Colony-forming Unit Counts Research Articles

Antimicrobial potency, prevention ability, and killing efficacy of daptomycin-loaded versus vancomycin-loaded β-tricalcium phosphate/calcium sulfate for methicillin-resistant Staphylococcus aureus biofilms.

Growing evidence has shown that the efficacy of systemic administration of daptomycin for the treatment of methicillin-resistant Staphylococcus aureus (MRSA)-related infections is satisfactory. However, the clinical efficacy of the local administration of daptomycin for the management of osteoarticular infections remains unclear. This in vitro study compared the efficacy of daptomycin and vancomycin against MRSA biofilms. The elution kinetics of daptomycin and vancomycin, combined with gentamicin and loaded with either β-tricalcium phosphate/calcium sulfate or calcium sulfate, in the presence of MRSA infection, was assessed. Their efficacy in preventing biofilm formation and killing pre-formed biofilms was assessed using colony-forming unit count and confocal laser scanning microscopy. In addition, the efficacy of daptomycin, vancomycin, and gentamicin in prophylaxis and eradication of MRSA biofilms was also evaluated. Daptomycin + gentamicin and vancomycin + gentamicin displayed similar antimicrobial potency against MRSA, by either β-tricalcium phosphate/calcium sulfate or calcium sulfate. In the prevention assays, both daptomycin + gentamicin and vancomycin + gentamicin showed similar efficacy in preventing bacterial colony formation, with approximately 6 logs lower colony-forming units than those in the control group at both 1 and 3 days. The killing effect on pre-formed biofilms showed significant decreases of approximately 4 logs at 1 and 3 days following treatment with daptomycin + gentamicin and vancomycin + gentamicin. In addition, the confocal laser scanning microscopy results support the colony-forming unit data. Moreover, single use of vancomycin and gentamicin showed similar efficacies in preventing and killing MRSA biofilms, both of which were better than that of gentamicin. Our study demonstrated that vancomycin + gentamicin and daptomycin + gentamicin loaded with β-tricalcium phosphate/calcium sulfate or calcium sulfate showed similar prophylactic and killing effects on MRSA biofilms, implying a potential indication of local administration daptomycin for the treatment of MRSA-associated osteoarticular infections, especially if vancomycin administration presents limitations.

Benefits and Challenges of the Use of Two Novel vB_Efa29212_2e and vB_Efa29212_3e Bacteriophages in Biocontrol of the Root Canal Enterococcus faecalis Infections.

E. faecalis ATCC 29212 strain was used for the trial. Two novel vB_Efa29212_2e and vB_Efa29212_3e virulent phages were isolated from urban wastewater and characterized. The E. faecalis biofilm was established in 15 bovine teeth for 21 days. Transmission (TEM) and scanning electron (SEM) microscopes with the colony-forming unit (CFU) counting were used for assessment. Isolated phages differed in morphology. Taxonomy for vB_Efa29212_2e (Siphoviridae, Efquatovirus) and for vB_Efa29212_3e (Herelleviridae, Kochikohdavirus) was confirmed. Both phages were stable at a temperature range of 4-50 °C and showed a different tolerance to chemicals: 15% EDTA, 1-3% sodium hypochlorite, and chlorhexidine. SEM analysis showed distortion of bacteria cells after phage inoculation, which proved the lytic activity against E. faecalis. A 54.6% reduction in the E. faecalis biofilm confirmed bacteriophage efficacy against isolates in the ex vivo model. Results strongly support the concept that phage therapy has a real therapeutic potential for the prevention and treatment of E. faecalis-associated infections.

Efficacy of Paracrine Factors from Mesenchymal Stem Cells for Treating Biofilm-Infected Wounds in a Murine Model

Background: Chronic wounds are a serious medical condition affecting over 6 million people in the United States. Biofilms, which alter the host immune response and establish a microenvironment that prevents wound healing, are intimately associated with the development of chronic wounds. Current treatment options do not specifically target the underlying molecular mechanisms of biofilm pathology. Paracrine factors secreted by mesenchymal stem cells (MSCs) have been demonstrated to play an important role in wound healing. The objective of this study was to examine the effects of the paracrine factors secreted from MSCs on reducing infection and accelerating wound closure in biofilm-infected wounds.Methods: MSCs were cultured by seeding on an extracellular matrix (ECM). The supernatant from MSCs containing paracrine factors were applied to mature <i>Pseudomonas aeruginosa</i> biofilms <i>in vitro</i> and the number of viable bacteria were quantitated. BALB/cJ mice were wounded and infected with <i>P. aeruginosa</i> biofilms and the paracrine factors from reprogrammed MSCs were applied topically. The wound surface area and colony forming units counts of the treatment group were compared to control (culture media) and biofilm (untreated) groups. Results: The paracrine factors from MSCs grown on ECM were found to reduce <i>P. aeruginosa</i> biofilm growth significantly (P<0.01). Wounds in the treatment group had lower bacterial counts and an increased rate of wound closure compared to non-treated mice wounds. Conclusion: The results indicate that paracrine factors from reprogrammed MSCs accelerated wound healing and reduced the bacterial burden in biofilm-infected wounds. Future studies are needed to further characterize this phenomenon.

