Colony-forming Unit Counts Research Articles

Contamination of Dental Surgical Masks by Aerosols Generated During Different Dental Treatments.

The COVID-19 pandemic raised the question about the extent of microbial exposure encountered by dentists during dental therapy. The purpose of this study was to quantify microbial counts on surgical masks related to duration and type of dental therapy, as well as patient oral health variables. Sterile filter papers were fixed on surgical masks used during routine daily dental therapy. Thereafter, the filter papers were pressed onto blood agar plates for 1 min, before the agar plates were incubated with 10% CO2. After 48 h, the colony forming units (CFU) were counted and microorganisms were identified. The dependence of the CFU counts on treatment and patient-related variables was analysed using linear regression. Filter papers obtained from 322 dental treatments (429 masks) were included in the final analysis. On average, 5.41 ± 9.94 CFUs were counted. While mostly oral bacteria were detected, Staphylococcus aureus was also identified on 16 masks. Linear regression, incorporating patient-related and treatment characteristics through step-wise inclusion, revealed statistical significance (p 0.001) only with the variable 'assistance during therapy'. The type of dental treatment exhibited a trend, with fewer CFUs observed in caries treatment compared to periodontal or prosthodontic therapy. Furthermore, after analysing filter papers from masks used by dental assistants in 107 dental treatments, fewer CFUs were found on the masks compared to those used by dentists (p 0.001). The mean number of CFUs observed consistently remained low, highlighting the efficacy of the implemented hygiene measures. Consequently, it is clinically recommended to support dental treatment with precise suction of the generated aerosols.

Remineralising enamel caries with a novel peptide: An in vitro study

ObjectiveTo evaluate the antibacterial and remineralising effects of GAPI peptide on artificial enamel caries. MethodsHuman enamel blocks were exposed to Streptococcus mutans biofilm to create artificial carious lesions. The blocks were randomly assigned to either the GAPI treatment group or the deionised water control group, treated twice daily for 21 days. The viability, growth kinetics, and morphology of S. mutans biofilms were assessed using confocal laser scanning microscopy (CLSM), colony-forming unit (CFU) counting, and scanning electron microscopy (SEM). Lesion depth, mineral loss, calcium-to-phosphorus ratio, Knoop hardness, enamel surface morphology, and crystal characteristics of enamel lesions were determined using micro-computed tomography (Micro-CT), SEM-energy dispersive spectroscopy (EDS), a microhardness tester, SEM, and X-ray diffraction (XRD). ResultsCLSM showed that the dead-to-live ratio of S. mutans was 0.8±0.1 in the GAPI group and 0.4±0.1 in the control group (p<0.001). The Log CFUs were 6.9±0.7 in the GAPI group and 8.1±0.5 in the control group (p=0.002). SEM revealed confluent growth of S. mutans in the control group but not in the GAPI group, which also exhibited cell damage. Micro-CT showed that the lesion depth (µm) was 142±11 in the GAPI group and 178±20 in the control group (p<0.001), with mineral loss (gcm⁻³) of 1.1±0.1 and 1.5±0.1, respectively (p<0.001). SEM-EDS indicated that the calcium-to-phosphorus ratio was 1.71±0.02 in the GAPI group and 1.67±0.03 in the control group (p=0.006). Additionally, Knoop hardness was 302±22 in the GAPI group and 242±17 in the control group (p<0.001). SEM revealed an orderly pattern of enamel rods in the GAPI group, and XRD showed better crystallisation of hydroxyapatite in the GAPI group compared to the control group. ConclusionGAPI exhibits antibacterial and remineralising properties against artificial enamel caries. Clinical SignificanceIf the anti-caries properties of GAPI are confirmed in clinical studies, it could be used for caries prevention.

Mycobacterial lipoarabinomannan negatively interferes with macrophage responses to Aspergillus fumigatus in-vitro.

Over 1 million people have chronic pulmonary aspergillosis (CPA) secondary to pulmonary tuberculosis. Additionally, Aspergillus fumigatus ( Af ) has been reported as one of the most common pathogens associated with mycobacteria in patients with cystic fibrosis. Mycobacterial virulence factors, like lipoarabinomannan, have been shown to interfere with host's intracellular pathways required for an effective immune response, however, the immunological basis for mycobacterial-fungal coinfection is still unknown. We therefore investigated the effect of lipoarabinomannan on macrophage responses against Af . Bone marrow-derived macrophages (BMDMs) were stimulated with non-mannose-capped lipoarabinomannan (LAM) from Mycobacterium smegmatis or mannose-capped lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis for 2 hours and then infected with swollen Af conidia. Cell death was assessed by lactate dehydrogenase release. Cytokine release was measured in supernatant using Enzyme Linked Immuno-Sorbent Assay (ELISA). Colony forming units counting and time-lapse fluorescence microscopy was performed for studying conidia killing by macrophages. BMDMs stimulated with LAM showed increased cell death and inflammatory cytokine release in a dose-dependent manner, characterised by a significant increase of IL-1β release. Time-lapse fluorescence microscopy and CFUs revealed that both LAM and ManLAM significantly decrease the capacity of macrophages to kill Af conidia within the first 6 hours of infection. The mycobacterial virulence factor, lipoarabinomannan, disrupts macrophage capacity to efficiently clear Af at early stages of infection in-vitro .

