Crystal Violet Assay Research Articles

Anti-Inflammatory and Anti-proliferative Role of Essential Oil of Leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry.

Phytochemicals have long remained an essential component of the traditional medicine system worldwide. Advancement of research in phytochemicals has led to the identification of novel constituents and metabolites from phytochemicals, performing various vital functions ranging from antimicrobial properties to anticarcinogenic roles. This plant is traditionally used by local people to manage inflammation. In this study, we aim to extract and chemically profile the essential oil from the leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry and study of the anti-inflammatory and anti-proliferative role of essential oil. The hydro distillation method was used for the extraction of essential oil, and the GC-MS was applied for the chemical profiling. The percentage of cell viability was calculated using a crystal violet assay, colony formation assay was performed using Semiquantitative PCR, Propodium iodite staining was used for cell death assay, and Western blotting was used to determine antibodies and proteins. Schrodinger 2015 software was used for molecular docking. Myrcene, a monoterpene, constitutes 56% of the oil and could be attributed to its anti-inflammatory potential. Treatment of LPS-challenged mouse macrophages RAW264.7 cells with essential oil resulted in a decline in the inflammatory markers, such as IL-1β, TNFα, iNOS, COX-2, and NFκB. Further, essential oil inhibited cancer PC-3, A431, A549, and MCF-7 cell lines at concentrations lower than normal PNT2 and HEK-293 cell lines. This decline in proliferative potential can be attributed to a decline in anti-apoptotic proteins, such as procaspase 3 and PARP, an increase in CKIs, such as p21, and a decline in the Akt signaling responsible for survival. The essential oil of the plant Cleistocalyx operculatus may be a potential lead for anti-inflammatory and anti-proliferative function.

Clinical laboratory evaluation of the Hologic Panther Aptima BV and CV/TV assays for the diagnosis of vaginitis in Dunedin, Aotearoa New Zealand.

Vaginitis presentations are common, but traditional diagnostic methods are imperfect. Molecular methods for bacterial vaginosis (BV) and vulvovaginal candidiasis (CV) are increasingly available but not commonly utilized in Aotearoa New Zealand. We evaluated the Hologic Aptima BV and CV/Trichomonas vaginalis (TV) assays against our current methods (Gram stain, yeast culture, and Hologic Aptima TV assay) and performed a retrospective BV clinical audit. The BV Aptima assay performed well with high sensitivity (97.5%) and specificity (96.3%) when the indeterminate BV category was excluded. BV indeterminate samples were almost evenly split between positive and negative results when tested on the Aptima BV assay. BV Gram stain interpretation was error prone, with 20% of samples discordant on duplicate examination. Although the Aptima CV assay was highly sensitive, it lacked specificity compared with Gram stain (83.5%) but was similar to culture (91.2%). Our BV clinical audit showed that patients with a BV indeterminate result were less likely to be treated for BV than those with a positive result, meaning more women may be treated for BV if this assay were implemented. Overall, implementation may improve laboratory workflow and consistency of reporting, but cost may be a barrier. The clinical impact of changing methods needs to be considered.IMPORTANCEIn this paper, we evaluate the performance of the Aptima molecular assays against current Gram stain and culture methods, as well as a clinical audit to determine the potential clinical impact of implementation. Although molecular methods are increasingly used in other countries, New Zealand has not yet adopted this approach. Importantly, we found Gram stain for bacterial vaginosis (BV) to be error prone, with 20% of Gram stain results discordant on repeat examination. We show the potential for molecular methods to increase BV diagnoses and improve reproducibility and consistency of reporting which, according to our clinical audit results, would lead to more women being treated for this dysbiosis condition overall.

SLFN12 Expression Significantly Effects the Response to Chemotherapy Drugs in Triple-Negative Breast Cancer.