Novel protein-repellent and antibacterial polymethyl methacrylate dental resin in water-aging for 6 months

BackgroundThe present study aimed to develop a novel protein-repellent and antibacterial polymethyl methacrylate (PMMA) dental resin with 2-methacryloyloxyethyl phosphorylcholine (MPC) and quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM), and to investigate the effects of water-aging for 6 months on the mechanical properties, protein adsorption, and antibacterial activity of the dental resin.MethodsFour groups were tested: PMMA control; PMMA + 3% MPC; PMMA + 1.5% DMAHDM; and PMMA + 3% MPC + 1.5% DMADDM in acrylic resin powder. Specimens were water-aged for 1 d, 3 months, and 6 months at 37 ℃. Their mechanical properties were then measured using a three-point flexure test. Protein adsorption was measured using a micro bicinchoninic acid (BCA) method. A human saliva microcosm model was used to inoculate bacteria on water-aged specimens and to investigate the live/dead staining, metabolic activity of biofilms, and colony-forming units (CFUs).ResultsThe flexural strength and elastic modulus showed a significant loss after 6 months of water-ageing for the PMMA control (mean ± SD; n = 10); in contrast, the new protein repellent and antibacterial PMMA resin showed no strength loss. The PMMA–MPC–DMAHDM-containing resin imparted a strong antibacterial effect by greatly reducing biofilm viability and metabolic activity. The biofilm CFU count was reduced by about two orders of magnitude (p < 0.05) compared with that of the PMMA resin control. The protein adsorption was 20% that of a commercial composite (p < 0.05). Furthermore, the PMMA–MPC–DMAHDM-containing resin exhibited a long-term antibacterial performance, with no significant difference between 1 d, 3 months and 6 months (p > 0.05).ConclusionsThe flexural strength and elastic modulus of the PMMA–MPC–DMAHDM-containing resin were superior to those of the PMMA control after 6 months of water-ageing. The novel PMMA resin incorporating MPC and DMAHDM exhibited potent and lasting protein-repellent and antibacterial properties.

Mycobacterium tuberculosis ketol-acid reductoisomerase down-regulation affects its ability to persist, and its survival in macrophages and in mice

Branched-chain amino acids (BCAAs) leucine, isoleucine and valine biosynthetic pathways have been reported from plants, fungi and bacteria including Mycobacterium tuberculosis (Mtb) but are absent in animals. This makes interventions with BCAAs biosynthesis an attractive proposition for antimycobacterial drug discovery. In the present study, Mycobacterium tuberculosis H37Ra (Mtb-Ra) ketol-acid reductoisomerase encoding ORF MRA_3031 was studied to establish its role in Mtb-Ra growth and survival. Recombinant knockdown (KD) and complemented (KDC) strains along with wild-type (WT) Mtb-Ra were studied under in-vitro and ex-vivo conditions. KD was defective for survival inside macrophages and showed time dependent decrease in its colony forming unit (CFU) counts, while, WT and KDC showed time dependent increase in CFUs, after macrophage infection. Also, KD showed reduced ability to form persister cells, had altered membrane permeability against ethidium bromide and nile red dyes, and had reduced biofilm maturation, compared to WT and KDC. The in-vivo studies showed that KD infected mice had lower CFU counts in lungs, compared to WT. In summary Mtb shows survival deficit in macrophages and in mice after ketol-acid reductoisomerase down-regulation.

Thermal Endurance by a Hot-Spring-Dwelling Phylogenetic Relative of the Mesophilic Paracoccus.

