Colony-forming Unit Counts Research Articles

Perspectives on antimicrobial properties of Paulownia tomentosa Steud. fruit products in the control of Staphylococcus aureus infections

Ethnopharmacological relevancePaulownia tomentosa Steud. (P. tomentosa) is a medium-sized tree traditionally used in Chinese folk medicine for the treatment of infectious diseases. It is a rich source of prenylated phenolic compounds that have been extensively studied for their promising biological activities. Aim of the studyDue to the increasing development of antibiotic resistance, our study investigated plant-derived natural products from the fruits of P. tomentosa that could control Staphylococcus aureus infections with novel targets/modes of action and reduce antimicrobial resistance. Materials and methodsThe ethanolic extract was fractionated and detected by liquid chromatography. The antistaphylococcal effects of the plant formulations were studied in detail in vitro by various biological methods, including microdilution methods for minimum inhibitory concentration (MIC), the checkerboard titration technique for synergy assay, fluorescence measurements for membrane disruption experiments, autoinducer-2-mediated bioassay for quorum sensing inhibition, and counting of colony-forming units for relative adhesion. Morphology was examined by transmission electron microscopy. ResultsTotal ethanolic extract and chloroform fraction showed MICs of 128 and 32 μg/mL, respectively. Diplacol, diplacone, and 3′-O-methyl-5′-hydroxydiplacone inhibited S. aureus growth in the range of 8–16 μg/mL. Synergistic potential was shown in combination with mupirocin and fusidic acid. The ethanolic extract and the chloroform fraction destroyed the cell membranes by 91.61% and 79.46%, respectively, while the pure compounds were less active. The ethanolic extract and the pure compounds reduced the number of adhered cells to 47.33–10.26% compared to the untreated control. All tested plant formulations, except diplacone, inhibited quorum sensing of S. aureus. Transmission electron microscopy showed deformation of S. aureus cells. ConclusionsThe products from the fruit of P. tomentosa showed antimicrobial properties against S. aureus alone and in combination with antibiotics. By affecting intracellular targets, geranylated flavonoids proposed novel approaches in the control of staphylococcal infections.

Kali carbonicum 6cH controls Malassezia pachydermatis growth in vitro

External otitis caused by Malassezia pachydermatis is a common disease in the veterinary practice routine, whose treatment has been challenging due to the risk of antimicrobial resistance. Thus, Homeopathy is seen as a possible alternative to control the infection. To screen potential medicines to be used in a non-individualized clinical homeopathy approach, a series of in vitro assays were performed, in which a suspension of M. pachydermatis colonies in 0.5 McFarland scale was diluted 1:1000 in sterile saline solution, and 10 µL of pre-selected homeopathic medicines was added. Sulphur 6cH, Dolichos pruriens 6cH, and Kali carbonicum 6cH were chosen from a pilot study, and the last potency was prepared in pure, sterile water. The controls were pure, sterile water and succussed pure, sterile water. An amount of 50 µL of each suspension has seemed in plates containing Sabouraud dextrose agar medium, with or without 1% Tween 80, for counting of colony-forming units (CFU) and cytomorphological analysis of M. pachydermatis samples taken from the same colonies. The analysis was made using Image J 1.53 by manually counting quiescent and germinative forms. Kali carbonicum 6cH treatment decreased the germinative/quiescent ratio in relation to the controls, independent of Tween 80 presence in the medium. The UFC counting, however, did not show statistically significant differences among groups, probably by the lack of cytotoxicity against the yeasts. In conclusion, Kali carbonicum 6cH was considered a promising medicine to be tested in clinical conditions, probably due to its capacity to control yeast growth without creating a selective pressure able to induce resistance mechanisms.