Schlafen12 (SLFN12) is an intermediate human Schlafen protein shown to correlate with survivability in triple-negative breast cancer (TNBC). SLFN12 causes differential expressions of significant cancer genes, but how they change in response to chemotherapy remains unknown. Our aim is to identify the effect of chemotherapy on genes that improve TNBC outcomes and other SLFN family members following SLFN12 knockout or overexpression. We overexpressed SLFN12 using a lentiviral vector and knocked out SLFN12 (AdvShSLFN12) using a hairpin adenovirus in MDA-MB-231 TNBC cells. Cells were treated with camptothecin, paclitaxel, zoledronic acid, or carboplatin to evaluate the SLFN12 signature cancer genes associated with improved TNBC outcomes using qPCR. Additionally, cells were treated alone and in combination with AdvShSLFN12, IFN-α2 (known SLFN12 stimulator), carboplatin, and paclitaxel. After treatment, the viable cell numbers were analyzed utilizing a colorimetric crystal violet assay for cell viability. The SLFN family and SLFN12 cancer signature gene mRNA expressions were analyzed by RT-qPCR. Treating SLFN12-overexpressing TNBC cells with chemotherapy agents resulted in the differential expressions of eight cancer-related genes. Notably, GJB3 was downregulated following treatment with each chemotherapeutic drug. Inducing SLFN12 with IFN-α2 resulted in decreased cell viability and increased SLFN12 mRNA levels following treatment with paclitaxel or carboplatin. These results suggest that SLFN12 overexpression significantly affects the expressions of genes driving phenotypic changes in response to chemotherapy and influences additional SLFN family members following IFN-α2 treatment. This may contribute to improving the survival of patients with SLFN12 overexpression. Additionally, patient SLFN12 levels can be used as a factor when pursuing personalized chemotherapy treatments.

Biofilm Inhibition Activity of Fennel Honey, Fennel Essential Oil and Their Combination.

The eradication of bacterial biofilms remains a persistent challenge in medicine, particularly because an increasing number of biofilms exhibit resistance to conventional antibiotics. This underscores the importance of searching for novel compounds that present antibacterial and biofilm inhibition activity. Various types of honey and essential oil were proven to be effective against a number of biofilm-forming bacterial strains. The current study demonstrated the effectiveness of the relatively unexplored fennel honey (FH), fennel essential oil (FEO), and their combination against biofilm-forming bacterial strains Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and Escherichia coli, with a series of in vitro experiments. The authenticity of FH and FEO was checked with light microscopy and gas chromatography-mass spectrometry, respectively. Minimum inhibitory concentrations were determined using the microdilution method, and antibiofilm activity was assessed with crystal violet assay. Structural changes in bacterial cells and biofilms, induced by the treatments, were monitored with scanning electron microscopy. FEO and FH inhibited the biofilm formation of each bacterial strain, with FEO being more effective compared to FH. Their combination was the most effective, with inhibitory rates ranging between 87 and 92%, depending on the bacterial strain. The most sensitive bacterium was E. coli, while P. aeruginosa was the most resistant. These results provide justification for the combined use of honey and essential oil to suppress bacterial biofilms and can serve as a starting point to develop an effective surface disinfectant with natural ingredients.

Exploring time-killing and biofilm inhibition potential of bioactive proteins extracted from two varieties of Pleurotus ostreatus.

Dental caries, caused by oral microbial pathogens, are a global health concern, further exacerbated by the presence of methicillin-resistant Staphylococcus aureus (MRSA). Bioactive proteins and peptides (BAPs) exhibit potent antimicrobial properties, targeting multiple cellular mechanisms within pathogens, reducing the likelihood of resistance development. Given the antimicrobial potential of BAPs, this study aimed to compare the efficacy of BAPs extracted from cultivated (Pleurotus ostreatus, PoC) and wild (Pleurotus ostreatus, PoW) mushrooms against pathogens responsible for dental caries. BAPs were extracted from both PoC and PoW using a TCA-acetone method. Antimicrobial activities were tested against seven bacteria and one fungus using agar well diffusion and MIC determination. Antibiofilm activity was assessed via modified CV assay, while DPPH and erythrocyte lysis tests evaluated free radical scavenging. PoC showed superior antimicrobial efficacy, with lower MIC and MBC values, and disrupted biofilm integrity at increasing concentrations. PoW exhibited better antioxidant activity with higher DPPH scavenging, though its antimicrobial efficacy was slightly lower than PoC. Both PoC and PoW BAPs inhibited dental pathogens, with PoC showing stronger inhibition against MRSA and nystatin-resistant Candida albicans. This suggests BAPs may target additional cellular mechanisms beyond membranes, PBPs, and ergosterols. Despite PoW's stronger antioxidant properties, both BAPs had comparable antibiofilm activity. These findings suggest complementary actions of BAPs from PoC and PoW both, in treating dental caries, offering broad-spectrum antimicrobial and antioxidant benefits.