High temperature growth/survival was revealed in a phylogenetic relative (SMMA_5) of the mesophilic Paracoccus isolated from the 78 to 85°C water of a Trans-Himalayan sulfur-borax spring. After 12 h at 50°C, or 45 min at 70°C, in mineral salts thiosulfate (MST) medium, SMMA_5 retained ~2% colony forming units (CFUs), whereas comparator Paracoccus had 1.5% and 0% CFU left at 50°C and 70°C, respectively. After 12 h at 50°C, the thermally conditioned sibling SMMA_5_TC exhibited an ~1.5 time increase in CFU count; after 45 min at 70°C, SMMA_5_TC had 7% of the initial CFU count. 1,000-times diluted Reasoner's 2A medium, and MST supplemented with lithium, boron, or glycine-betaine, supported higher CFU-retention/CFU-growth than MST. Furthermore, with or without lithium/boron/glycine-betaine, a higher percentage of cells always remained metabolically active, compared with what percentage formed single colonies. SMMA_5, compared with other Paracoccus, contained 335 unique genes: of these, 186 encoded hypothetical proteins, and 83 belonged to orthology groups, which again corresponded mostly to DNA replication/recombination/repair, transcription, secondary metabolism, and inorganic ion transport/metabolism. The SMMA_5 genome was relatively enriched in cell wall/membrane/envelope biogenesis, and amino acid metabolism. SMMA_5 and SMMA_5_TC mutually possessed 43 nucleotide polymorphisms, of which 18 were in protein-coding genes with 13 nonsynonymous and seven radical amino acid replacements. Such biochemical and biophysical mechanisms could be involved in thermal stress mitigation which streamline the cells' energy and resources toward system-maintenance and macromolecule-stabilization, thereby relinquishing cell-division for cell-viability. Thermal conditioning apparently helped inherit those potential metabolic states which are crucial for cell-system maintenance, while environmental solutes augmented the indigenous stability-conferring mechanisms. IMPORTANCE For a holistic understanding of microbial life's high-temperature adaptation, it is imperative to explore the biology of the phylogenetic relatives of mesophilic bacteria which get stochastically introduced to geographically and geologically diverse hot spring systems by local geodynamic forces. Here, in vitro endurance of high heat up to the extent of growth under special (habitat-inspired) conditions was discovered in a hot-spring-dwelling phylogenetic relative of the mesophilic Paracoccus species. Thermal conditioning, extreme oligotrophy, metabolic deceleration, presence of certain habitat-specific inorganic/organic solutes, and potential genomic specializations were found to be the major enablers of this conditional (acquired) thermophilicity. Feasibility of such phenomena across the taxonomic spectrum can well be paradigm changing for the established scopes of microbial adaptation to the physicochemical extremes. Applications of conditional thermophilicity in microbial process biotechnology may be far reaching and multifaceted.

CD14 signaling mediates lung immunopathology and mice mortality induced by Achromobacter xylosoxidans.

Objective and designOur research aimed to investigate the role of CD14 in pulmonary infection by Achromobacter xylosoxidans in an experimental murine model.MethodsC57Bl/6 or CD14-deficient mice were infected intratracheally with non-lethal inoculum of A. xylosoxidans. At times 1, 3 and 7 days after infection, lungs, bronchoalveolar lavage and blood were collected. CD14 gene expression was determined by RT-PCR. The bacterial load in the lungs was assessed by counting colony forming units (CFU). Cytokines, chemokines, lipocalin-2 and sCD14 were quantified by the ELISA method. Inflammatory infiltrate was observed on histological sections stained with HE, and leukocyte subtypes were assessed by flow cytometry. In another set of experiments, C57Bl/6 or CD14-deficient mice were inoculated with lethal inoculum and the survival rate determined.ResultsCD14-deficient mice are protected from A. xylosoxidans-induced death, which is unrelated to bacterial load. The lungs of CD14-deficient mice presented a smaller area of tissue damage, less neutrophil and macrophage infiltration, less pulmonary edema, and a lower concentration of IL-6, TNF-α, CXCL1, CCL2 and CCL3 when compared with lungs of C57Bl/6 mice. We also observed that A. xylosoxidans infection increases the number of leukocytes expressing mCD14 and the levels of sCD14 in BALF and serum of C57Bl/6-infected mice.ConclusionsIn summary, our data show that in A. xylosoxidans infection, the activation of CD14 induces intense pulmonary inflammatory response resulting in mice death.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-022-01641-8.

