Colony-forming Unit Counts Research Articles

A Comparative Evaluation of the Antimicrobial Properties of 50% Grape Seed Extract, N-acetyl Cysteine and 5.25% Sodium Hypochlorite against Enterococcus faecalis (ATCC 19433) – An In vitro Study

Abstract Introduction: Endodontic infections are complex and polymicrobial, presenting significant challenges in dental treatment. Eliminating bacteria is the main objective of endodontic preparation, ensuring a tight seal to prevent reinfection. A commonly isolated bacterium, Enterococcus faecalis, is Gram positive and facultative anaerobic. It thrives in low-nutrient environments, forming resilient biofilms. Due to its tissue dissolving properties and broad-spectrum antimicrobial, sodium hypochlorite (NaOCl) is the most sought after irrigant, but its cytotoxicity is a concern. Alternatives such as grape seed extract (GSE) and N-acetyl cysteine (NAC) are being explored for their potential antimicrobial effects and biocompatibility. Materials and Methods: Forty single-rooted, non-carious premolars extracted for orthodontic reasons were selected. Teeth were cleaned, sectioned and prepared, ensuring apical patency. Specimens were autoclaved and contaminated with E. faecalis (ATCC 19433) for 30 days. Four groups (n = 10) were formed: Group I – GSE 50%, Group II – NAC 200 mg/ml, Group III – NaOCl 5.25% and Group IV – sodium chloride (0.9%). Manufacturer’s instructions were followed precisely to prep the solutions, and root canals were treated using the Reciproc R25 instrument. Samples were collected before (F1) and after (F2) instrumentation and irrigation. Colony-forming units (CFUs) were quantified and analysis was done using two-way analysis of variance and Tukey’s honestly significant difference post hoc tests, with significance set at P < 0.05. Results: NaOCl significantly reduced CFU counts, demonstrating superior antimicrobial efficacy compared to NAC and GSE (P < 0.01). In comparison to negative control and GSE (P < 0.01), NAC showed significant antimicrobial activity, but not significantly different from GSE (P > 0.05). GSE displayed potential antimicrobial properties but was less effective than NaOCl and NAC. Conclusion: NaOCl remains the most effective irrigant; however, NAC and GSE also demonstrated significant antimicrobial properties and could serve as adjuncts in endodontic therapy. Further research is needed to optimise their formulations and validate clinical applications.

Study of the Microbiological Efficiency of a Hospital Ventilation System With Hepa Filtration in the Prevention of Airborne Pathogens

Objective: To evaluate the microbiological efficiency in air conditioning systems with HEPA filtration in an infectious disease hospital. Theoretical Framework: Air conditioning in intensive care units and wards must consider the spread of microorganisms through respiratory pathways, as both patients and healthcare professionals are exposed to infectious agents transmitted by bioaerosols. Due to the possibility of environmental infection, there is a growing recognition that poorly designed air conditioning systems enhance the transmission of pathogens in areas with constant flow of individuals, particularly immunocompromised patients, who are more susceptible to infections. Air conditioning with HEPA filtration is a hospital engineering technique that shows great potential for filtering airborne contaminants, thereby reducing their dispersion in the environment. Method: Air samples were collected using the active impaction method over a 32-week period in the intensive care unit and wards of a reference hospital for infectious diseases. Results and Discussion: In the hospital environment, 67% of fungi and 33% of bacteria were identified, along with their respective subcategories. The colony-forming unit count exceeded the standards established by current regulations. The study highlights that hospital air is a pathway for the transmission and persistence of pathogenic microorganisms. Research Implications: The importance of ensuring a safe and healthy hospital environment is emphasized. Originality/Value: There are few studies addressing pathogens transmitted by bioaerosols. This study contributes to expanding knowledge on this topic and suggests the need for a review of current regulatory standards.

Comparison of three methods for generating the coccoid form of Helicobacter pylori and proteomic analysis