Diverse antifungal potency of terbinafine as a therapeutic agent against Exophiala dermatitidis in vitro

Exophiala dermatitidis (E. dermatitidis), which causes skin or respiratory disease, is occasionally fatal in immunocompromised patients. Here, we report the unique antifungal potency of terbinafine (TRB), which targets squalene epoxidase, against E. dermatitidis (SQLEED) using various in vitro approaches. The versatile antifungal activities, including fungicidal activity, biofilm formation inhibition, biofilm eradication activity, and the combination effect of TRB, posaconazole (PSC), and amphotericin B (AmB) with great antifungal potency against E. dermatitidis were evaluated using crystal violet and cell viability assay. TRB formed an H-bond through Y102 in SQLEED in the binding model. E. dermatitidis hyphae elongated and attached to a cell scaffold, forming a membrane-like biofilm. TRB and PSC showed more potent antibiofilm activities than AmB, and exhibited post-antifungal effects without incubation against E. dermatitidis conidia, reducing growth at lower concentrations. In contrast, AmB exhibited strong dose- and time-dependent killing and biofilm-eradication activities. The combination of TRB and PSC was more effective than that of TRB and AmB or PSC and AmB. Although the tissue migration of TRB must be considered, these data suggest that TRB and PSC may be useful agents and a potent combination in severely immunocompromised patients with refractory and systemic E. dermatitidis infection.

Surface-functionalized UIO-66-NH2 for dual-drug delivery of vancomycin and amikacin against vancomycin-resistant Staphylococcus aureus

BackgroundConventional antibacterial compounds can inhibit the growth of microorganisms, but their adverse effects and the development of drug limit their widespread use. The current study aimed to synthesize PEG-coated UIO-66-NH2 nanoparticles loaded with vancomycin and amikacin (VAN/AMK-UIO-66-NH2@PEG) and evaluate their antibacterial and anti-biofilm activities against vancomycin-resistant Staphylococcus aureus (VRSA) clinical isolates.MethodsThe VAN/AMK-UIO-66-NH2@PEG were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) to determine their size, polydispersity index (PDI), encapsulation efficiency (EE%), zeta-potential, drug release profile, and physical stability. Antibacterial activity was evaluated using minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays. Biofilm formation by VRSA was assessed using the crystal violet (CV) and minimum biofilm eradication concentration (MBEC) assays. The effect of sub-MIC concentrations of the formulations on the expression of biofilm-related genes (icaA, icaD) and resistance-related genes (mecA, vanA) was investigated using quantitative real-time polymerase chain reaction (RT-qPCR).ResultsAs demonstrated by MIC, MBC and time-kill assay, the VAN/AMK-UIO-66-NH2@PEG nanoparticles exhibited enhanced antibacterial activity against VRSA isolates compared to free drugs and prepared formulations. Furthermore, CV and MBEC tests indicated that the VAN/AMK-UIO-66@NH2/PEG can reduce biofilm formation dramatically compared to VAN/AMK and VAN/AMK-UIO-66@NH2, due to its great drug release properties. This study also found that the expression level of the mecA, vanA, icaA, and icaD genes in VAN/AMK-UIO-66@NH2/PEG treated VRSA isolates was substantially decreased compared to other groups.ConclusionsThese findings highlighted the efficiency of VAN/AMK-UIO-66@NH2/PEG in combating antimicrobial resistance and biofilm formation in VRSA isolates. Future studies, particularly in vivo models, are necessary to evaluate the safety, efficacy, and clinical applicability of these nanoparticles for the treatment of bacterial infections.

Anti-biofilm potential of red pitahaya betacyanins against Streptococcus mutans, Actinomyces viscosus and Aggregatibacter actinomycetemcomitans

This study investigated the antibiofilm effects of betacyanin fraction (BF) from red pitahaya against Streptococcus mutans, Actinomyces viscosus, and Aggregatibacter actinomycetemcomitans, key pathogens in oral biofilm formation. Betacyanin purification employed column chromatography, and characterization was performed using liquid chromatography-mass spectrometry. Crystal violet assay and scanning electron microscopy demonstrated significant inhibition of early biofilm formation and disruption of preformed biofilms using BF (0.94–15 mg/mL). Anti-adhesion assays on artificial teeth indicated potent betacyanin-mediated inhibition of both sucrose-dependent and -independent bacterial attachment. Notably, growth curve and pH drop assays revealed that BF hindered pH reduction for all tested bacteria without suppressing their growth. Tetrazolium-based cytotoxicity assays showed minimal toxicity towards normal human gingival fibroblasts (HGF-1) at 0.78–12.5 mg/mL. These findings demonstrate the potential of red pitahaya betacyanins as promising antibiofilm agents for oral health, targeting both initial and mature stages of biofilm development.