Bioinspired Anti-demineralization Enamel Coating for Orthodontics

White spot lesions and enamel cracks are the 2 most prominent diseases that occur after orthodontic treatment and are caused by enamel demineralization from accumulated bacterial biofilms and/or enamel damage caused by the removal of residual adhesive after bracket debonding. Inspired by the self-assembled amelogenin nanoribbons in enamel, we developed an enamel coating with a self-assembling antimicrobial peptide, D-GL13K, to simultaneously reduce demineralization and residual adhesive. The self-assembled amphiphilic nanoribbons significantly increased the hydrophobicity of the etched enamel, which reduced the permeability of the coated enamel surfaces as desired. The antimicrobial activity of this coating was evaluated against Streptococcus mutans by colony-forming unit counting and live/dead assays. The anti-demineralization effect was demonstrated by the reduced demineralization depth analyzed by optical coherence tomography and the increased Vickers hardness. The coatings did not reduce the shear bond strength but significantly reduced the adhesive remnant index score. This bioinspired enamel coating may provide a new strategy for preventing white spot lesions and enamel cracks after orthodontic treatment.

Benzyldimethyldodecyl Ammonium Chloride Doped Dental Adhesive: Impact on Core's Properties, Biosafety, and Antibacterial/Bonding Performance after Aging.

Current dental adhesives lack antibacterial properties. This study aimed to explore the effect of incorporating benzyldimethyldodecyl ammonium chloride (BDMDAC) on the degree of conversion, contact angle, ultimate tensile strength (UTS), microtensile bond strength (µTBS), cytotoxicity, antibacterial and bonding performance after artificial aging. A dental adhesive was doped with BDMDAC in the concentration range of 1-5 wt.%. For antibacterial assays, the BDMDAC compound was subject to planktonic cells of Streptococcus mutans. Then, after incorporation into the dental adhesive, an S. mutans biofilm model was used to grow 48 h-mature biofilms. The biofilms grown over the formulated materials were assessed by colony-forming unit (CFU) counting assay and fluorescence microscopy staining. In addition, the cytotoxicity was evaluated. Samples were subjected to 10,000 thermal cycles for aging and evaluated by UTS, µTBS, and CFU. Incorporating BDMDAC did not increase the cytotoxicity or change the physical properties when the mass fraction of the BDMDAC was 1-5 wt.%. The UTS of BDMDAC-doped adhesives was not impaired immediately or over time. A significant bacterial reduction was obtained for the mass fraction of the BDMDAC greater than 3 wt.%. However, the BDMDAC-doped adhesives did not offer an antibacterial effect after artificial aging. The overall results indicate that the BDMDAC strategy has the potential to control of microbial growth of cariogenic planktonic cells and biofilms. However, other new technological approaches are needed to overcome the deleterious effect of BDMDAC release over time such as those based on the principle of drug delivery systems whereby the BDMDAC is transported on microparticles or core shells, providing tangible benefits to oral health over time.

Secondary metabolite from &lt;em&gt;Pseudomonas aeruginosa&lt;/em&gt; LV strain exhibits antibacterial activity against &lt;em&gt;Staphylococcus aureus&lt;/em&gt;

This study aimed to evaluate the antibacterial activity of the dichloromethane/ethyl acetate fraction (named F4a), obtained from the culture of Pseudomonas aeruginosa LV strain in presence of copper chloride, against planktonic and sessile cells of Staphylococcus aureus, including those presenting multidrug resistance. First, the minimal inhibitory concentrations (MIC) of F4a for twenty-six clinical isolates were determined and the values ranged from 1.56 to 6.25 µg/mL. Minimal bactericidal concentration (MBC) of 3.13 µg/mL was detected in 84.6% of the isolates. The time-kill curve analysis revealed a significant decreased in colony-forming unit counts after 4 h of treatment with the MIC/MBC of F4a. Moreover, the MIC/MBC of the fluopsin C, a copper-containing compound present in F4a, were 1.56/3.13 µg/mL, indicating that this compound seems to be one of the active components related to the antibacterial activity against S. aureus. Images of transmission electron microscopy showed significant ultrastructural alterations in planktonic cells treated with the MIC/MBC of F4a. A significant reduction in the metabolic activity of established biofilms of all S. aureus isolates was observed after treatment with F4a. No hemolytic activity to human erythrocytes was detected for F4a, and the cytotoxic concentration to LLC-MK2 cells was 3.44 µg/mL. In conclusion, F4a exhibited a bactericidal activity against planktonic cells and inhibited the metabolic activity of biofilms of S. aureus. F4a can be promising for the development of new strategies for the treatment of infections caused by S. aureus.

Culture Supernatant of Enterococcus faecalis Promotes the Hyphal Morphogenesis and Biofilm Formation of Candida albicans.