BackgroundHelicobacter pylori changes from spiral to coccoid depending on the host state, environmental factors, and surrounding microbial communities. The coccoid form of H. pylori still maintains its complete cellular structure, retains virulence genes, and thus plays a role in pathogenicity. To understand the coccoid form, it is crucial to establish the in vitro generation of the coccoid H. pylori. Although some conditions have been studied for the generation of the coccoid form, few studies have compared these conditions for coccoid generation. Here, we generated coccoid forms via three methods and compared the differences in morphology, viability, culturability, and protein expression.ResultsThe coccoid H. pylori was generated in vitro via three methods: a starvation method, a method using amoxicillin, and a method using the culture supernatant of Streptococcus mitis. The morphology and viability of the cells were examined by fluorescence microscopy after staining with SYTO9 and propidium iodide. The culturability of H. pylori was examined by counting colony-forming units on chocolate agar plates. In the starvation group, no colonies formed after 7 days, but viable coccoids were continuously observed. In the amoxicillin-treated group, the culturability decreased rapidly after 12 h, and showed a viable but non culturable (VBNC) state after the third day. Most cells treated with S. mitis supernatant changed to coccoid forms after 7 days, but colonies were continuously formed, probably due to living spiral forms. We performed proteomics to analyse the differences in protein profiles between the spiral and coccoid forms and protein profiles among the coccoid forms generated by the three methods.ConclusionAmoxicillin treatment changed H. pylori to VBNC cells faster than starvation. Treatment with the S. mitis supernatant prolonged the culturability of H. pylori, suggesting that the S. mitis supernatant may contain substances that support spiral form maintenance. Proteomic analysis revealed that the expression of proteins differed between the spiral form and coccoid form of H. pylori, and this variation was observed among the coccoid forms produced via three different methods. The proteins in the coccoid forms produced by the three methods differed from each other, but common proteins were also observed among them.

Comparative evaluation of chlorhexidine gluconate with alcohol and polyhexamethylene biguanide with Tris-EDTA as antiseptic solutions for pre-operative skin preparation in dogs

Background and Aim: Skin antisepsis plays a crucial role in pre-operative skin preparation, with chlorhexidine gluconate and alcohol being historically the preferred choice. However, concerns have risen regarding the development of bacterial resistance to chlorhexidine. Polyhexamethylene biguanide (PHMB) combined with Tris-ethylenediaminetetraacetic acid (Tris-EDTA) has recently emerged as a skin and wound antiseptic. This study aimed to compare the antibacterial efficacy and local safety of 2% chlorhexidine gluconate with 70% alcohol (CG+Alc) and 0.3% PHMB with 6% Tris and 1.86% EDTA (PHMB+Tris-EDTA) for pre-operative skin preparation in dogs. Materials and Methods: Twenty-four adult dogs underwent aseptic preparation on both sides of their ventral abdomens, with one side receiving CG+Alc and the other side receiving PHMB+Tris-EDTA, assigned randomly. Skin swab samples were collected pre-antisepsis and at 3-, 10-, and 60-min post-antisepsis to quantify bacterial colony-forming units (CFUs). Local skin reactions (erythema and edema) were evaluated after hair clipping, pre-antisepsis, and at 3-, 10-, 30-, and 60-min post-antisepsis. Results: There was no significant difference in bacterial CFU counts between the two antiseptic groups pre-antiseptic. Both solutions significantly reduced CFU counts (p < 0.05) at all post-antisepsis sampling times compared with pre-antisepsis. However, dogs treated with PHMB+Tris-EDTA showed a significantly higher incidence of edema at 10 min (p = 0.02) and 30 min (p = 0.003) and a higher incidence of erythema at 10 min (p = 0.043) post-antisepsis compared with CG+Alc. No skin reactions were observed in either group at 60 min post-antisepsis. Conclusion: CG+Alc and PHMB+Tris-EDTA reduced bacterial counts in pre-operative skin preparation in dogs. However, acute transient skin reactions were observed more frequently following the application of PHMB+Tris-EDTA. Keywords: alcohol, antisepsis, chlorhexidine gluconate, dogs, polyhexamethylene biguanide, skin preparation, tris-ethylenediaminetetraacetic acid.

Tooth-Binding Graphene Quantum Dots Silver Nanocomposites for Prevention of Dental Caries.

The objectives of this study were to develop a tooth-binding graphene quantum dots silver nanocomposites (ALN-GQDs-Ag) and evaluate their antibacterial, mineralising, and discolouring properties for the prevention of dental caries. In this study, ALN-GQDs-Ag were developed by synthesising nano silver (Ag) with graphene quantum dots (GQDs) and functionalised GQDs with alendronate (ALN). ALN-GQDs-Ag were characterised by transmission electron microscopy (TEM), zeta potential analysis, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. The cytotoxicity of ALN-GQDs-Ag against human gingival fibroblasts (HGF-1) and stem cells from human exfoliated deciduous teeth (SHED) was examined using a colorimetric assay with reference to silver nitrate solution. The affinity of ALN-GQDs-Ag for hydroxyapatite particles was investigated using inductively coupled plasma spectroscopy (ICP). The antibacterial properties of ALN-GQDs-Ag against Streptococcus mutans were evaluated by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and colony-forming units counting (CFUs). The mineralisation properties of ALN-GQDs-Ag on human dentine were assessed using micro-computed tomography (micro-CT), scanning electron microscopy (SEM), and Fourier transform infrared in a biochemical cycling model. The discolouring properties of ALN-GQDs-Ag on artificial dentine caries were determined using spectrophotometry. TEM, Zeta potential, XPS, FTIR, and Raman spectroscopy confirmed the synthesis of stable spherical ALN-GQD-Ag nanocomposites with a 10.3 ± 5.5 nm diameter. The colorimetric assay demonstrated that ALN-GQDs-Ag were less cytotoxic than silver nitrate to HGF-1 and SHED (p<0.001). ICP showed that ALN-GQDs-Ag were bound to hydroxyapatite. SEM, CLSM, and CFUs showed that ALN-GQDs-Ag was bactericidal and inhibited biofilm growth of Streptococcus mutans. Micro-CT, SEM, and FTIR showed that ALN-GQDs-Ag repressed dentine demineralisation under a cariogenic challenge. Spectrophotometry revealed no significant discolouration of dentine caries in the ALN-GQDs-Ag. This study developed a biocompatible and tooth-binding ALN-GQDs-Ag with promising antibacterial, mineralising, and non-discolouring properties. ALN-GQDs-Ag could be a novel anti-caries agent for preventing dentine caries if translated for clinical use.