Educational Intervention on Cleaning and Sanitation of Surfaces in a Pediatric Hospitalization Unit

Introduction: Environmental surfaces may serve as a reservoir for various microorganisms and consequently, they represent a potential risk for the spread of healthcare-associated infections. Objective: This study aimed to assess the cleaning and sanitation of surfaces (CSS) before and after implementing a Standardization Program for Cleaning and Sanitation of Surfaces (SPCSS). Methods: An analytical, comparative, and intervention study was conducted from 2020 to 2021 in a pediatric hospitalization unit in Midwest Brazil. Four frequently touched surfaces were monitored before and after the cleaning and sanitation process using the following methods: Adenosine Triphosphate (ATP) quantification, Colony-Forming Unit (CFU) count, and visual inspection. The study consisted of three stages: stage I (situational diagnosis of the CSS process), stage II (implementation of the SPCSS), and stage III (assessment 60 days after implementing the program). A total of 576 assessments were performed in all three study stages. Results: The CSS process was effective in all three study stages by using the ATP and CFU methods. In stage I, statistically significant results were obtained for four surfaces using the ATP method, and two by the CFU count. In stages II and III, all surfaces presented lower ATP and CFU results (p<0.05). In the visual inspection, only the bathroom door handle (stage I: p=0.041; stage III: p=0.007) and toilet flush handle (stage I: p=0.026; stage III: p=0.007) passed the test. Implications for Nursing: This study presents subsidies to evaluate the cleaning and disinfection process carried out by the nursing and hygiene team. Conclusion: The SPCSS exerted a positive impact on the CSS process.

A Cross-Over Randomized Controlled Clinical Trial for Evaluation of Two Hygiene Protocols on Microbial Load, Tissue Health, and Opinion of Ocular Prosthesis Wearers

The recommendations for the wear and hygiene of ocular prostheses can vary among practitioners, and it is still a controversial theme in the literature. This clinical trial evaluated the microbial load, tissue health of the socket, and the participants’ opinions before and after the use of two hygiene protocols. Thirty ocular prosthesis wearers used either a Daily Protocol (DPt: hygiene once a day) or Weekly Protocol (WPt: hygiene once a week) for 5 weeks with a washout of 7 days. The microbial load was quantified by the colony-forming unit count of the aerobic bacteria, Candida spp., Staphylococcus spp., and Gram-negative bacteria. The tissue health of the socket was evaluated by scores, and patients’ opinion of the protocols was evaluated using the analogic visual scale (VAS). Data were analyzed by ANOVA Repeated Measures, Friedman, Cochran’s Q Test, Wilcoxon, Fisher, and Pearson’s chi-square tests considering p < 0.05. There was no difference in the microbial load of the microorganisms (p > 0.05). Both protocols improved socket inflammation (p = 0.005) and discharge (p < 0.001); DPt improved edema (p = 0.021) and crusting (p = 0.020). There was no difference in patients’ rating responses (VAS) for all the questions of patients’ opinion (Q1: p = 1.0; Q2: p = 1.0; Q3: p = 1.0; Q4: p = 1.0; Q5: p = 1.0; Q6: p = 0.317; Q7: p = 1.0; Q8: p = 0.159). There was a correlation between eye drops/edema (p = 0.030), eye drops/pain (p = 0.016), microbial load with discharge, inflammation, eyelid edema, and pain. Inflammation was correlated with edema at baseline (p < 0.001) and after DPt (p = 0.018), and with crusting at baseline (p = 0.003); edema was correlated with crusting at baseline (p < 0.001); crusting was correlated with discharge after WPt (p < 0.001). The protocols showed no effects on the microbial load of the anophthalmic socket and ocular prosthesis. However, better tissue health and patient acceptance were observed after both regimens.

Is ultraviolet light disinfection fit to be the future standard for the disinfection of flexible endoscopes without a working channel?

To investigate colony-forming unit (CFU) reduction on contaminated flexible endoscopes (FEs) without a working channel after UV-C light disinfection, compared to the current disinfection method with the endoscope washer disinfector (EWD). After pharyngolaryngoscopy, a manual pre-cleaning with tap water was performed. A culture was then collected by rolling the distal 8-10 cm of the FE over an Agar plate. The FE was disinfected using the D60 (60-s disinfection process with UV-C light) or the EWD (gold standard reprocessing process with water and chemicals). Another culture was then taken. After incubation, a CFU count was performed. A total of 200 FEs without a working channel were divided equally between the two disinfection groups. After clinical use and manual pre-cleaning, 84 of the 100 (84.0%) (UV-C light group) and 79 of the 100 (79.0%) (EWD) FEs were contaminated with at least 1 CFU. FEs that showed no contamination after use were excluded from further analysis. After disinfection with UV-C light, 72 (85.7%) FEs showed no contamination (i.e., 0 CFUs) versus 66 (83.5%) FEs after reprocessing with the EWD. There is no difference in CFUs reduction on contaminated FEs without a working channel between UV-C light disinfection and the current gold standard, the EWD.