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 &lt; 0.05. Results: NaOCl significantly reduced CFU counts, demonstrating superior antimicrobial efficacy compared to NAC and GSE (P &lt; 0.01). In comparison to negative control and GSE (P &lt; 0.01), NAC showed significant antimicrobial activity, but not significantly different from GSE (P &gt; 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.

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 C. tropicalis of approximately 1 μm of radius following aPDT treatment. Methylene blue served as the photosensitizer at 20 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.

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 × 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.

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 &lt; 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.

Photodynamic therapy reduces viability, enhances itraconazole activity, and impairs mitochondrial physiology of Sporothrix brasiliensis

Sporothrix brasiliensis is the main agent of sporotrichosis in Brazil, with few therapeutic options. This study aimed to investigate the in vitro efficacy of photodynamic therapy using a diode laser (InGaAIP) in combination with the photosensitizer methylene blue against S. brasiliensis yeasts. Additionally, we evaluated the underexplored mitochondrial activity of S. brasiliensis and the impact of laser treatment on the fungal mitochondrial aspects post-treatment. Three strains of S. brasiliensis were used, including a non-wild-type strain to itraconazole. Yeast viability was determined by counting colony-forming units. For a comprehensive analysis of irradiated versus non-irradiated cells, we assessed combined therapy with itraconazole, scanning electron microscopy of cells, and mitochondrial activity. The latter included high-resolution respirometry, membrane potential analysis, and reactive oxygen species production. Methylene blue combined with photodynamic therapy inhibited the growth of the isolates, including the non-wild-type strain to itraconazole. Photodynamic therapy induced the production of reactive oxygen species, which negatively affected mitochondrial function, resulting in decreased membrane potential and cell death. Photodynamic therapy altered the ultrastructure and mitochondrial physiology of S. brasiliensis, suggesting a new therapeutic approach for sporotrichosis caused by this species.

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.

Mycobacterium tuberculosis short mutant H37Rv-S with reduced growth adaptability is more readily recognized by the host immune system.

Mycobacterium tuberculosis (Mtb) is the bacterium responsible for causing Tuberculosis (TB) and understanding its mechanisms of virulence, persistence, and pathogenesis is a global research priority. Attenuated strains of Mtb are valuable tools for investigating the genes and proteins involved in these processes. In this study, we identified an Mtb mutant, H37Rv-S, which exhibits a shorter mycelium, smoother colony, slower growth, and reduced antibiotics resistance compared to the wild-type strain H37Rv. Genomic sequencing revealed 34 mutation events in the coding regions of H37Rv-S, affecting 31 genes. We conducted TMT-labeling quantitative proteomics to compare the expression differences between H37Rv-S and H37Rv, as well as their infected bone marrow derived macrophages (BMDMs). The results showed that 716 protein groups (23.96%) in H37Rv-S and 115 protein groups (2.99%) in the infected BMDMs were differentially expressed. The dysregulated proteins in H37Rv-S correspond with its phenotype characteristics. Among the 31 affected genes in H37Rv-S, 10 showed upregulation and 1 showed downregulation at the protein level. Notable, 16 associated network proteins in the phoP/phoR system were significantly dysregulated due to a frameshift mutation in phoP, altering its protein sequence after Phe45. The dysregulated host proteins in H37Rv-S were associated with immune response, necroptosis, and ferroptosis. Additionally, H37Rv-S demonstrated reduced survival capability in strain fluorescence labeling and colony-forming unit (CFU) counting post-infection of BMDM cells. These findings suggest that H37Rv-S is an attenuated strain exhibiting defective phenotype characteristics and is more readily recognized and eliminated by the host. This enhanced understanding of the differences between virulent and attenuated strains could facilitate the development of new targets and therapeutics for TB prevention and treatment.

Preparation and characterization of microcapsules and tablets for probiotic encapsulation via whey protein isolate-nanochitin complex coacervation

This research delved into the feasibility of utilizing three nanochitin—chitin nanocrystal (CNC), chitin nanofiber (CNF), and chitin nanosphere (CNS) in complexation with whey protein isolate (WPI) to fabricate complex coacervation and create microcapsules for probiotic encapsulation. The results showed that CNC, CNF, and CNS exhibited notable differences in morphologies, dimensions, and properties due to the respective synthesis methodologies. Nevertheless, all of them maintained a positive charge and were capable of assembling into microcapsules with WPI via electrostatic interactions at optimal pHs. The inclusion of Lactobacillus casei (L. casei) during the complex coacervation phase engendered a shell-like formation around the bacterium within the microcapsule, which enhanced probiotic viability and increased colony-forming unit count. Additionally, these probiotic-loading microcapsules were also processed into tablets, displaying robust structural integrity, augmented protective capabilities, and a distinctive sustained-release profile compared to the microcapsules alone. In summary, this study pioneered the employment of nanochitin formulations in complex coacervation to encapsulate L. casei, spearheading an innovative approach to the creation of a compressed probiotic supplement and contributing to the advancement in the design and fabrication of encapsulation vehicles for active ingredients.