Rosmarinic Acid Exhibits Antifungal and Antibiofilm Activities Against Candida albicans: Insights into Gene Expression and Morphological Changes.

Candida species, opportunistic pathogens that cause various infections, pose a significant threat due to their ability to form biofilms that resist antifungal treatments and immune responses. The increasing resistance of Candida spp. and the limited availability of effective treatments have prompted the research of natural compounds as alternative therapies. This study assessed the antifungal properties of RA against Candida species, focusing on its impact on C. albicans biofilms and the underlying mechanisms. The antifungal efficacy of RA was evaluated using the CLSI M27-A3 microdilution method on both fluconazole-susceptible and -resistant strains. Biofilm formation by C. albicans was assessed through a crystal violet assay, while its antibiofilm activity was analyzed using an MTT assay and field emission scanning electron microscopy (FESEM). Gene expression related to biofilm formation was studied using quantitative real-time PCR (qRT-PCR), and statistical analysis was performed with an ANOVA. Among the 28 Candida strains tested, RA exhibited minimum inhibitory concentration (MIC) values ranging from 160 to 1280 μg/mL. At a 640 μg/mL concentration, it significantly reduced the expression of genes associated with adhesion (ALS3, HWP1, and ECE1), hyphal development (UME6 and HGC1), and hyphal cAMP-dependent protein kinase regulators (CYR1, RAS1, and EFG1) in RAS1-cAMP-EFG1 pathway (p < 0.05). FESEM analysis revealed a reduction in hyphal networks and disruptions on the cell surface. Our study is the first to demonstrate the effects of RA on C. albicans adhesion, hyphae development, and biofilm formation through gene expression analysis with findings supported by FESEM. This approach distinguishes our study from previous studies on the effect of RA on Candida. However, the high MIC values of RA limit its antifungal potential. Therefore, more extensive research using innovative methods is required to increase the antifungal effect of RA.

Multi-metal resistance and antibiotic susceptibility of biofilm forming bacteria isolated from Kuzhikandam Creek in Kochi, southwest part of India

The purpose of this work was to characterize and identify the bacteria isolated from highly polluted locations that could potentially be able to withstand extremely toxic heavy metals. The study area selected for bacterial isolation from sediment samples was Kuzhikandam Creek, a tributary of the Periyar River and one of the world’s most hazardous hotspots, located in Kochi’s Eloor industrial region. Heavy metal analysis of chromium, copper, and lead in sediment samples was performed, and the results showed that the concentrations were greater than permissible limits. Several metal-resistant bacteria were isolated from sediments, which, upon further screening in solid media at different concentrations of chromium, copper, and lead, found three highly resistant species (S1B1, S2B2, and S3B3), one from each site. The phenotypic and biochemical characterization of these bacteria was examined using standard procedures. The ability of these species to generate biofilm was examined using methods like the crystal violet assay (qualitative assessment) and also using the scanning electron microscope. The antibiotic susceptibility of these species was tested with six different antibiotics, which are used in various disease treatments. These isolates, sequenced using the 16S rRNA Sanger dideoxy method, have been identified as Staphylococcus arlettae, Bacillus paramycoides, and Cellulosimicrobium funkei.

Single-agent Adavosertib Shows Anticancer Effects Against Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer-related deaths worldwide. Adavosertib (AZD1775), a small molecule inhibitor of WEE1 kinase, abrogates G2/M cell cycle arrest and induces double-stranded DNA breaks. According to previous findings, adavosertib, in combination with other DNA-damaging agents, causes premature mitosis and cell death in p53-mutated cancer cells mainly via abrogation of the G2/M cell cycle checkpoint. This study aims to evaluate the inhibition of WEE1 kinase by adavosertib as monotherapy in the TP53-wildtype human CRC cell line HCT116. In this study, HCT116 cells were treated with different concentrations of adavosertib for 24 to 72 hours. Cell viability was assessed by Water-Soluble Tetrazolium 1 (WST-1) assay and crystal violet assays. Cell migration was evaluated by the wound healing assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The IC50 value of adavosertib for the HCT116 cell line was 0.1310 μM. Adavosertib monotherapy (both 0.125 and 0.250 μM) significantly reduced cell viability, inhibited cell migration and abrogated intra-S phase cell cycle arrest. In addition, 0.250 μM of adavosertib significantly induced apoptosis in HCT116 cells. Adavosertib effectively inhibits the TP53-wildtype HCT116 cells via the abrogation of intra-S phase cell cycle arrest. Our findings suggest that adavosertib monotherapy may be a potential targeted therapy for CRC.