Nanostructured silver vanadate gel: Evaluation of physicochemical, mechanical, and antibiofilm properties against a five-species oral model

The aim of this study was to develop a gel with nanostructured silver vanadate decorated with silver nanoparticles (β-AgVO3) and to evaluate its physicochemical, mechanical, and antibiofilm properties against a five-species oral model composed of Candida albicans, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa. The formulations were prepared with 0 % w/v (G0), 0.02 % w/v (G1), 0.05 % w/v (G2), and 0.12 % w/v (G3) β-AgVO3. The physicochemical properties of pH (n = 5), viscosity (n = 5), spreadability (n = 10) and syringability (n = 10) were evaluated, as well as the mechanical properties of hardness, cohesiveness, compressibility, elasticity, and adhesiveness using the texture profile assay (TPA) (n = 5) and ex vivo mucoadhesion (n = 5). The antibiofilm effect was assessed by counting colony-forming units (CFU/mL) (n = 8), metabolic activity (XTT) (n = 8), and scanning electron microscopy (SEM) (n = 1), using 0.12 % chlorhexidine gel (CG) as a positive control and G0 as a negative control. All groups demonstrated a neutral pH. G2 and G3 exhibited reduced viscosity and syringeability, along with increased spreadability. The incorporation of β-AgVO3 did not impact the gel's elasticity or cohesiveness but led to a decrease in hardness, compressibility, and adhesiveness. Ex vivo mucoadhesion was not altered by the addition of the nanomaterial. G3 showed a higher microbial reduction compared to GC and G0. It was concluded that the formulations with β-AgVO3 showed compatible physicochemical and mechanical properties for application and maintenance in the oral cavity. G3 showed superior antimicrobial effect against all microorganisms compared to GC and G0.

The anti-caries effects of copper tetraamine fluoride on enamel: An in vitro study

ObjectiveTo investigate the antibacterial, remineralising, and discolouring effects of copper tetraamine fluoride (CTF) on artificial enamel caries. MethodHuman enamel blocks with artificial caries were treated with CTF, silver diamine fluoride (SDF, positive control) and water (negative control) before being challenged with Streptococcus mutans. The morphology, viability, and growth kinetics of biofilm were evaluated using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and colony-forming unit (CFU) counting. The lesion depths, mineral loss, micro-hardness, and crystal characteristics were assessed using micro-computed tomography, Knoop Hardness Tester, and X-ray diffraction (XRD), respectively. The discolouring property was assessed by spectrophotometry. ResultsSEM showed that bacteria completely covered the enamel surface treated with water, but not the enamel surface treated with CTF and SDF. CLSM showed dead-to-live ratio of biofilm treated with CTF, SDF and water were 0.8 ± 0.1, 0.9 ± 0.2 and 0.5 ± 0.1 (p < 0.001, CTF, SDF>Water). Log10 CFU values of biofilm treated with CTF, SDF and water were 7.7 ± 0.2, 7.7 ± 0.1 and 8.1 ± 0.1 (p < 0.001, CTF, SDF<Water). Lesion-depths (μm) of the CTF, SDF and water groups were 93 ± 6, 92 ± 7 and 152 ± 29 (p < 0.001, CTF, SDF<Water). Mineral loss (gHApcm−3) of the CTF, SDF and water groups were 0.4 ± 0.1, 0.4 ± 0.2 and 1.0 ± 0.2 (p < 0.001, CTF, SDF<Water). Knoop hardness of the CTF, SDF and water groups were 70 ± 23, 68 ± 16 and 21 ± 10 (p < 0.05, CTF, SDF>Water). XRD revealed well-crystallised hydroxyapatite in enamel treated with CTF and SDF, but not water. Spectrophotometry showed ΔE values of the CTF, SDF and water groups were 5 ± 3, 54 ± 6 and 6 ± 2 (p < 0.001, CTF, water<SDF). ConclusionCTF inhibited Streptococcus mutans biofilm and remineralised artificial enamel caries without discolouration. Clinical significanceIf CTF is successfully translated into clinical use, it can be a simple agent for clinicians to arrest enamel caries.