A Review of Bacterial Colonization on Dental Implants With Various Hygiene Instruments.

Peri-implant diseases can still develop despite oral hygiene practices being maintained. Consequently, regular debridement must be carried out to ensure the implant is sustained. This review evaluated bacterial colonization on implants following the use of different hygiene instruments. A literature search was conducted in PubMed, ScienceDirect, and Scopus databases for articles published from 2012 to 2022. A total of 19 full-text papers were selected. The number of bacteria colonized was most commonly evaluated with a scanning electron microscope (SEM) or by colony-forming unit (CFU) counts, crystal violet assays, plaque index, probing depth, bleeding on probing, turbidity test, and live-dead assays. Rubber cup polishing with an abrasive paste showed a significantly greater reduction in biofilm formation compared with air abrasion with glycine powder, while the air abrasion treatment was found to be more efficient than piezoelectric, carbon, and stainless steel scalers. Surface treatment with Er, Cr: YSGG laser, and Er: YAG laser resulted in statistically significant superior dental biofilm removal compared with titanium curettes and photodynamic therapy. Air abrasion, plastic curette, titanium curette, and ultrasonic scaler showed no significant differences in bacterial colonization, but air abrasion and plastic curette were safer for zirconia implant decontamination. Furthermore, the titanium brush showed better results in decontaminating the implant surface than the Er: YAG laser. Although no single instrument or method could be considered as offering a gold standard in treating peri-implant diseases, the use of air abrasion with glycine powder, laser therapies, rubber cup polishing with an abrasive paste, and a titanium brush had high levels of cleaning efficacy and acceptance by patients.

Characterization of potential probiotic starter cultures of lactic acid bacteria isolated from Ethiopian fermented cereal beverages, Naaqe and Cheka.

To test the in vitro probiotic potential and starter culture capacity of lactic acid bacteria (LAB) isolated from Naaqe and Cheka, cereal-based Ethiopian traditional fermented beverages. 44 strains were isolated from spontaneously fermented Ethiopian cereal-based beverages, Naaqe and Cheka, with 24 putatively identified as LAB and 14 identified up to the species level. The species Limosilactobacillus fermentum (6/12; 50%) and Weissella confusa (5/12, 41.67%) were the predominant species identified from Naaqe, while the two Cheka isolates were Limosilactobacillus fermentum and Pediococcus pentosaceus. Six LAB strains inhibited eight of the nine gastrointestinal indicator key pathogens in Ethiopia, including Escherichia coli, Salmonella enterica subsp. enterica var. Typhimurium, Staphylococcus aureus, Shigella flexneri, and Listeria monocytogenes. Three of the LAB isolates exhibited strain-specific immunostimulation in human monocytes. Based on these probiotic properties and growth, six strains were selected for in situ evaluation in a mock fermentation of Naaqe and Cheka. During primary fermentations, L. fermentum 73B, P. pentosaceus 74D, L. fermentum 44B, Weissella confusa 44D, L. fermentum 82C and Weissella cibaria 83E and their combinations demonstrated higher pH-lowering properties and colony-forming unit counts compared to the control spontaneous fermentation. The same pattern was also observed in the secondary mock fermentation by the Naaqe LAB isolates. In this study, we selected six LAB strains with antipathogenic, immunostimulatory and starter culture potentials that can be used as autochthonous probiotic starters for Naaqe and Cheka fermentations, once their health benefit is ascertained in a clinical trial as a next step.

