxicity of the resin was detected. The mechanical properties were significantly influenced by the addition of the fluoride-releasing complex. ConclusionsThe present 3D printing UA resin incorporating a fluoride complex effectively inhibited the growth of S. mutans and demonstrated the ability to slowly release fluoride over an extended period of time. Clinical significanceThis study provided informative composition of a fluoride-releasing UA-based 3D printing resin, ideal for dental applications such as crowns, bridges, removable partial dentures, and orthodontic appliances, which can benefit from sustained fluoride release and antimicrobial properties. Further modifications to the resin composition can be easily achieved to enhance the resin qualities.

Reduction of Multispecies Biofilms on an Acrylic Denture Base Model by Antimicrobial Photodynamic Therapy Mediated by Natural Photosensitizers.

The aim of this study is to investigate the antimicrobial efficacy of antimicrobial photodynamic therapy (PDT) using natural photosensitizers (curcumin, riboflavin, and phycocyanin) and light-emitting diode (LED) irradiation against multispecies biofilms in an acrylic denture base model. Forty-five acrylic specimens were fabricated using heat-curing acrylic resin. The specimens were then infected with a mixed culture of bacterial and fungal species (including Streptococcus mutans, Streptococcus sanguinis, Candida albicans, and Candida glabrata) for 4 days. The acrylic discs were divided into nine groups, with each group containing five discs: control, 0.2% chlorhexidine, 5.25% sodium hypochlorite, curcumin, riboflavin, phycocyanin alone or along with LED. After treatment, the number of colony-forming units (CFUs) per milliliter was counted. In addition, the extent of biofilm degradation was assessed using the crystal violet staining method and scanning electron microscopy. All experimental groups exhibited a significant reduction in colony numbers for both bacterial and fungal species compared to the control (p < 0.001). The PDT groups exhibited a statistically significant reduction in colony counts for both bacteria and fungi compared to the photosensitizer-only groups. The results of this in vitro study show that PDT with natural photosensitizers and LED devices can effectively reduce the viability and eradicate the biofilm of microorganisms responsible for causing denture infections.

Comparison of the Antimicrobial Efficacy of Conventional Versus Chitosan Re-inforced Heat-Polymerized Polymethylmethacrylate Dental Material: An In Vitro Study.

Polymethylmethacrylate (PMMA) is widely used in the fabrication of dentures due to its aesthetic appeal and mechanical strength. However, PMMA's susceptibility to microbial colonization often leads to oral infections such as denture stomatitis. Enhancing the antimicrobial properties of denture materials is crucial for improving patient outcomes.Chitosan, a natural biopolymer, possesses inherent antimicrobial properties and could potentially enhance the microbial resistance of PMMA. This study has investigated thepotential of chitosan-reinforced heat-polymerized PMMA denture material to reduce microbial colonization. The aim of the study was to evaluate and assess the anti-bacterial and antifungal properties of chitosan-reinforced heat-polymerized PMMA with conventional heat-polymerized PMMA Materials and methods:Chitosan-reinforced PMMA samples were fabricated with varying chitosan concentrations (0% control, 5%, 10%, and 15% by weight). The fabrication involved mixing chitosan powder with PMMA powder, adding monomer liquid, followed by mixing, packing, and curing using the conventional heat polymerization technique. The antimicrobial efficacy was assessed in vitro using two common oral pathogens: Streptococcus mutans and Candida albicans. Blood agar plates were used for S. mutans and Sabouraud agar plates were used for C. albicans. Each sample was placed on the respective agar plates inoculated with a standardized microbial suspension and incubated at 37°C for 24 hours. The number of colony-forming units (CFUs) was counted to quantify microbial growth. Statistical analyses, including linear regression analysis, one-way ANOVA test, and Pearson correlation were performed to evaluate the relationship between chitosan concentration and antimicrobial efficacy. The p-value was calculated to determine the statistical significance of the results. The chitosan-reinforced PMMA samples showed significantly greater antimicrobial efficacy compared to the conventional PMMA samples. The CFU counts for both S. mutans and C. albicans decreased with increasing chitosan concentration. Linear regression analysis indicated a strong negative correlation between chitosan concentration and CFU counts, with Pearson correlation coefficients of -0.97 for S. mutans and -0.98 for C. albicans. ANOVA analysis revealed a statistically significant difference in antimicrobial efficacy across different chitosan concentrations (p < 0.001). Incorporating chitosan into heat-polymerized PMMA significantly enhances its antimicrobial properties against S. mutans and C. albicans. The antimicrobial efficacy improves with higher concentrations of chitosan, with the 15% chitosan-reinforced samples showing the most substantial reduction in microbial growth. These results suggest that chitosan-reinforced PMMA dentures could be a superior alternative to conventional PMMA dentures, potentially reducing denture-related infections and improving oral health outcomes for denture wearers.

Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life.Key points• Innovative “bioreactor-in-a-granule” system for T. asperelloides is devised.• Dry granules of aerial conidia remain highly viable for 24 months at 4 °C.• Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Catching some air: a method to spatially quantify aerial triazole resistance in Aspergillus fumigatus.

Airborne triazole-resistant spores of the human fungal pathogen Aspergillus fumigatus are a significant human health problem as the agricultural use of triazoles has been selecting for cross-resistance to life-saving clinical triazoles. However, how to quantify exposure to airborne triazole-resistant spores remains unclear. Here, we describe a method for cost-effective wide-scale outdoor air sampling to measure both spore abundance as well as antifungal resistance fractions. We show that prolonged outdoor exposure of sticky seals placed in delta traps, when combined with a two-layered cultivation approach, can regionally yield sufficient colony-forming units (CFUs) for the quantitative assessment of aerial resistance levels at a spatial scale that was up to now unfeasible. When testing our method in a European pilot sampling 12 regions, we demonstrate that there are significant regional differences in airborne CFU numbers, and the triazole-resistant fraction of airborne spores is widespread and varies between 0 and 0.1 for itraconazole (∼4 mg/L) and voriconazole (∼2 mg/L). Our efficient and accessible air sampling protocol opens up extensive options for fine-scale spatial sampling and surveillance studies of airborne A. fumigatus.IMPORTANCEAspergillus fumigatus is an opportunistic fungal pathogen that humans and other animals are primarily exposed to through inhalation. Due to the limited availability of antifungals, resistance to the first choice class of antifungals, the triazoles, in A. fumigatus can make infections by this fungus untreatable and uncurable. Here, we describe and validate a method that allows for the quantification of airborne resistance fractions and quick genotyping of A. fumigatus TR-types. Our pilot study provides proof of concept of the suitability of the method for use by citizen-scientists for large-scale spatial air sampling. Spatial air sampling can open up extensive options for surveillance, health-risk assessment, and the study of landscape-level ecology of A. fumigatus, as well as investigating the environmental drivers of triazole resistance.

Oral Colonization by Different Candida Species: First Comparative Study between Denture and Nondenture Wearers in Tunisia.

This study aimed to compare different Candida species present in patients with and without removable dentures to identify alterations in biofilm composition following denture wear within a Tunisian population. A cross-sectional study was conducted, comprising a group of patients wearing removable dentures (test group) and a control group without dentures. In the test group, two mycological samples were obtained: one from the prosthetic intaglio and another from the osteomucosal area bearing the denture. For the control group, mycological samples were collected from the oral mucosa. The collected swabs were cultured on CHROMagar Candida medium, and yeast counts were quantified as colony forming units (CFUs). Candida species were identified through chromogenic analysis. The normality of quantitative variables was evaluated using the Kolmogorov-Smirnov's test. To compare means and ranks between the test and control groups, the independent samples t-test and the Mann-Whitney's U test were employed, respectively. Qualitative variables were compared using Fisher's exact test. Statistical significance was determined at a critical uncertainty value of p < 0.05. A total of 150 participants were involved in this study, with 75 patients in each group. Wearing an acrylic removable denture was found to increase the number of detected Candida species (p < 0.001) and significantly increases the overall growth of Candida spp. (p = 0.001). Specifically, the numbers of CFUs of Candida tropicalis and Candida glabrata were elevated in denture wearers (p < 0.001). Findings stemming from this study indicate that removable dentures promote the growth of Candida species. This can be a predisposing factor for Candida-associated denture stomatitis in cases of poor oral hygiene or compromised immunity. Therefore, it is imperative to emphasize the fabrication of high-quality dentures and the implementation of rigorous postdenture maintenance protocols to prevent or limit Candida infection.

The treatment of bacterial biofilms cultivated on knee arthroplasty implants using the bioelectric effect.