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.

Antibacterial and mineralising properties of copper tetraamine fluoride on dentine caries: A laboratory study

ObjectiveTo investigate the antibacterial, mineralising, and discolouring properties of copper tetraamine fluoride (CTF) on artificial dentine caries. MethodDemineralised human dentine blocks were treated with CTF, silver diamine fluoride (SDF, positive control) and water (negative control) before they were subjected to cariogenic challenge with Streptococcus mutans biofilm. The morphology, viability, and growth kinetics of the biofilm were assessed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and colony-forming unit (CFU) counting. The lesion depths, mineral loss, chemical structure, and crystal characteristics were measured using micro-computed tomography, Fourier transform infrared (FTIR), and X-ray diffraction (XRD). The discolouring property on demineralised dentine was assessed by spectrophotometry. One-way analysis of variance with the Bonferroni post hoc test was performed to assess and compare the data. ResultsSEM revealed confluent bacterial growth covering the surface of dentine treated with Water but not CTF and SDF. CLSM showed the dead-to-live ratio of the biofilm treated with CTF, SDF and Water were 0.7 ± 0.1, 0.8 ± 0.1 and 0.5 ± 0.1 (p < 0.001, CTF, SDF>Water). The Log10 CFU values of the biofilm treated with CTF, SDF and Water were 7.8 ± 0.1, 7.8 ± 0.1 and 8.1 ± 0.2 (p < 0.001, CTF, SDF<Water). Micro-computed tomography showed the Δlesion-depth (μm) of dentine treated with CTF, SDF and water were 33 ± 5, 40 ± 7 and 169 ± 17 (p < 0.001, CTF, SDF<Water). The Δmineral loss (gHApcm−3) of dentine treated with CTF, SDF and Water were 0.19 ± 0.04, 0.18 ± 0.04 and 0.30 ± 0.04 (p < 0.001, CTF, SDF<Water). FTIR showed the amide I-to-hydrogen phosphate ratios of dentine treated with CTF, SDF and Water were 0.23 ± 0.02, 0.24 ± 0.05, and 0.44 ± 0.06 (p < 0.001, CTF, SDF<Water). XRD revealed well-crystallised hydroxyapatite in the CTF and SDF groups. Spectrophotometry showed the ΔE values of the CTF, SDF and Water groups were 6 ± 4, 49 ± 3 and 10 ± 3 (p < 0.001, CTF, water<SDF). ConclusionCTF inhibited Streptococcus mutans. It mineralised but not discoloured artificial dentine caries. Clinical significanceIf CTF is successfully translated into clinical application, it may be a non-discolouring anti-caries agent for clinicians to arrest dentine caries.

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.

Bio-based polylactic acid labware as a sustainable alternative for microbial cultivation in life science laboratories

Single-use plastics (SUPs) in life science laboratories account for approximately 5.5 million tonnes of waste per year globally. Of SUPs used in life science laboratories, Petri dishes, centrifuge tubes, and inoculation loops are some of the most common. In order to reduce the reliance on petrochemical-based SUPs in the life science research laboratory and minimize the negative environmental impacts associated with SUPs, this research investigates the applicability of polylactic acid (PLA) in single-use labware as a replacement for petrochemical-based plastics. PLA is one of the most well-studied biodegradable plastics that can be produced from sustainable resources. Commercially available PLA was used to 3D print a select range of labware to test the suitability of PLA-based material for routine microbiology work. An injection moulded PLA-based Petri dish was also designed and produced, for increased optical clarity. The biocompatibility was tested against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus epidermidis) strains of bacteria. The PLA-based labware did not negatively impact the cell growth, viability, and metabolic activity of the bacterial cultures. The injection moulded PLA Petri dish showed a reduced colony forming unit count for the Gram-negative E. coli strain compared to the polystyrene Petri dish, ∼1.5 × 109 CFU/mL and ∼3.0 × 109 CFU/mL respectively, during late-exponential growth. The colony counts were, however, in the same order of magnitude. This observed difference may be due to the internal environment inside the Petri dish, hence the internal O2 concentration, humidity, and temperature during bacterial growth were investigated. This work demonstrates, for the first time, a full successful workflow of bacterial growth using a sustainable bioplastic, providing a pathway to reducing the environmental impacts of SUPs in life science laboratories.