Multispecies oral biofilm and identification of components as treatment target

Endodontic infections involve a multispecies biofilm, making it difficult to choose an antimicrobial treatment. Characteristics such as the pathogens involved and number of microorganisms, nutrients, material surface to develop the biofilm, flow and oxygenation conditions are important for biofilm development using in vitro models. ObjectiveTo develop a standardized biofilm model, which replicates the main features (chemical, microbiological, and topographical) of an infected root canal tooth to detect components as treatment target. DesignClinical strains of Enterococcus faecalis, Candida albicans, and Actinomyces israelii were isolated, and a multispecies biofilm was developed using continuous laminar flow reactors under anaerobic conditions in human dental roots. The microbiological composition was determined by counting colony-forming units and scanning electron microscope micrographs. In addition, the chemical composition of the exopolymeric matrix was determined by vibrational Raman spectroscopy and liquid chromatography of biofilm supernatant treated with enzyme. ResultsE. faecalis turned out to be the main microorganism in mature biofilm, this was related to the presence of β-galactosidase detected by vibrational Raman spectroscopy. After the enzymatic treatment of the extracellular polymeric substance, the presence of mannose and glucose was established. ConclusionsThe present work contributes to better understanding of standard conditions to develop a multispecies biofilm in human dental roots, which could have an impact on the generation of new root canal disinfection techniques in endodontic pathologies.

Effect of Graphene Oxide and Silver Nanoparticle Hybrid Composite on Acinetobacter baumannii Strains, Regarding Antibiotic Resistance and Prevalence of AMP-C Production.

Background and Objectives: Growing antibiotic resistance among bacteria is a global issue that is becoming harder and more expensive to solve. Traditional treatment options are becoming less effective, causing more fatal outcomes of nosocomial infections. Since the development of new antibiotics has stagnated in the last decade, a novel approach is needed. Materials and Methods: Graphene-based materials are being developed and tested for various applications, and the medical field is no exception. We tested 98 clinical A. baumannii strains for antibiotic resistance, AMP-C production and the effectiveness of a graphene oxide and silver nanoparticle hybrid nanocomposite. The disc diffusion method was used to determine antibiotic susceptibility results. Antibiotic discs containing cefotaxime, cloxacillin and clavulanate were used to detect AMP-C production. The effectiveness of the GO-Ag hybrid nanocomposite was determined by counting colony forming units (CFUs) after a suspension of A. baumannii and the GO-Ag hybrid nanocomposite was plated on MH agar and incubated overnight to grow colonies. Results: In our research, we found that A. baumannii strains are resistant to the majority of commonly used antibiotics. Antibiotic resistance levels and AMP-C production can be factors, indicating the better effectiveness of the graphene oxide and silver nanoparticle hybrid nanocomposite. Conclusions: In this study, a GO-Ag hybrid nanocomposite was shown to have the potential to fight even the most problematic bacteria like A. baumannii.

Hybrid Approach to Colony-Forming Unit Counting Problem Using Multi-Loss U-Net Reformulation.

Colony-Forming Unit (CFU) counting is a complex problem without a universal solution in biomedical and food safety domains. A multitude of sophisticated heuristics and segmentation-driven approaches have been proposed by researchers. However, U-Net remains the most frequently cited and used deep learning method in these domains. The latter approach provides a segmentation output map and requires an additional counting procedure to calculate unique segmented regions and detect microbial colonies. However, due to pixel-based targets, it tends to generate irrelevant artifacts or errant pixels, leading to inaccurate and mixed post-processing results. In response to these challenges, this paper proposes a novel hybrid counting approach, incorporating a multi-loss U-Net reformulation and a post-processing Petri dish localization algorithm. Firstly, a unique innovation lies in the multi-loss U-Net reformulation. An additional loss term is introduced in the bottleneck U-Net layer, focusing on the delivery of an auxiliary signal that indicates where to look for distinct CFUs. Secondly, the novel localization algorithm automatically incorporates an agar plate and its bezel into the CFU counting techniques. Finally, the proposition is further enhanced by the integration of a fully automated solution, which comprises a specially designed uniform Petri dish illumination system and a counting web application. The latter application directly receives images from the camera, processes them, and sends the segmentation results to the user. This feature provides an opportunity to correct the CFU counts, offering a feedback loop that contributes to the continued development of the deep learning model. Through extensive experimentation, the authors of this paper have found that all probed multi-loss U-Net architectures incorporated into the proposed hybrid approach consistently outperformed their single-loss counterparts, as well as other comparable models such as self-normalized density maps and YOLOv6, by at least 1% to 3% in mean absolute and symmetric mean absolute percentage errors. Further significant improvements were also reported through the means of the novel localization algorithm. This reaffirms the effectiveness of the proposed hybrid solution in addressing contemporary challenges of precise in vitro CFU counting.