Laser NIR Irradiation Enhances Antimicrobial Photodynamic Inactivation of Biofilms of Staphylococcus aureus.

Photodynamic inactivation (PDI) is a powerful technique for eradicating microorganisms, and our group previously demonstrated its effectiveness against planktonic cultures of Staphylococcus aureus bacteria using 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and visible light irradiation. However, biofilms exhibit a lower sensitivity to PDI, mainly due to limited penetration of the photosensitizer (PS). In the context of emerging antibacterial strategies, near-infrared treatments (NIRTs) have shown promise, especially for combating resistant strains. NIRT can act either through photon absorption by water, causing a thermal effect on bacteria, or by specific chromophores without a significant temperature increase. Our objective was to enhance biofilm sensitivity to TAPP-PDI by pretreatment with NIRT. This combined approach aims to disrupt biofilms and increase the efficacy of TAPP-PDI against bacterial biofilms. In vitro biofilm models of S. aureus RN6390 were utilized. NIRTs involved a 980 nm laser (continuous mode, 7.5 W/cm2, 30 s, totaling 225 J/cm2) post-TAPP exposure to enhance photosensitizer accumulation. Subsequent visible light irradiation at 180 J/cm2 was employed to perform PDI. Colony-forming unit counts evaluated the synergistic effect on bacterial viability. Scanning electron microscopy visualized the architectural changes in the biofilm structure. TAPP was extracted from bacteria to estimate the impact of NIRT on biofilm penetration. Using in vitro biofilm models, NIRT application following biofilm exposure to TAPP increased PS accumulation per bacteria. Under these conditions, NIRT induced a transient increase in the temperature of PBS to 46.0 ± 2.6°C (ΔT = 21.5°C). Following exposure to visible light, a synergistic effect emerged, yielding a substantial 4.4 ± 0.1-log CFU reduction. In contrast, the PDI and NIRT treatments individually caused a decrease in viability of 0.9 ± 0.1 and 0.8 ± 0.2-log respectively. Interestingly, preheating TAPP-PBS to 46°C had no significant impact on TAPP-PDI efficacy, suggesting the involvement of thermal and nonthermal effects of NIR action. In addition to the enhanced TAPP penetration, NIRT dispersed the biofilms and induced clefts in the biofilm matrix. Our findings suggest that NIR irradiation serves as a complementary treatment to PDI. This combined strategy reduces bacterial numbers at lower PS concentrations than standalone PDI treatment, highlighting its potential as an effective and resource-efficient antibacterial approach.

Surface characteristics, cytotoxicity, and microbial adhesion of 3D-printed hybrid resin-ceramic materials for definitive restoration

ObjectiveThis study investigated the surface properties, cytotoxicity, and microbial adhesion of 3D-printed specimens made from hybrid resin-ceramic materials intended for use in definitive crowns. MethodsDisc-shaped specimens were 3D-printed using six different hybrid resin-ceramic materials recommended for definitive restorations: Crowntec (CT), VarseoSmile Crown Plus (VS), Tera Harz TC-80DP Graphy (TH), C&B Permanent ODS (CB), Formlabs Permanent Crown (FP), and HeyGears (HG). Surface topography, surface roughness, and water contact angle values were measured for each material (n = 6). Cytotoxicity was assessed using direct contact and extract tests on human gingival fibroblasts (n = 4). Additionally, the adhesion of mixed oral bacteria to the surfaces of the specimens was evaluated by counting colony-forming units (CFUs) after a 2-hour incubation period (n=6). ResultsThe TH group exhibited significantly lower surface roughness (Ra: 0.28 ± 0.13 μm) compared to the other materials (CT: 1.87 ± 0.34 μm; VS: 1.13 ± 0.09 μm; CB: 2.91 ± 0.27 μm; FP: 2.50 ± 0.08 μm; HG: 1.50 ± 0.55 μm). The VS group had the highest water contact angle (129.5 ± 1.1°), indicating greater hydrophobicity, in contrast to the other groups (CT: 72.6 ± 2.1°; TH: 75.0 ± 0.3°; CB: 69.1 ± 0.2°; FP: 93.0 ± 1.6°; HG: 77.7 ± 0.3°). Cytotoxicity testing showed no harmful effects, as relative cell viability exceeded 70%, and lactate dehydrogenase (LDH) release remained below 30% for all materials. The TH specimens also demonstrated the lowest bacterial adhesion. ConclusionsThe surface characteristics of the tested resin-ceramic materials varied significantly, with TH showing the smoothest surface and the least bacterial adhesion. All materials were found to be non-toxic. Therefore, TH material has the potential to provide definitive restorations with less microbial adhesion. Clinical SignificanceThe type of resin-ceramic material significantly affects the surface properties of 3D-printed specimens. These findings are crucial for selecting the appropriate resin-ceramic material for definitive restorations.