(1) Background: Enterococcus faecalis and Candida albicans are often isolated from infected root canals. The interaction between these two species is not clear enough. Therefore, the objective of this study was to investigate the effect of E. faecalis on the biofilm formation, hyphal morphogenesis and virulence gene expression of C. albicans. (2) Methods: We used the culture supernatant of E. faecalis (CSE) to treat the biofilms of C. albicans. Then, crystal violet staining and colony-forming unit (CFU) counting were performed to evaluate biofilm biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscope (CLSM) were applied to observe fungal morphology. Subsequently, exopolymeric substances (EPS) production, cellular surface hydrophobicity (CSH) and adhesion force of biofilms were investigated by CLSM, water–hydrocarbon two-phase assay and atomic force microscopy (AFM), respectively. Finally, the expression of C. albicans virulence genes (ALS1, ALS3, HWP1 and EFG1) were measured by RT-qPCR assay. (3) Results: The exposure of CSE promoted the biofilm formation and hyphal morphogenesis of C. albicans, increased the EPS production, CSH and adhesion force of C. albicans biofilms, and increased the expression level of EFG1. (4) Conclusions: Our data indicated that CSE promoted the hyphal morphogenesis and biofilm formation of C. albicans.

Therapeutic efficacy of rifalazil (KRM-1648) in a M. ulcerans-induced Buruli ulcer mouse model.

Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans infection that requires long-term antibiotic treatment and/or surgical excision. In this study, we investigated the therapeutic efficacy of the rifamycin derivative, rifalazil (RLZ) (also known as KRM-1648), in an advanced M. ulcerans infection model. Six-week-old female BALB/c mice were infected with 3.25 x 104 colony-forming units (CFU) of M. ulcerans subcutaneously into the bilateral hind footpads. At 33 days post-infection, when the footpads exhibited significant redness and swelling, mice were treated orally with 5 or 10 mg/kg of RLZ for up to 15 weeks. Mice were followed for an additional 15 weeks following treatment cessation. Untreated mice exhibited a progressive increase in footpad redness, swelling, and erosion over time, and all untreated mice reached to endpoint within 5–8 weeks post-bacterial injection. In the RLZ-treated mice, footpad redness and swelling and general condition improved or completely healed, and no recurrence occurred following treatment cessation. After 3 weeks of treatment, the CFU counts from the footpads of recovered RLZ-treated mice showed a 104 decrease compared with those of untreated mice. We observed a further reduction in CFU counts to the detection limit following 6 to 15 weeks of treatment, which did not increase 15 weeks after discontinuing the treatment. Histopathologically, bacteria in the treated mice became fragmented one week after RLZ-treatment. At the final point of the experiment, all the treated mice (5mg/kg/day; n = 6, 10mg/kg/day; n = 7) survived and had no signs of M. ulcerans infection. These results indicate that the rifamycin analogue, RLZ, is efficacious in the treatment of an advanced M. ulcerans infection mouse model.

Exposure of multidrug-resistant Klebsiella pneumoniae biofilms to 1,8-cineole leads to bacterial cell death and biomass disruption

Klebsiella pneumoniae is a common cause of health-care associated infections. The rise of antibiotic resistance and the ability to form biofilm among K. pneumoniae strains are two key factors associated with antibiotic treatment failure. The present study investigates the antibiofilm activity of 1,8-cineole against preformed biofilms of multidrug-resistant extended-spectrum β-lactamase-producing K. pneumoniae clinical isolates. To evaluate the antibiofilm activity, cellular viability was analyzed by colony-forming units counting and live/dead staining. In addition, biofilm biomass was evaluated by crystal violet and the biofilm matrix was stained with calcofluor white and observed by confocal laser scanning microscopy. A time- and concentration-dependent effect of the phytochemical over biofilm cell viability was observed revealing that 1% (v/v) 1,8-cineole during 1 h was the optimal treatment condition displaying a significant reduction of cell viability in the preformed biofilms (2.5–5.3 log cfu/cm2). Furthermore, confocal laser scanning microscopy after SYTO-9 and propidium iodide staining showed that 1,8-cineole was capable of killing bacteria throughout all layers of the biofilm. The compound also caused a biofilm disruption (30–62% biomass reduction determined by crystal violet staining) and a significant decrease in biofilm matrix density. Altogether, our results demonstrate that 1,8-cineole is a promising candidate as a novel antibiofilm agent against multidrug-resistant K. pneumoniae strains producing extended-spectrum β-lactamases, given its capability to disrupt the structure and to kill cells within the biofilm.