Microbial adhesion and biofilm formation by Candida albicans on 3D-printed denture base resins.

This study evaluated surface properties and adhesion/biofilm formation by Candida albicans on 3D printed denture base resins used in 3D printing. Disc-shaped specimens (15 mm x 3 mm) of two 3D-printed resins (NextDent Denture 3D+, NE, n = 64; and Cosmos Denture, CO, n = 64) and a heat-polymerized resin (Lucitone 550, LU, control, n = 64) were analyzed for surface roughness (Ra μm) and surface free energy (erg cm-2). Microbiologic assays (90-min adhesion and 48-h biofilm formation by C. albicans) were performed five times in triplicate, with the evaluation of the specimens' surface for: (i) colony forming units count (CFU/mL), (ii) cellular metabolism (XTT assay), and (iii) fluorescence and thickness of biofilm layers (confocal laser scanning microscopy). Data were analyzed using parametric and nonparametric tests (α = 0.05). LU presented higher surface roughness Ra (0.329±0.076 μm) than NE (0.295±0.056 μm) (p = 0.024), but both were similar to CO (0.315±0.058 μm) (p = 1.000 and p = 0.129, respectively). LU showed lower surface free energy (47.47±2.01 erg cm-2) than CO (49.61±1.88 erg cm-2) and NE (49.23±2.16 erg cm-2) (p<0.001 for both). The CO and NE resins showed greater cellular metabolism (p<0.001) and CO only, showed greater colonization (p = 0.015) by C. albicans than LU in the 90-min and 48-hour periods. It can be concluded that both 3D-printed denture base resins are more prone to colonization by C. albicans, and that their surface free energy may be more likely associated with that colonization than their surface roughness.

Antibiofilm activity of promethazine, deferiprone, and Manuka honey in an ex vivo wound model.

This study evaluated the antibiofilm activity of promethazine, deferiprone, and Manuka honey against Staphylococcus aureus and Pseudomonas aeruginosa in vitro and ex vivo in a wound model on porcine skin. The minimum inhibitory concentrations (MICs) and the effects of the compounds on biofilms were evaluated. Then, counting colony-forming units (CFUs) and confocal microscopy were performed on biofilms cultivated on porcine skin for evaluation of the compounds. For promethazine, MICs ranging from 97.66 to 781.25µg/ml and minimum biofilm eradication concentration (MBEC) values ranging from 195.31 to 1562.5µg/ml were found. In addition to reducing the biomass of both species' biofilms. As for deferiprone, the MICs were 512 and>1024µg/ml, the MBECs were≥1024µg/ml, and it reduced the biomass of biofilms. Manuka honey had MICs of 10%-40%, MBECs of 20 to >40% and reduced the biomass of S. aureus biofilms only. Concerning the analyses in the ex vivo model, the compounds reduced (P<.05) CFU counts for both bacterial species, altering the biofilm architecture. The action of the compounds on biofilms in in vitro and ex vivo tests raises the possibility of using them against biofilm-associated wounds. However, further studies are needed to characterize the mechanisms of action and their effectiveness on biofilms in vivo.

Dynamic interactions between Candida albicans and different streptococcal species in a multispecies oral biofilm.