Cytocompatibility, Antibacterial, and Anti-Biofilm Efficacy of Grape Seed Extract and Quercetin Hydrogels Against a Mature Endodontic Biofilm Ex Vivo Model.

Background/Objectives: To assess the cytocompatibility, antibacterial and anti-biofilm efficacy of grape seed extract (GSE) and quercetin hydrogels versus calcium hydroxide (CH) as intracanal medications (ICMs) against an endodontic ex vivo biofilm model. Methods: Single-rooted teeth (n = 50) were prepared and sterilized before being infected with E. faecalis to develop a mature biofilm. They were divided into five equal groups according to the ICM used: G1: medicated with CH paste, G2: medicated with GSE hydrogel, G3: medicated with quercetin hydrogel, G4: positive control group that was infected and not medicated, and G5: negative control group that was neither infected nor medicated. After 1 week, the ICM was removed, and the root canals were cultured to assess the antibacterial efficacy by counting the colony-forming units and the anti-biofilm efficacy by the crystal violet assay. Dead/live bacterial viability was assessed by CFLSM examination, while the cytocompatibility was assessed using the MTT assay. Results: CH had the best antibacterial efficacy, followed by GSE and quercetin hydrogels (p < 0.001). Regarding the anti-biofilm efficacy, GSE was superior, followed by quercetin and CH (p < 0.001). CFLSM examination showed CH and GSE hydrogel to be highly effective in comparison to the positive control (p < 0.0001), with no statistical difference between them (p > 0.05). CH showed significantly higher cell viability percentages using a 500 μg/mL, while quercetin and GSE started to show cell viability > 70% at concentrations of 125 μg/mL and 62.5 μg/mL. Conclusions: CH fulfilled the ideal requirements of ICM as being both antibacterial and non-cytotoxic compared to the other materials tested.

Biofilm formation and drug susceptibility of biofilm Candida spp. clinically isolated from nasopharyngeal cancer patients in Vietnam.

The biofilm formation has been widely recognized as one of the main mechanisms of antimicrobial resistance development in microorganisms. However, few studies are focusing on this phenomenon in Candida spp. in clinical settings, especially on immuno-compromised patients. In this study, both the rate of biofilm formation in those patients and its drug susceptibility in initial and mature biofilm were assessed using crystal violet assay and dilution method. The results demonstrated that the biofilm formation rate was similar between albicans and non-albicans Candida. However, the biofilm formation capacity was more pronounced in non-albicans Candida, especially, C. glabrata. As expected, there was a significant relationship between biofilm formation and drug resistance. In addition, our study reconfirmed that the age of high concentration of antifungal agents only affected Candida before its biofilm formation regardless of its biofilm formation capacity. In the contrary, once the biofilm was formed even elevated drug concentrations did not show sufficient efficacy, highlighting a need for high dosage at the early stage of treatment for those patients. The results of this study highlighted the importance of using appropriate antifungal agents for Candida treatment before the formation of biofilm.

In vitro and computational investigation of antioxidant and anticancer properties of Streptomyces coeruleofuscus SCJ extract on MDA-MB-468 triple-negative breast cancer cells

This study aimed to explore the antioxidant potential of the ethyl acetate extract of Streptomyces coeruleofuscus SCJ strain, along with its inhibitory effects on the triple-negative human breast carcinoma cell line (MDA-MB-468). The ethyl acetate extract’s total phenolic and flavonoid contents were quantified, and its antioxidant activity was investigated using DPPH (1,1-Diphenyl-2-picrylhydrazyl), ABTS (2,2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid), and FRAP (Ferric Reducing Antioxidant Power) assays. Furthermore, the cytotoxic effect of the organic extract from Streptomyces coeruleofuscus SCJ on MDA-MB-468 cancer cells was assessed via the crystal violet assay. In tandem, a thorough computational investigation was conducted to explore the pharmacokinetic properties of the identified components of the extract, utilizing the SwissADME and pKCSM web servers. Additionally, the molecular interactions between these components and Estrogen Receptor Beta, identified as a potential target, were probed through molecular docking studies. The results revealed that ethyl acetate extract of SCJ strain exhibited remarkable antioxidant activity, with 39.899 ± 1.56% and 35.798 ± 0.082% scavenging activities against DPPH and ABTS, respectively, at 1 mg/mL. The extract also displayed significant ferric reducing power, with a concentration of 1.087 ± 0.026 mg ascorbic acid equivalents per mg of dry extract. Furthermore, a strong positive correlation (p < 0.0001) between the antioxidant activity, the polyphenol and the flavonoid contents. Regarding anticancer activity, the SCJ strain extract demonstrated significant anticancer activity against TNBC MDA-MB-468 cancer cells, with an inhibition percentage of 62.76 ± 0.62%, 62.67 ± 0.93%, and 58.07 ± 4.82% at 25, 50, and 100 µg/mL of the extract, respectively. The HPLC-UV/vis analysis revealed nine phenolic compounds: gallic acid, sinapic acid, p-coumaric acid, cinnamic acid, trans-fereulic acid, syringic acid, chloroqenic acid, ellagic acid, epicatechin. Streptomyces coeruleofuscus SCJ showed promise for drug discovery, exhibiting antioxidant and anticancer effects.