Development and evaluation of a standardised sampling protocol to determine the effect of cleaning in the pig sty

BackgroundAll-in, all-out with strict hygienic routines is necessary in modern pig production. Furthermore, a standardised, validated method is needed to quantitatively control the effect of these hygiene protocols. This study aimed to establish a reproducible and reliable sampling method to assess cleaning of the pig pen.MethodsSterilised pig faeces were mixed with indicator bacteria (i.e. Enterococcus hirae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus) and spread out in a controlled environment. The retrieval rate of three different sampling methods were evaluated; swabbing by (i) a cloth and (ii) a sponge, analysed by standardised bacterial culture and counting of colony-forming units, and (iii) a cotton swab analysed by adenosine triphosphate (ATP) bioluminescence. Two time-points were evaluated during the study; after drying overnight and after manual scraping of the surfaces. To determine sample-to-sample variability, sampling by the cloth and the cotton swab was carried out after manual scraping and further, after high-pressure washing with cold water.ResultsSampling by the cloth and the sponge showed few differences in in the number of CFU obtained before and after the manual scraping (retrieval rate), whereas the swabs, measuring ATP bioluminescence, showed a very high retrieval rate. Sample-to-sample variability was low for all three methods.ConclusionsIn conclusion, to sample pens for the presence of bacteria, the cloth was assessed as the preferable material, being cheap, easy, specific, and approachable, and with a low sample-to-sample variability. The ATP measurement could have potential for use when evaluating the cleaning of stables, however, threshold values for evaluating the cleaning of a pig sty needs to be developed.

Effect of fumigants on inoculum of Neopestalotiopsis spp. in strawberry crowns and soil.

Florida's strawberry industry has been facing an emerging threat after several outbreaks of an aggressive Neopestalotiopsis sp. affecting fruit, leaf, and crown caused severe yield losses. Our studies found Neopestalotiopsis sp. can survive from one production season to the next on crop debris in soil, which led us to evaluate the effect of fumigants on resident inoculum in strawberry crowns and soil. Different rates of 1,3-dichloropropene, chloropicrin, and metam potassium were applied to pasteurized soil inside glass jars containing infected strawberry crowns. Soil samples were taken after broadcast and bed fumigation treatments with a combination of 1,3-dichloropropene/chloropicrin at ratios of 63:35 were applied at four commercial fields. Crowns and soil samples were processed and plated on a semi-selective medium for Neopestalotiopsis spp. and colony-forming units (CFU) were counted 5 days after plating. CFU counts in crowns treated with 1,3-dichloropropene, chloropicrin, and metam potassium decreased significantly as rates increased and were described by exponential decay models. CFUs were not recovered in most of the soil samples from fumigated strawberry beds after broadcast or bed fumigation. However, CFUs were found in non-fumigated row middles between fumigated beds which can serve as a source of inoculum to start new epidemics. Chloropicrin, 1,3-dichloropropene, and metam potassium were effective on reducing Neopestalotiopsis spp. inoculum in strawberry crowns and soil, providing new evidence on the fungicidal activity of 1,3-dichloropropene. Broadcast fumigation with 1,3-dichloropropene/chloropicrin at ratios of 63:35 could potentially be used to reduce inoculum of Neopestalotiopsis sp. in severely infested fields.