Comparative analysis of Candida spp. isolated from complete denture wearers with and without an HIV diagnosis

Statement of problemDenture-related stomatitis (DRS), an inflammation frequently present in human immunodeficiency virus-positive (HIV+) individuals, can be attributable to colonization by Candida spp., which is considered a main factor. The virulence factors of these species are often modulated by the systemic condition of their hosts. PurposeThe purpose of this clinical study was to evaluate the incidence, virulence, and morphology of Candida spp. isolated from biofilms of complete denture wearers with DRS, with and without an HIV diagnosis. In addition, the interaction of the systemic condition with the ability of Candida spp. to colonize was evaluated. Material and methodsFifty-five complete denture wearers diagnosed with DRS were divided into 2 groups: experimental (HIV+) and control (human immunodeficiency virus-noninfected participants [HIV-]). Biofilm was collected by a standardized method of ultrasonification of prostheses. The incidence was evaluated by a chromogenic method and polymerase chain reaction (PCR). The virulence factors were assessed by using the capacity for biofilm formation by counting colony-forming units (CFUs/mL), biofilm metabolism by tetrazolium salt metabolization, and proteinase and phospholipase production by using a fluorimetric kit. Morphology was verified by using the hyphae-inducing test, and participants’ health data were collected with a form. Data were analyzed by using the Student t, Mann-Whitney U, Spearman, and Fisher tests (α=.05). ResultsThe results of incidence were related to 55 participants (22 experimental and 33 control); in total, 63 Candida spp. samples were isolated, showing 28 Candida albicans and 36 nonalbicans strains. No significant difference was found between groups in baseline CFU/mL counts, biofilm formation capacity, cell metabolism, and phospholipase production. Proteinase production was higher for C. albicans in the control (P=.031) and for nonalbicans in the experimental (P=.016) groups. Relative to health data, the experimental group showed a moderate negative correlation between the CFU count/mL at baseline for nonalbicans and DRS classification (P=.020). ConclusionsC. albicans was the most prevalent species. No difference was found in the Candida spp. of complete denture wearers with DRS, with and without an HIV diagnosis, with regard to virulence factors (except for proteinase production) and morphology.

Middlebrook 7h11 reduces invalid results and turnaround time of phenotypic drug-susceptibility testing of M. tuberculosis.

Phenotypic drug-susceptibility testing (pDST), which relies on growth inhibition in the drug-containing media, remains a challenge for fastidious Mycobacterium tuberculosis complex (MTBc) isolates due to insufficient growth on the growth controls (GC). Middlebrook 7H11 (M7H11) medium contains casein hydrolysate, which may favor the growth of such strains. In this study, we tested whether M7H11 reduces invalid results due to insufficient growth on the GCs and the turnaround time (TAT) of pDST for MTBc compared to Middlebrook 7H10 (M7H10) without affecting the accuracy of the pDST results and how it differs between rifampicin- and isoniazid-susceptible non multi-drug resistant (non-MDR), MDR and MDR with additional resistance to fluoroquinolones (Pre-XDR) MTBc isolates. We compared the proportions of invalid pDST results due to lack of growth on the GCs, TATs of valid parallel drug-susceptibility testings as an indicator of speed of MTBc growth, and colony-forming unit (CFU) count on the most diluted GC of the parallel pDSTs after equal incubation periods as an indicator of growth abundance on M7H11 and M7H10. We also analyzed the agreement between the pDST results of the same drug or drugs in the same drug class, tested in parallel on both media. For MDR and pre-XDR isolates, relative to M7H10, M7H11 significantly reduced the occurrence of invalid pDST results due to insufficient growth on the GCs (odds ratio [OR] = ∞ [95% confidence interval (CI) 1.9-∞], P = 0.004 for MDR, OR = ∞ [95% CI 3.3-∞], P = 0.0001 for pre-XDR) and the TAT of pDSTs (OR = 17 [95% CI 2.6-710.4], P = 0.0001 for MDR, OR = 9.3 [95% CI 4.0-26.5], P < 0.0001 for pre-XDR). The growth abundance of MTBc on M7H11 was significantly higher compared to M7H10 (17 CFU on M7H10 vs. 28 on M7H11), irrespective of drug-resistance profiles. The agreement between the pDST results between the two media was high (Cohen's k > 0.98). Our study findings suggest that M7H11 is preferred over M7H10 for pDSTs of MTBc isolates.