The Biofilm Removal and Bactericidal Effect of an 810-nm High-Power Laser on an Orthodontic Bracket Surface: An In Vitro Study.

Objective: The present study aimed to analyze the biofilm removal and bactericidal effect of laser treatment alone and laser combined with ultrasonic scaling on orthodontic brackets. It also assessed whether the use of a laser can improve the efficiency of biofilm removal and bactericidal effect compared with traditional ultrasonic instrumentation. Background Data: Streptococcus mutans (S. mutans) can lead to white spots and dental caries. Orthodontic brackets make teeth cleaning more difficult, and biofilms or bacteria on the surface of brackets worsen the oral environment, which may cause some oral diseases. Laser can be used for biofilm removal and killing bacteria on the surface of an object through thermal, photochemical, and pressure effects, which is widely used in the treatment of oral diseases. Methods: A total of 600 mandibular incisor brackets were collected for this study. Among these, 320 unused brackets were used for the S. mutans crystal violet assay (n = 160) and for S. mutans live/dead bacterial staining (n = 160). Another 280 brackets, obtained from patients who had undergone therapy for over two years, were used for the mature multispecies biofilms removal assay (n = 120) and multispecies bacterial live/dead bacterial staining (n = 160). Ultrasonic scaling, laser, and laser combined with ultrasonic scaling were applied to the labial surface of brackets covered by S. mutans biofilm or mature multispecies biofilms. Specifically, we used the following three methods: ultrasonic scaling for 10 sec without laser; 810-nm laser (Doctor Smile, Italy, LA5D0 001.1) with 0.3-mm spot size at total 21.2 kJ/cm2 for 10 sec; and 810-nm laser at total 10.6 kJ/cm2 for 5 sec, followed by ultrasonic scaling for 5 sec. The 810-nm diode laser removed biofilms with a power of 1.5 W and a power density of 2.12 kW/cm2. The S. mutans biofilm was examined using crystal violet assay, and scanning electron microscopy (SEM) was used for mature multispecies biofilms to evaluate the effect of the three methods on biofilm removal. Live/dead bacterial staining was used to examine the bactericidal effect on remaining biofilms by confocal laser scanning microscopy (CLSM). Results: For S. mutans biofilm, the optical density (OD) value and live/dead bacterial ratio in the laser and the laser combined with ultrasonic scaling groups were significantly lower than those in the ultrasonic scaling group (p < 0.05); moreover, the OD value and the live/dead bacterial ratio in laser treatment combined with ultrasonic scaling and laser treatment alone showed no significant difference (p > 0.05). For mature multispecies biofilms, the percentage of biofilm coverage after treatment was higher in the laser group than in the ultrasonic scaling group (p < 0.05) and lower in the laser combined with ultrasonic scaling group than in the ultrasonic scaling group (p < 0.05), and live/dead bacterial staining showed that laser treatment alone killed the most bacteria, followed by laser treatment combined with ultrasonic scaling, while ultrasonic scaling alone seldom killed bacteria. Conclusions: Laser treatment alone has a better bactericidal effect and can also remove more S. mutans biofilm than ultrasonic scaling alone, but it fails to remove more mature multispecies biofilms. Laser treatment combined with ultrasonic scaling can remove more S. mutans biofilm and mature multispecies biofilms than ultrasonic scaling alone and also has a better bactericidal effect than ultrasonic scaling alone on a bracket surface.