69 Urinary tract infections in preterm infants: Review in two Canadian tertiary centers

Abstract Background Urinary tract infections (UTI) in premature infants are commonly seen in the neonatal intensive care unit (NICU). The reported prevalence of UTI in this population ranges from 1.9-12.2% due to the lack of standardized criteria for UTI among infants less than two months of age. Enterococcus and Enterobacteriaceae are well known to cause UTI and their resistance pattern has been reported to be rising in the neonatal nosocomial infections worldwide. Objectives To describe the epidemiology, microbiology, and antimicrobial treatment for UTI in premature infants based on different diagnostic criteria. Design/Methods This retrospective study (2019-2022) included infants born at &amp;lt;33 weeks of gestational age (GA) with positive urine cultures and ≥5 days of antimicrobials treatment. We collected data from two tertiary level NICUs in Canada, including demographics, microbial susceptibility patterns, urinalysis and imaging results, and antibiotic treatment. Results We identified 43 infants managed as UTI based on physicians’ discretions. The median GA at birth was 26-27 weeks, while the median onset of first UTI ranged 39-80 days, with male predominance [Table 2]. Mixed growth of organisms constituted 21-42% of cases of UTI [Table 1]. Among UTI cases with only single growth organism, we found 53.5% and 18.6% cases fulfilled UTI diagnostic criteria based on the concentration of colony forming units (CFU) of &amp;gt;104/mL or &amp;gt;107/L and &amp;gt;105/mL or &amp;gt;108/L, respectively [Table 1]. Of these cases, 67.4% of infants had urinalysis sent with the culture, and the higher the concentration of CFU, the more likely the samples showed positive nitrites on urinalysis, with 100% correlation among those with CFU &amp;gt;105/mL or &amp;gt;108/L. [Table 1] The most common pathogens were Enterococcus species, E.Coli, Klebsiella species, Citrobacter species and Enterobacter cloacae complex [Table 2]. Among gram negative bacilli (E. Coli and Klebsiella species), greater than 42-80% were resistant to 3rd generation cephalosporins (3GC). We did not identify any vancomycin-resistant Enterococcus strains. Median duration of antimicrobial treatment ranged from 8-13 days, depending on the diagnostic criteria applied. At least 10% of infants with UTI showed abnormalities on kidney ultrasound [Table 2]. Conclusion There exists a large variation in UTI prevalence according to different diagnostic criteria. Abnormal urinalysis with presence of nitrites were frequently associated with UTI of high CFU count. High resistance to 3GC among gram negative bacilli was concerning and carried an impact on the appropriate empirical treatment choice.

Effectiveness of Activated Sodium Hypochlorite Irrigation by Shock Wave-Enhanced Emission Photoacoustic Streaming, Sonic and Ultrasonic Devices in Removing Enterococcus faecalis Biofilm From Root Canal System.

Aim: To compare shock wave-enhanced emission photoacoustic streaming (SWEEPS) with sonic- and ultrasonically activated irrigation systems in removing Enterococcus faecalis biofilm from the root canal system. Methodology: Fifty human single-canalled mandibular premolars were included in the study. After access cavity preparation, the root canals were prepared to a standardized size and taper. Then, the entire root surface was covered with two layers of resin, and the root apices were sealed before sterilization. All root canals were inoculated with E. faecalis biofilm, and the samples were incubated aerobically for 2 weeks at 37 °C. Biofilm formation was confirmed by scanning electron microscopy. All samples were randomly divided into five groups (n = 10 each) based on their irrigation activation method as A (no treatment or negative control), B (no irrigation or positive control), C (sonically activated irrigation (SAI)), D (ultrasonically activated irrigation (UAI)), and E (needle irrigation activated by an Er: YAG laser device using a SWEEPS quartz tip (SWEEPS)). Then, dentine chips were retrieved, vortexed, and diluted for colony-forming unit counts. Data were analysed using analysis of variance and post-hoc Tukey tests (α = 5%). Results: All methods could significantly reduce E. faecalis biofilm compared with control so that the UAI, SWEEPS, and SAI groups indicated a 23.54%, 14.89%, and 7.81% biofilm reduction, respectively. UAI demonstrated a significantly more effective reduction of E. faecalis biofilm than SAI (p = 0.004). Conclusions: All irrigation activation methods significantly reduced E. faecalis biofilm, with ultrasonic use being the most effective.

Bacterial Adhesion and In Situ Biodegradation of Preheated Resin Composite Used as a Luting Agent for Indirect Restorations.

To evaluate surface roughness and bacterial adhesion after in situ biodegradation of the cementation interface of indirect restorations cemented with preheated resin composite. Resin composite blocks (Z250XT/3M ESPE) were cemented to bovine enamel (7 × 2.5 × 2 mm) using preheated microhybrid resin composites: (1) Filtek Z100 (3M ESPE) (Z100); (2) Gradia Direct X (GC America) (GDX); and (3) Light-cured resin cement RelyX Veneer (3M ESPE) (RXV) (n = 21). The resin composites were preheated on a heating device (HotSet, Technolife) at 69°C for 30 minutes. Disk-shaped specimens (7 x 1.5 mm) were made for biodegradation analysis with the luting agents (n = 25). The in situ phase consisted of 20 volunteers' using an intraoral palatal device for 7 days. Each device had six cylindrical wells for the blocks and the disk-shaped specimens. Biodegradation was evaluated through surface roughness (Ra), scanning electron microscopy (SEM) micromorphological analysis, and colony-forming unit (CFU) count. The film thickness of the luting agents was also measured under stereomicroscopy. Increased surface roughness was observed after the cariogenic challenge without differences between the luting agents. Higher variation and surface flaws suggestive of particulate detachment were observed for Z100. No differences were observed in CFU counts. All materials underwent surface biodegradation, and the surface roughness of the resin cements was similar to or lower than that of the preheated resin composites. The resin composites' film thickness was thicker than that of the resin cement. Clinicians should be aware of these factors when choosing the use of preheated resin composite since it can lead to reduced longevity of the cementation interface and, therefore, restorations.