Caspofungin alone or combined with polymyxin B are effective against mixed biofilm of Aspergillus fumigatus and carbapenem-resistant Pseudomonas aeruginosa

Aspergillus fumigatus and Pseudomonas aeruginosa biofilms are associated to the recalcitrant and persistent infections due to resistance to antimicrobials. Here, we evaluated the effect of antimicrobials on single and mixed biofilms of A. fumigatus and P. aeruginosa (carbapenem-resistant and susceptible strains) determining total biomass by crystal violet, cell viability by colony forming unit count, and microscopy. Polymyxin B (PMB) had the best action on P. aeruginosa biofilms inhibiting the biomass (2–4 μg/mL) and it was efficient reducing the viable bacterial cells. Amphotericin B (AMB) and caspofungin (CAS) were the best antifungal at inhibiting A. fumigatus biofilms and reducing fungal viability at concentration ≥1 and ≥ 16 μg/mL, respectively. In addition, CAS was able to significantly reduce P. aeruginosa viability in mixed biofilms. CAS combined with PMB also significantly reduced the mixed biofilm biomass and fungal and bacterial viability mainly against carbapenem-resistant bacterium. The light and fluorescence microscopy showed alterations on hyphae morphology and confirmed the increase of fungal and bacterial death cells after combined therapy of mixed biofilms. Taken together, our work showed that CAS alone and its combination with PMB showed better potential in reducing mixed biofilm biomass and fungal and bacterial viability, even for the carbapenem-resistant P. aeruginosa strain.

Effects of immunoglobulin Y-loaded amorphous calcium phosphate on dentinal tubules occlusion and antibacterial activity.

Aim: This study aimed to evaluate the effects of immunoglobulin Y (IgY)-loaded amorphous calcium phosphate (ACP) (IgY@ACP) on dentinal tubule occlusion and antibacterial activity. Methodology: IgY@ACP was synthesized based on a biomimetic mineralization strategy. The structure was examined by transmission electron microscopy and Fourier transform infrared spectroscopy. The IgY release property was assessed in vitro. The cell biocompatibility of IgY@ACP was evaluated by CCK-8. The dentin disks were prepared using healthy human molars, and their dentinal tubules were exposed to EDTA. Subsequently, they were randomly selected and treated with or without IgY@ACP for 7 days. The tubule occlusion morphologies and newly formed layers were observed by scanning electron microscopy (SEM) and x-ray diffraction, respectively. To evaluate the acid resistance and abrasion resistance of IgY@ACP, dentin disks that were treated for 1 day were immersed in acid solution or subjected to a toothbrush. The antibacterial effects against Streptococcus mutans (S. mutans) were evaluated by colony-forming unit (CFU) counting, adhesion property assessment, and crystal violet and live/dead bacterial staining. Finally, the occlusion effect was evaluated in rat incisors in vivo. One-way analysis of variance (ANOVA) was performed for statistical analysis. The level of significance was set at 0.05. Results: IgY@ACP presented an amorphous phase with a nanosize (60–80 nm) and sustained release of protein within 48 h. The CCK-8 results showed that IgY@ACP had good biocompatibility. After treatment with IgY@ACP for 1 day, the majority of dentinal tubules were occluded by a 0.3-μm-thick mineralized layer. Seven days later, all dentinal tubules were occluded by mineralization with a thickness of 1.4 μm and a depth of 16 μm. The newly mineralized layer showed hydroxyapatite-like diffraction peaks. In addition, IgY@ACP had good acid and abrasion resistance. After treatment with IgY@ACP, the CFU counting and adhesion rate of S. mutans were significantly reduced, the crystal violet staining was lighter, and the S. mutans staining revealed more dead cells. Most importantly, IgY@ACP had a certain occluding property in rat incisors in vivo. Conclusion: IgY@ACP can effectively occlude dentinal tubules with acid-resistant stability and has prominent anti-S. mutans effects, rendering it a potentially suitable desensitization material in the clinic.

Effect of N-2-methyl-pyrrolidone on Enterococcus faecalis biofilms.

This study aimed to investigate the effect of N-2-methyl-pyrrolidone (NMP) on the removal of Enterococcus faecalis biofilm. Colony-forming unit (CFU) counting, crystal violet staining, and extracellular DNA (eDNA) measurements were performed to analyze removal of the biofilms formed in a bovine root canal. A confocal laser scanning microscope (CLSM) assay was used to measure the volume of the biofilms. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to investigate the biofilm-associated genes. The morphologic feature of the biofilms was observed under a scanning electron microscope (SEM). NMP decreased CFU numbers, eDNA levels, and biofilm biomass significantly compared to control. qRT-PCR showed that NMP increased the expression of some virulence-associated genes, but downregulated genes related to colonization and persistency. SEM showed that the numerous dentinal tubules were exposed as a result of removal of the biofilm. Collectively, NMP has the potential to be used as a vehicle for endodontic intracanal medicaments.