Introduction: The formation of bacterial biofilms on knee arthroplasty implants can have catastrophic consequences. The aim of this study was to analyze the effectiveness of the bioelectric effect in the elimination of bacterial biofilms on cultivated knee arthroplasty implants. Methods: A novel device was designed to deliver a bioelectric effect on the surface of knee arthroplasty implants. 4-femoral prosthetic implants were cultivated with a staphylococcus aureus inoculum for 15days. The components were divided into four different groups: A (not treated), B (normal saline 20-minutes), C (bioelectric effect 10-minutes), D (bioelectric effect 20-minutes). The implants were sonicated, and the detached colonies were quantified as the number of colony-forming unit (CFUs). The implants were sterilised and the process was repeated in a standardized manner four more times, to obtain a total of five samples per group. Results: The number of the CFUs after a 10-minute exposure to the bioelectric effect was of 208.2 ± 240.4, compared with 6,041.6 ± 2010.7 CFUs in group A, representing a decrease of 96.5% ± 4.3 (p = 0.004). And a diminution of 91.8% ± 7.9 compared with 2,051.0 ± 1,364.0 CFUs in group B (p = 0.109). The number of bacterial colonies after a 20-minute exposure to the bioelectric effect was 70 ± 126.7 CFUs, representing a decrease of 98.9% ± 1.9 (p = 0.000) compared with group A. And a decrease of 97.8% ± 3.0 (p = 0.019) compared with group B. Conclusions: The bioelectric effect was effective in the elimination of bacterial biofilm from knee arthroplasty implants. This method could be used in the future as part of conventional surgical procedures.

A Novel Device for the Evaluation of In Vitro Bacterial Colonization in Membranes for Guided Tissue and Bone Regeneration.

Purpose: To evaluate, in vitro, the efficiency of a novel apparatus to test the adherence and penetration of bacteria on different membranes for guided regeneration. Methodology: To create the 3D device, Computer Aided Design/Computer Aided Manufacturing (CAD/CAM) systems were used. Three types of biomaterials were tested (n = 6): (DT) a collagen membrane; (DS) a polymer membrane; and (LP) a dense polytetrafluoroethylene barrier. The biomaterials were adapted to the apparatuses and challenged with two different monospecies bacterial culture of A. actinomycetemcomitans b and S. mutans. After 2 h, bacterial adherence and penetration were quantified by counting the number of colony-forming units (CFUs). Two specimens from each group were used for image analysis using Confocal Laser Scanning Microscopy. Statistical analysis was performed. Findings: The DS group had a higher adherence of S. mutans compared to A. actinomycetemcomitans b (p = 0.05). There was less adherence of A. actinomycetemcomitans b in the DS group, compared to the LP (p = 0.011) and DT (p < 0.001) groups. Only the membranes allowed penetration, which was blocked by barriers. The DT group allowed a greater penetration of S. mutans to occur compared to A. actinomycetemcomitans b (p = 0.009), which showed a higher penetration into the DS membranes compared to S. mutans (p = 0.016). The penetration of A. actinomycetemcomitans b through DS was higher compared to its penetration through DT and LP (p < 0.01 for both). DT and DS allowed a greater penetration of S. mutans to occur compared to LP, which prevented both bacterial species from penetrating. Conclusion: The apparatus allowed for the settlement and complete sealing of the biomaterials, enabling standardization.

Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis.

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

Rapid bacterial evaluation beyond the colony forming unit in osteomyelitis

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony forming unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

Effect of silica-sprayed collection tubes on synovial fluid bacterial culture.

Silica-sprayed tubes (SSTs) are often used to transport synovial fluid samples in equine practice. They promote the coagulation of the sample. The objective of the study is to evaluate the effect of SST on bacterial culture. The study was divided into two parts: sterile saline (Part A) and synovial fluid (Part B). Four common bacteria associated with equine synovial sepsis were used: Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). Three collection tubes were used: STT, plain (no-additives) and brain and heart infusion (BHI) broth. Bacteria were cultured in horse blood agar plates for 48h. Outcome variables were negative culture, positive culture and total number of colony-forming units (CFUs). Statistical analysis was performed using Mann-Whitney U test, and significance was set at p<0.05. The total number of agar plates read was 1557 (779 saline; 778 synovial fluid). Total negative cultures were 25/779 on saline and 3/778 on synovial fluid. In broth, maximum growth CFU was achieved after 8h for both saline and synovial fluid for all bacteria. S. pyogenesand E. coli produced a significantly lower number of CFU when in SST compared to plain or broth after 4h, whereas S. aureus (American Type Culture Collection [ATCC] and MRSA) only after 24h. Silica-containing tubes reduced bacterial proliferation, whereas the use of a BHI broth provided the highest bacterial load in the sample. The use of SST may have a negative effect on bacterial proliferation in samples obtained from clinical cases.

Antimicrobial Efficacy of Saline, Chlorhexidine, and Zataria Multiflora and Mentha Piperita Essential Oils in Root Canal Irrigation of Primary Molars.