A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor

BackgroundFrancisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.MethodsIn order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.ResultsAs a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.ConclusionsIn this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.

Optical trapping of lipid bodies reveals increased cytoplasm viscosity in Candida tropicalis post antimicrobial photodynamic therapy

Abstract Antimicrobial photodynamic therapy (aPDT) is a common treatment for large cell colonies, but its effectiveness is typically assessed through colony-forming unit counting, which lacks microscopic details about cell death. This study monitors the trap stiffness of optically trapped lipid bodies of Candida tropicalis of approximately 1 μm of radius following aPDT treatment. Methylene blue served as the photosensitizer at 10 μM concentration, with a lethal light dose of 60 J cm-². The results revealed a significant increase in viscosity after aPDT treatment. Additionally, image analysis confirmed substantial morphological changes indicative of cell death. These findings demonstrate the potential of optical tweezers as a non-invasive tool for assessing cellular health by providing both functional (viscosity) and morphological data on the response to aPDT.&amp;#xD;

Synthesis of metal nanoparticles on graphene oxide and antibacterial properties.

Pathogen-induced infections and the rise of antibiotic-resistant bacteria, such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), pose significant global health challenges, emphasizing the need for new antimicrobial strategies. In this study, we synthesized graphene oxide (GO)-based composites functionalized with silver nanoparticles (AgNPs) and copper nanoparticles (CuNPs) as potential alternatives to traditional antibiotics. The objective is to assess the antibacterial properties of these composites and explore their efficacy against E. coli and S. aureus, two common bacterial pathogens. The composites are prepared using eco-friendly and conventional methods to ensure effective nanoparticle attachment to the GO surface. Structural and morphological characteristics are confirmed through SEM, AFM, EDS, XRD, UV-vis, FTIR, and Raman spectroscopy. The antibacterial efficacy of the composites is tested through disk diffusion assays, colony-forming unit (CFU) counts, and turbidimetry analysis, with an emphasis on understanding the effects of different nanoparticle concentrations. The results demonstrated a dose-dependent antibacterial effect, with GO/AgNP-1 showing superior antibacterial activity over GO/AgNP-2, particularly at lower concentrations (32.0μg/mL and 62.5μg/mL). The GO/CuNP composite also exhibited significant antibacterial properties, with optimal performance at 62.5μg/mL for both bacterial strains. Turbidimetry analysis confirmed the inhibition of bacterial growth, especially at moderate concentrations, although slight nanoparticle aggregation at higher doses reduced efficacy. Lastly, both GO/AgNP and GO/CuNP composites demonstrated significant antibacterial potential. The results emphasize the need to fine-tune nanoparticle concentration and refine synthesis techniques to improve their efficacy, positioning these composites as strong contenders for antimicrobial use.

Rapid growth rate of Enterobacter sp. SM3 determined using several methods

BackgroundBacterial growth rate, commonly reported in terms of doubling time, is frequently determined by one of two techniques: either by measuring optical absorption of a growing culture or by taking samples at different times during their growth phase, diluting them, spreading them on agar plates, incubating them, and counting the colonies that form. Both techniques require measurements of multiple repeats, as well careful assessment of reproducibility and consistency. Existing literature using either technique gives a wide range of growth rate values for even the most extensively studied species of bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. This work aims to apply several methods to reliably determine the growth rate of a recently identified species of Enterobacteriaceae, called Enterobacter sp. SM3, and to compare that rate with that of a well-known wildtype E. coli strain KP437.ResultsWe extend conventional optical density (OD) measurements to determine the growth rate of Enterobacter sp. SM3. To assess the reliability of this technique, we compare growth rates obtained by fitting the OD data to exponential growth, applying a relative density method, and measuring shifts in OD curves following set factors of dilution. The main source of error in applying the OD technique is due to the reliance on an exponential growth phase with a short span. With proper choice of parameter range, however, we show that these three methods yield consistent results. We also measured the SM3 division rate by counting colony-forming units (CFU) versus time, yielding results consistent with the OD measurements. In lysogeny broth at 37oC, SM3 divides every 21 ± 3 min, notably faster than the RP437 strain of E. coli, which divides every 29 ± 2 min.ConclusionThe main conclusion of this report is that conventional optical density (OD) measurements and the colony-forming units (CFU) method can yield consistent values of bacterial growth rate. However, to ensure the reproducibility and reliability of the measured growth rate of each bacterial strain, different methods ought to be applied in close comparison. The effort of checking for consistency among multiple techniques, as we have done in this study, is necessary to avoid reporting variable values of doubling time for particular species or strains of bacteria, as seen in the literature.