Effects of a 445 nm diode laser and silver diamine fluoride in preventing enamel demineralisation and inhibiting cariogenic bacteria

ObjectiveTo study the effects of a 445 nm diode laser (L) and silver diamine fluoride (F) on preventing enamel demineralisation and inhibiting cariogenic bacteria. MethodsThirty-three enamel slices were sectioned each into four blocks for four groups to receive L with F (LF), F, L and Water (W, control). Ten blocks from each group were used to evaluate demineralisation. Surface morphology, lesion depth and nanohardness of the blocks after pH-cycling were studied by scanning electron microscopy (SEM), nanohardness test, and micro-computed tomography, respectively. Twenty-three blocks per group were used for biofilm assessment. Morphology, viability, and growth kinetics of the Streptococcus mutans biofilm were assessed by SEM, confocal laser scanning microscopy, and the counting of colony-forming units (CFUs), respectively. ResultsSEM images of LF-treated enamel showed an intact surface compared with other groups. Nanohardness (GPa) for LF, F, L and W were 1.43±0.17, 1.01±0.11, 1.04±0.13 and 0.73±0.14, respectively (p < 0.001; LF>F, L>W). Their lesion depths (µm) were 46±8, 52±6, 88±13 and 111±9, respectively (p < 0.001; LF, F<L<W). SEM showed few bacteria for LF and F compared with other groups. Their dead-live ratio were 1.67±0.13, 1.60±0.15, 0.39±0.05 and 0.32±0.05, respectively (p < 0.001; LF, F>L>W). Log CFUs for LF, F, L and W were 4.2±0.3, 4.5±0.2, 7.9±0.3 and 9.4±0.2, respectively (p < 0.05; LF<F<L<W). Two-way ANOVA analysis revealed an interaction effect on nanohardness and Log CFUs between the laser irradiation and SDF treatment (p < 0.001). ConclusionThis study showed a superior caries preventive effect of a combined treatment of the diode laser and SDF. Because diode laser and SDF are affordable and readily available, clinicians can provide this treatment to their patients for caries prevention. Clinical significance statementDiode lasers are handy, afforable and readily avaliable to clinicians. This study provides information of use of 445 nm diode laser for caries prevetion. The laser irradiation hopefully can be added before conventional topical SDF application.

P341 Evaluation of anti-Candida activity of Cinnamaldehyde using mice model

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectiveVaginal candidiasis is a frequent infection afflicting the female population. Candida co-infections are reported in escalating frequency and can be associated with severe health hazards or even death. People with severe COVID-19, malignancies, AIDS, and organ transplants are particularly vulnerable to invasive vaginal candidiasis. Due to the high frequency of infections associated with recurrence, vaginal candidiasis poses a significant medical problem worldwide. Treatments of vaginal candidiasis are limited due to drug resistance, side effects, and toxicity. CIN is a natural compound and its antifungal activity is widely reported. The introduction of cinnamaldehyde (CIN) as the anti-Candidal agent will revolutionize the treatment of vaginal candidiasis.In this study, we investigated the anti-Candida activity of CIN against vaginal candidiasis in Swiss albino mice (C3HHC-Strain).MethodsVaginal candidiasis in mice (Swiss albino) was induced under conditions of pseudo-estrus. Persistent vaginal infection was found in estrogenized mice after vaginal challenge with C. albicans. The mice were treated orally after confirmation of infection in mice. The efficacy of CIN treatment was investigated phenotypically by colony-forming unit (CFU) counts in the vaginal smear, fungal load determination in the blood, the ovarian and vaginal tissues and periodic acid-Schiff (PAS) staining of histopathological sections of the vaginal tissues. The hematological parameters of the experimental mice were also evaluated.ResultsThe pseudohyphae and spores of C. albicans were present in the vaginal smear of experimentally infected mice. After treatment, no C. albicans colonies were found in the vaginal lavage of infected mice. The fungal burden was significantly higher in the vagina and the ovaries of infected mice. However, a dose of 262.5 mg/kg BW of CIN reduced the number of CFU in the vagina and ovaries in treated mice. The histopathology revealed the absence of C. albicans in the vaginal tissue of the treated mice. Nonetheless, the vaginal sections of infected mice exhibited pathological changes. The hematological parameters such as RBC count, WBC count, and percentage of hemoglobin showed significant differences in the treatment groups compared to the infected group.ConclusionCinnamaldehyde showed good in vivo antifungal potential against vaginal candidiasis. However, evaluation of its pharmacodynamic and pharmacokinetic parameters and complete elucidation of its mode of action are desirous.