Antisalmonella Activities of Essential Oil of Psidium guajava Leaves: An In vitro and In vivo Study

Background: Life-threatening typhoidal salmonella infection is becoming more difficult to treat due to rising resistance to commonly used antibiotics. This resistance is aided by the biofilm-forming ability of the bacteria. This study investigated the in vitro and in vivo antisalmonella activities of essential oil of Psidium guajava leaves. Methods: Essential oil was extracted from the leaves of Psidium guajava with solvents. The sensitivity of Salmonella Typhi to the essential oil of Psidium guajava (EOPG) was assessed via the agar well diffusion method. Minimum inhibitory, bactericidal and biofilm-inhibitory concentrations (MIC, MBC and MBIC respectively) were measured using micro-broth dilution and crystal violet assay respectively. The effect of EOPG on the bacterial cell membrane was evaluated by measuring the efflux of potassium ions, inorganic phosphate and pyruvic acid. Bioautography and GC-MS were employed to identify the constituents of the active fraction of the EOPG. Effects of EOPG on the level of bacteremia and bone marrow infection in male Wistar rats were determined by plating caudal blood on salmonella-shigella agar. Selected serum cytokines were measured using ELISA kits. Effect on hepatic DNA was measured by comet assay while histological evaluation of the liver of infected rats was done by Hematoxylin and Eosin staining. Results: Essential oil of Psidium guajava leaves (EOPG) inhibited the growth of S. Typhi with a zone of inhibition of 13±0.2mm compared to 31±0.3mm recorded by ciprofloxacin. Minimum inhibitory, minimum bactericidal and minimum biofilm inhibitory concentrations were 25, &gt;50 and 12.5 mg/mL respectively. Exposure to EOPG caused efflux of potassium ions, inorganic phosphate and pyruvic acid in S. Typhi. Oral administration of 50 mg/mL of EOPG reduced bacterial load in the blood and bone marrow of S. Typhi-infected rats significantly (P&lt;0.05) when compared to untreated animals. Infection-induced reduction of serum IL-1β and rise in serum IFN-γ were ameliorated considerably in treated animals. The hepatic DNA of infected animals were protected from breakage in the group administered 50 mg/mL/day of EOPG compared with untreated, ciprofloxacin-treated as well as those administered 100 mg/mL/day of EOPG. Conclusion: EOPG has both in vitro and in vivo anti-salmonella activities, this could be exploited in developing new drug leads for treatment of typhoid fever

Transcriptomic Analysis of the Effect of Glabridin on Biofilm Formation in Staphylococcus Aureus.

Staphylococcus aureus (S. aureus) is among the major skin infection-causing pathogens in animals and humans. Its ability to form biofilms has become a foremost cause of bacterial infections and the extensive spread of drug resistance, which poses a great difficulty in clinical treatment. Glabridin (Glb), an extract of licorice with antibacterial and anti-infective properties, has a partially understood biofilm-inhibitory mechanism. This study investigated the inhibitory and antibiofilm activities of subinhibitory concentrations of Glb against S. aureus. The crystal violet assay revealed that Glb significantly suppressed biofilm expression. Scanning electron microscopy observations unveiled that Glb reduced S. aureus adhesion and accumulation by disrupting the spatial structure of the biofilm. In vitro extracellular DNA (eDNA) inhibition assays demonstrated that Glb inhibited biofilm formation by S. aureus by suppressing eDNA secretion. In total, 184 differentially expressed genes were obtained through transcriptomic (RNA-seq) sequencing, of which 81 and 103 genes were upregulated and downregulated, respectively. Glb regulated the transcript levels of biofilm-related genes through the phosphatase transfer system, two-component regulatory system, and nitrogen metabolism. The qPCR analysis was performed to confirm whether Glb interfered with the expression of regulatory genes involved in S. aureus biofilm formation (SarA, ArlR, FnbA, ClfA, icaD, and icaR) as well as the virulence gene Hla. In conclusion, this study demonstrates that Glb has a significant inhibitory effect on biofilm activity and is expected to be a good antibiofilm drug.

Zidovudine in synergistic combination with nitrofurantoin or omadacycline: in vitro and in murine urinary tract or lung infection evaluation against multidrug-resistant Klebsiella pneumoniae.