Objectives: This study aimed to compare the antimicrobial efficacy of saline, 0.5% and 2% Zataria multiflora (Z. multiflora) essential oil, 0.5% and 2% Mentha piperita (M. piperita) essential oil, and 0.2% chlorhexidine (CHX) as root canal irrigants for primary molar teeth. Materials and Methods: A total of 64 primary molars were used in this in vitro study. The teeth were randomly assigned to six groups (N=10). The root canals were prepared up to file #35, and all teeth were sterilized before contamination with Enterococcus faecalis (E. faecalis; ATCC 29212) suspension. After 48 hours of incubation, the root canals in each group were irrigated with the respective irrigants. Sterile paper points were then used to collect microbial samples from the root canals. A colony counter was used to count the number of colony-forming units (CFUs). Data were analyzed by SPSS version 20 (alpha=0.05). Results: The colony count was significantly different among the groups (P<0.001), and 2% M. piperita (P=0.009), 0.5% Z. multiflora (P=0.021), and 0.2% CHX (P=0.002) were significantly more effective than saline in elimination of E. faecalis. The ascending order of microbial count after irrigation was as follows: saline > 0.5% M. piperita > 0.2% CHX > 2% M. piperita > 0.5% Z. multiflora. Conclusion: The current study showed the optimal antibacterial activity of 0.5% Z. multiflora essential oil and 2% M. piperita essential oil against E. faecalis, and indicated their possible efficacy for use as an irrigant for root canal irrigation of primary molars.

Antifungal bioactivity of Sarcococca hookeriana var. digyna Franch. against fluconazole-resistant Candida albicans in vitro and in vivo

Ethnopharmacological relevanceSarcococca hookeriana var. digyna Franch. has been widely utilized in folk medicine by the Miao people in the southwestern region of China for treating skin sores which may be associated with microbial infection. Aim of the studyTo investigate the antifungal bioactivity of S. hookeriana var. digyna against fluconazole-resistant Candida albicans in vitro and in vivo, as well as its underlying mechanism and the key bioactive component. Materials and methodsThe antifungal bioactivity of 80% ethanol extract of S. hookeriana var. digyna (SHE80) was investigated in vitro using the broth microdilution method, time-growth curve, and time-kill assay. Its key functional component and antifungal mechanism were explored with combined approaches including UPLC-Q-TOF-MS, network pharmacology and metabolomics. The antifungal pathway was further supported via microscopic observation of fungal cell morphology and examination of its effects on fungal biofilm and cell membranes using fluorescent staining reagents. In vivo assessment of antifungal bioactivity was conducted using a mouse model infected with C. albicans on the skin. ResultsS. hookeriana var. digyna suppressed fluconazole-resistant C. albicans efficiently (MIC = 16 μg/mL, MFC = 64 μg/mL). It removed fungal biofilm, increased cell membrane permeability, induced protein leakage, reduced membrane fluidity, disrupted mitochondrial membrane potential, induced the release of reactive oxygen species, promoted cell apoptosis, and inhibited the transformation of fungi from the yeast state to the hyphal state significantly. In terms of mechanism, it affected sphingolipid metabolism and signaling pathway. Moreover, the predicted bioactive component, sarcovagine D, was supported by antifungal bioactivity evaluation in vitro (MIC = 4 μg/mL, MFC = 16 μg/mL). Furthermore, S. hookeriana var. digyna promoted wound healing, reduced the number of colony-forming units, and reduced inflammation effectively in vivo. ConclusionsThe traditional use of S. hookeriana var. digyna for fungal skin infections was supported by antifungal bioactivity investigated in vitro and in vivo. Its mechanism and bioactive component were predicted and confirmed by experiments, which also provided a new antifungal agent for future research.

Globospiramine Exhibits Inhibitory and Fungicidal Effects against Candida albicans via Apoptotic Mechanisms.

Candidiasis is considered an emerging public health concern because of the occurrence of drug-resistant Candida strains and the lack of an available structurally diverse antifungal drug armamentarium. The indole alkaloid globospiramine from the anticandidal Philippine medicinal plant Voacanga globosa exhibits a variety of biological activities; however, its antifungal properties remain to be explored. In this study, we report the in vitro anticandidal activities of globospiramine against two clinically relevant Candida species (C. albicans and C. tropicalis) and the exploration of its possible target proteins using in silico methods. Thus, the colony-forming unit (CFU) viability assay revealed time- and concentration-dependent anticandidal effects of the alkaloid along with a decrease in the number of viable CFUs by almost 50% at 60 min after treatment. The results of the MIC and MFC assays indicated inhibitory and fungicidal effects of globospiramine against C. albicans (MIC = 8 µg/mL; MFC = 8 µg/mL) and potential fungistatic effects against C. tropicalis at lower concentrations (MIC = 4 µg/mL; MFC > 64 µg/mL). The FAM-FLICA poly-caspase assay showed metacaspase activation in C. albicans cells at concentrations of 16 and 8 µg/mL, which agreed well with the MIC and MFC values. Molecular docking and molecular dynamics simulation experiments suggested globospiramine to bind strongly with 1,3-β-glucan synthase and Als3 adhesin-enzymes indirectly involved in apoptosis-driven candidal inhibition.