The oral cavity is colonized by a plethora of bacteria, fungi, and archaea, including streptococci of the mitis group (MSG) and the yeast Candida albicans. This study aims to investigate the role of streptococcal species in the development of oral biofilm and the cross-kingdom interactions between some of the members of the commensal MSG and the pathogen yeast C. albicans using a multispecies supragingival biofilm model. A total of nine different in vitro biofilms were grown, quantified with culture analyses, and visually examined with confocal laser scanning microscopy (CLSM). A four-species biofilm without any streptococcal species was used as a basic biofilm. In each subsequent inoculum, one species of MSG was added and afterward combined with Streptococcus mutans. The eight-species biofilm contained all eight strains used in this study. Culture analyses showed that the presence of S. mutans in a four-species biofilm with Streptococcus oralis or S. oralis subsp. tigurinus did not differ significantly in C. albicans colony-forming unit (CFU) counts compared to biofilms without S. mutans. However, compared to other mitis species, Streptococcus gordonii combined with S. mutans resulted in the lowest CFUs of C. albicans. Visual observation by CLSM showed that biofilms containing both S. mutans and one species of MSG seemed to induce the formation of filamentous form of C. albicans. However, when several species of MSG were combined with S. mutans, C. albicans was again found in its yeast form.

Konvansiyonel, CAD/CAM frezeleme ve 3D baskı yöntemleriyle üretilmiş geçici materyallerin yüzey özelliklerinin Streptococcus mutans ve Candida albicans tutunumuna etkileri

Objectives: The purpose of this in vitro study was to compare conventionally manufactured, CAD/CAM milled, and 3D-printed interim materials based on their susceptibility to adherence of Streptococcus mutans and Candida albicans, and examine the influence of surface roughness and hydrophobicity. Materials and Methods: Eighty disc-shaped specimens fabricated from autopolymerized polymethyl methacrylate (A-PMMA), bis-acryl composite (Bis-acrylate), CAD/CAM PMMA-based polymer (Milled-PMMA), and 3D-printed resin (Printed) were subjected to 10,000 thermal cycles (5-55 °C) and divided into two groups (n=10) according to microbial suspension used: Streptococcus mutans and Candida albicans. Surface roughness (Ra) and hydrophobicity (WCA) of specimens were measured. An adhesion test was performed by incubating the specimens in Streptococcus mutans and Candida albicans suspensions at 37 °C for 24 hours, and the adherent cells were evaluated by counting colony-forming units (CFU/ml). Scanning electron microscopy (SEM) was performed to analyze the surfaces (n=2). Data were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Spearman’s correlation analysis was used to determine correlation among the measurements (α=.05). Results: Type of restorative material significantly influenced Ra and WCA. The highest adhesion of Streptococcus mutans was observed in Printed, followed by Bis-acrylate, A-PMMA, and Milled-PMMA (p=.001). The highest adhesion of Candida albicans was noted on A-PMMA, followed by Printed, Bis-acrylate, and Milled-PMMA (r=.001). The adhesion of Streptococcus mutans (r=.660) and Candida albicans (r=.413) showed a positive correlation with Ra. A negative correlation was found between WCA of the materials and Streptococcus mutans adhesion (r=-.373). Conclusions: Surface roughness plays an important role in the adherence of microorganisms. CAD/CAM PMMA-based polymers may be a better choice to reduce microbial adhesion in long-term use.

Biofilm formation on endovascular aneurysm repair (EVAR) grafts—a proof of concept in vitro model

ObjectivesAn endovascular aneurysm repair (EVAR) graft is a catheter-implanted vascular prosthesis and is the preferred treatment for patients with aortic aneurysm. If an EVAR graft becomes the focus of infection, the treatment possibilities are limited because it is technically difficult to remove the graft to obtain source control. This study examines whether Pseudomonas aeruginosa and Staphylococcus aureus form biofilm on EVAR prostheses. MethodsEVAR graft sections were exposed to bacteria at 102 or 108 colony forming units (CFU)/mL in lysogeny broth and Krebs-Ringer at 37°C, bacterial biofilm formation was evaluated by scanning electron microscopy and counting CFU on the graft sections after antibiotic exposure at × 10 minimal inhibitory concentration. Bacteria were tested for tolerance to benzylpenicillin, tobramycin, and ciprofloxacin. ResultsBacterial exposure for 15 minutes established biofilms on all prosthesis fragments (6/6 replicates). After 4 hours, bacteria were firmly attached to the EVAR prostheses and resisted washing. After 18–24 hours, the median CFU/g of EVAR graft reached 5.2 × 108 (1.15 × 108–1.1 × 109) for S. aureus and 9.1 × 107 (3.5 × 107–6.25 × 108) for P. aeruginosa. Scanning electron microscopy showed bacterial attachment to the graft pieces. There was a time-dependent development of tolerance with approximately 20 (tobramycin), 560 (benzylpenicillin), and 600 (ciprofloxacin) times more S. aureus surviving antibiotic exposure in 24- compared with 0-hour-old biofilm. Five (tobramycin) and 170 times (ciprofloxacin) more P. aeruginosa survived antibiotic exposure in 24- compared with 0-hour-old biofilms. DiscussionOur results show that bacteria can rapidly adhere to and subsequently form antibiotic-tolerant biofilms on EVAR graft material in concentrations equivalent to levels seen in transient bacteraemia in vivo. Potentially, the system can be used for identifying optimal treatment combinations for infected EVAR prosthesis.