Limited treatment options and multidrug-resistant (MDR) Klebsiella pneumoniae present a significant therapeutic challenge, underscoring the need for novel approaches. Drug repurposing is a promising tool for augmenting the activity of many antibiotics. This study aimed to identify novel synergistic drug combinations against K. pneumoniae based on drug repurposing. We used the clinically isolated GN 172867 MDR strain of K. pneumoniae to determine the reversal resistance activity of zidovudine (AZT). The combined effects of AZT and various antibiotics, including nitrofurantoin (NIT) and omadacycline (OMC), were examined using the checkerboard method, growth curves, and crystal violet assays to assess biofilms. An in vitro combination activity testing was carried out in 12 isolates of K. pneumoniae. In vivo murine urinary tract and lung infection models were used to evaluate the therapeutic effects of AZT + NIT and AZT + OMC, respectively. The fractional inhibitory concentration index and growth curve demonstrated that AZT synergized with NIT or OMC against K. pneumoniae strains. In addition, AZT + NIT inhibited biofilm formation and cleared mature biofilms. In vivo, compared with untreated GN 172867-infected mice, AZT + NIT and AZT + OMC treatment decreased colony counts in multiple tissues (P < 0.05) and pathological scores in the bladder and kidneys (P < 0.05) and increased the survival rate by 60% (P < 0.05). This study evaluated the combination of AZT and antibiotics to treat drug-resistant K. pneumoniae infections and found novel drug combinations for the treatment of acute urinary tract infections. These findings suggest that AZT may exert significant anti-resistance activity.

Transfer of beef bacterial communities onto food-contact surfaces.

Food spoilage and pathogenic bacteria on food-contact surfaces, especially biofilm-forming strains, can transfer to meats during processing. The objectives of this study were to survey the bacterial communities of beef cuts that transfer onto two commonly used food-contact surfaces, stainless steel (SS) and high-density polyethylene (HDPE) and identify potentially biofilm-forming strains. Top round, flank, chuck, and ground beef were purchased from 3 retail stores. SS and HDPE coupons (approximately 2cm × 5cm) were placed on beef portions (3h, 10°C), after which, the coupons were submerged halfway in PBS (24h, 10°C). Bacteria from the beef cuts and coupon surfaces (n = 3) were collected, plated on tryptic soy agar plates and incubated (5 days, 25°C). Bacterial isolates were identified by 16S rRNA gene amplicon sequencing and assayed for biofilm formation using a crystal violet binding (CV) assay (72h, 10°C). Additionally, beef and coupon samples were collected for bacterial community analysis by 16S rRNA gene amplicon sequencing. Sixty-one of 972 beef isolates, 29 of 204 HDPE isolates, and 30 of 211 SS isolates were strong biofilm-formers (Absorbance>1.000 at 590 nm in the CV assay). Strong-binding isolates identified were of the genera Pseudomonas, Acinetobacter, Psychrobacter, Carnobacterium, and Brochothrix. Coupon bacterial communities among stores and cuts were distinct (p < 0.001, PERMANOVA), but there was no distinction between the communities found on HDPE or SS coupons (p > 0.050, PERMANOVA). The bacterial communities identified on the coupons may help determine the communities capable of transferring and colonizing onto surfaces, which can subsequently cross-contaminate foods.

Biofilm Formation among Staphylococcus aureus Strains from Food Contact Surfaces in Households: A Public Health Concern

Staphylococcus aureus is a foodborne pathogen of serious public health concern due to its high biofilm formation capability. One of the contributing factors to foodborne illnesses is improper cleaning and sanitization of food contact surfaces This study aimed to determine the prevalence of S. aureus with biofilm formation potential (BFP) from various food contact surfaces. A total of 100 samples were obtained, 20 from each of stainless steel spoons, plastic plates, plastic eating bowls, plastic cutting boards and glass cups. Samples were obtained by surface swabbing. The cotton swabs were streaked onto nutrient agar plates and incubated overnight. Colonies obtained were subjected to Gram staining, coagulase, catalase and DNAse tests for the identification of S. aureus. The identified S. aureus strains were subjected to biofilm formation potential assay using crystal violet assay (CV-assay) in polystyrene 96-well microtitre plates. Out of the 100 samples obtained, 26 were found to harbour S. aureus. Moreover, out of the 20 isolates, 2 (10%) were from stainless steel spoons, 8 (40%) from plastic plates, 5 (25%) from plastic-eating bowls, 10 (50%) from plastic cutting boards and 1 (5%) from glass cups. Additionally, 11 (42%) of the strains had strong BFP, 11 (42%) had moderate BFP, 3 (12%) and 1 (4%) had no BFP. Presence of S. aureus with BFP on food contact surfaces should be checked to avoid foodborne outbreaks due to this organism.