Antimicrobial evaluation of root canal irrigants of natural sources with and without gamma radiation activation (in vitro study)

ABSTRACT Aims: The present study aimed to evaluate the antibacterial effectiveness of newly formulated root canal irrigants derived from natural sources. In particular, this study investigated how these irrigants’ antimicrobial properties were affected by gamma radiation activation. Materials and Methods: Fifty-three single-rooted extracted human teeth with straight canals were selected, fully prepared, and sterilized. The roots were then infected with Escherichia coli, Enterococcus faecalis, and Candida albicans for 21 days at 37°C. Based on the irrigating solution utilized, the roots were divided into seven groups: Group I: chitosan irrigant, Group II: chitosan irrigant gamma irradiated, Group III: curcumin irrigant, Group IV: curcumin irrigant gamma irradiated, Group V: sodium hypochlorite (NaOCl) 5% irrigant, Group VI: positive control (infected roots without treatment), and Group VII: negative control (uncontaminated roots). After cleaning and shaping, microbiological sampling was performed. The samples were then transferred to sterile centrifuge tubes containing 1 mL of saline to determine the microbial growth. Colony-forming unit counts were employed for assessing the antimicrobial action. Statistical analysis was conducted to compare the percentage reduction in the microbial count from the initial count. Results: Group V and Group IV showed significantly higher values in microbial reduction than Group III and Group I. In addition, the results showed that Group II and Group III have significantly higher values than Group I. Conclusion: Natural irrigating solutions exposed to gamma radiation exhibited promising antimicrobial activity that is comparable to that of NaOCl and may be used as effective antimicrobial agents in endodontic treatment.

A-243 Quantitative Bacteriology Parameters for Seal-Packaged Agar Plates Challenged with Experimental Low-resource Tropical Field Clinic Stress

Abstract Background InTray® Mueller Hinton Chocolate (MHC) and Colorex™ Screen (CS) plates are in vitro diagnostic devices combining prepared bacteriological agar-media with Biomed’s ‘InTray®’ format (a double sealed-packaging cassette system that provides extended shelf-life 12 months past the date of manufacture). The devices are intended for use with clinical specimens to culture, isolate and identify potentially pathogenic bacteria. This study evaluated MHC and CS device performance at 7 and 13 months past the date of manufacture after product was experimentally challenged with stress replicating a low-resource tropical field clinic scenario. Methods MHC and CS plates were exposed to high temperature (40.5°C ± 0.5) and high humidity (74.5 ± 2.5% relative humidity) for an extended time-period (18 days). After the 18 days of heat treatment, the stress-group plates were stored in the lab at working room temperature (20-25°C) for the remainder of the study. Quantitative differences between control-groups (stored under refrigeration: 2-8°C) and heat-stress treatment groups for both MHC and CS were examined via CFU/mL measurements comparing three different lab-strain bacteria chosen based on clinical sub-culturing prevalence from blood bottles; Escherichia coli (ATCC 25922), Streptococcus pneumoniae (ATCC 6305), and Staphylococcus aureus (ATCC 25923). Briefly, 24-hour (18-30 h) old bacterial colony suspensions (grown on MHC) were prepared in 0.85% saline solution, adjusted to a 0.5 McFarland standard (here defined as 0.08-0.96 Absorbance @ 625 nm), measured in triplicate and diluted. A 20 μL volume from the lowest dilution (2.5 x 103 CFU/mL) was then spread-plated to each plate in order to count colony-forming units (CFU) in the acceptable range of 25-250. Results Quantitative CFU/mL differences between control-groups (stored under refrigeration: 2-8°C) and heat-stress-treatment groups (&amp;gt;40°C and high humidity) for MHC and CS for three different test bacteria (listed above) were not statistically significant when tested at 13 months. The CS stress and control group comparison for S. pneumoniae with a p value &amp;lt; 0.05 at 7 months had a p value &amp;gt; 0.05 at 13 months, suggesting that the difference between the CFU counts may not be significant in biological terms. Conclusions In conclusion, control and heat-stress treatment groups for both MHC and CS met acceptable performance criteria as expected for the products, 13 months after the date of manufacture.