Antimicrobial Efficacy of a Novel Automated Irrigation Device As Compared to Conventional Needle Irrigation Against Enterococcus faecalis: An In Vitro Study.

Aim The present study aims to compare the antibacterial efficacy of a novel automated endodontic irrigation device with that of a manual irrigation technique. Materials and methods The present study considered 45 extracted single-rooted teeth. After sectioning the teeth, the specimens were inoculated with Enterococcus faecalis (E. faecalis) bacteria for three weeks. Instrumentation was done using ProTaper Gold rotary files to size 50 and 5% taper. Based on the irrigation protocol, the experimental samples were divided into Group I: control (normal saline irrigation) (n = 15); Group II: conventional needle irrigation (n = 15); and Group III: automated irrigation (n = 15). The irrigation in Group III was done using the automated irrigation device. After the experimental irrigation, the pre-weighted dentinal shaving was collected in Eppendorf tubes, diluted 10 times, and incubated in the Petri dish with tryptone soy agar (TSA) for 48 hours. Finally, the colony-forming unit (CFU) counts were assessed. IBM SPSS Statistics for Windows version 23.0 (Armonk, NY, USA, IBM Corp.) was used for data analysis. Intergroup comparisons were made using the non-parametric Kruskal-Wallis test. Results The mean CFU count (CFU/ml) for the groups was: Group 1 (normal saline irrigation): 3.67x101; Group 2 (manual irrigation): 2.69× 101; Group 3 (automated irrigation): 1.57× 101. A statistically significant reduction in E. faecalis CFU count was seen among the groups assessed (p<0.01). The automated irrigation group exhibited the most substantial reduction in E. faecalis CFU count. Group 2 showed a significant difference compared to Group 1 (p<0.05). Conclusions The novel automated endodontic irrigation device was superior to manual irrigation in reducing E. faecalis CFU counts. This indicates that the automated irrigation device has the potential to enhance bacterial elimination efficacy during endodontic treatment.

Microbial Detection and Quantification of Low-Biomass Water Samples Using an International Space Station Smart Sample Concentrator.

The pressing need to safeguard the health of astronauts aboard the International Space Station (ISS) necessitates constant and rigorous microbial monitoring. Recognizing the shortcomings of traditional culture-based methods, NASA is deliberating the incorporation of molecular-based techniques. The challenge, however, lies in developing and validating effective methods for concentrating samples to facilitate this transition. This study is dedicated to investigating the potential of an ISS Smart Sample Concentrator (iSSC) as an innovative concentration method. First, the iSSC system and its components were tested and optimized for microgravity, including various testing environments: a drop tower, parabolic flight, and the ISS itself. Upon confirming the system's compatibility with microgravity, we further evaluated its proficiency and reliability in concentrating large volumes (i.e., 1 L) of water samples inoculated with different microbes. The samples carried 102 to 105 colony-forming units (CFUs) of Sphingomonas paucimobilis, Ralstonia pickettii, or Cupriavidus basilensis per liter, aligning with NASA's acceptable limit of 5 × 104 CFU/L. The average retrieved volume post-concentration was ≈450 µL, yielding samples that were ≈2200 times more concentrated for subsequent quantitative PCR (qPCR) and CFU analysis. The average microbial percent recovery, as assessed with CFU counts, demonstrated consistency for C. basilensis and R. pickettii at around 50% and 45%, respectively. For S. paucimobilis, the efficiency oscillated between 40% and 80%. Interestingly, when we examined microbial recovery using qPCR, the results showed more variability across all tested species. The significance of these findings lies not merely in the successful validation of the iSSC but also in the system's proven consistency, as evidenced by its alignment with previous validation-phase results. In conclusion, conducted research underscored the potential of the iSSC in monitoring microbial contamination in potable water aboard the ISS, heralding a paradigm shift from culture-based to molecular-based monitoring methods.

In vitro efficacy of synthetic lawsone derivative disinfectant solution on removing dual-species biofilms and effect on acrylic denture surface properties

Candida albicans (C. albicans) and Streptococcus mutans (S. mutans) biofilms involve in denture stomatitis. This study compared compound 1 to 2% chlorhexidine gluconate (CHX), Polident, and distilled water (DW) in biofilms reduction and effect on polymethylmethacrylate acrylic (PMMA) properties. The structure of lawsone (naphthoquinone derivative) was modified by the addition of an alkylnyloxy group to yield compound 1. Dual-species biofilms of C. albicans and S. mutans were developed on PMMA discs. The colony-forming unit count measured the number of residual biofilm cells after exposure to the test agents. PMMA discs were examined for color stability, surface roughness, hardness, and chemical structure after 28 days. At 3 min, compound 1 was less effective than CHX in reducing C. albicans (p = 0.004) and S. mutans (p = 0.034) but more effective than Polident in reducing C. albicans (p = 0.001). At 15 min, no viable cells were detectable for compound 1 and its effectiveness was comparable to CHX (p = 0.365). SEM showed fungal cell surface damages in CHX, compound 1 and Polident groups. Only color change was affected by time (p < 0.001) and type of test agent (p = 0.008), and only CHX reached a clinical perception level. Compound 1 is a promising agent for removing biofilm from the PMMA surface without substantially degrading surface properties.

A High-Throughput Microtiter Plate Screening Assay to Quantify and Differentiate Species in Dual-Species Biofilms.

Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that Pseudomonas aeruginosa and Burkholderia cenocepacia strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.

In vitro evaluation of a novel fluoride-coated clear aligner with antibacterial and enamel remineralization abilities.

To investigate the antibacterial and enamel remineralization performances as well as physicochemical properties and biocompatibility of a fluoride-coated clear aligner plastic (FCAP). FCAP and normal clear aligner plastic (CAP) was bought from the manufacturer (Angelalign Technology Inc, China). The FCAP was observed under scanning electron microscopy. Its element composition, resistance to separation, contact angle, and protein adhesion performance were characterized. Colony-forming unit (CFU) count and 3-(4,5)-dimethylthiazol(-z-y1)-3,5-diphenyltetrazolium bromide (MTT) assay were used to evaluate the antibacterial ability of Streptococcus mutans. Fluoride release-recharge patterns were obtained. Apatite formation was evaluated after immersing FCAP in artificial saliva. Enamel remineralization capability was evaluated in the demineralization model (immersing samples in demineralization solution for 36h) and pH cycling model (immersing samples in demineralization solution and remineralization solution in turns for 14days). Cell Counting Kit-8 (CCK-8) and live/dead cell staining kits were used for cytotoxicity assay. The FCAP showed uniformly distributed fluoride and did not compromise protein adhesion performance. CFU count (5.47 ± 0.55 for CAP, 3.63 ± 0.38 for FCAP) and MTT assay (0.41 ± 0.025 for CAP, 0.28 ± 0.038) indicated that the FCAP had stronger antibacterial activity compared with normal CAP (P < 0.05 for both evaluations). The FCAP could release fluoride continuously for 14days and could be recharged after immersing in NaF solution. The FCAP could induce the formation of hydroxyapatite in artificial saliva and could reduce the microhardness decrease, color change, and mineral loss of enamels in both two models (P < 0.05 for all evaluations). CCK-8 and live/dead cell staining analyses showed that the coating did not compromise the biocompatibility of the clear aligner (P > 0.05 for CCK-8 evaluation). The FCAP had antibacterial, fluoride recharge, and enamel remineralization abilities while it did not compromise physicochemical properties and biocompatibility. The FCAP has the potential to prevent enamel demineralization during clear aligner treatment.