Objective: To explore the relationship between extracellular enzymes activity and virulence of Candida glabrata clinical isolates based on the infection model of Galleria mellonella larvae. Methods: Using experimental research methods, 71 strains of non-repetitive Candida glabrata were collected from Qinghai Provincial People's Hospital from June 2021 to January 2022. Bovine serum protein agar medium, egg yolk agar medium, sheep blood agar medium, Tween-80 agar medium and triglyceride agar medium were used to detect the aspartyl protease activity, phospholipase activity, hemolysis activity, esterase activity and lipase activity of Candida glabrata. Median lethal concentration (LC₅₀) was calculated by using 1.25×10⁸ CFU/ml,2.50×10⁸ CFU/ml,3.75×10⁸ CFU/ml,5.00×10⁸ CFU/ml suspension of Candida glabrata ATCC2001 to infect Galleria mellonella larvae. Histopathological and etiological analysis was performed to determine whether the infection model was successfully established. The clinical isolates of Candida glabrata were configured to infect Galleria mellonella larvae with LC₅₀ concentration to detect the pathogenicity of Galleria mellonella larvae.Spearman test or Pearson test were used to analyze the correlation between the extracellular enzyme activity of Candida glabrata clinical isolates and the pathogenicity of Galleria mellonella larvae. Results: 71 strains of Candida glabrata isolated clinically were detected to have low hemolytic activity after 2 days of culture. Aspartyl protease was detected after 4 days of culture, among which 7 strains (9.86%), 19 strains (26.76%) and 45 strains (63.38%) showed low, medium and high aspartyl protease activity. After 7 days of culture, 71 strains did not detect phospholipase, esterase and lipase activities. Candida glabrata on Galleria mellonella larvae of LC₅₀=2.5×10⁸ CFU/ml Fungal spore were found in the intestinal tissue pathological section of Galleria mellonella larvae in the experimental group, and Candida glabrata was identified by the microbial Mass Spectrometry after culture, while no fungi were found in the pathological section and culture of the control group. Spearman test shows that, there was a linear positive correlation between aspartyl protease activity and the survival rate of Galleria mellonella larvae (r = 0.73, P<0.01), the difference was statistically significant.Pearson test shows that, there was no significant linear relationship between hemolytic activity and survival rate of Galleria mellonella larvae (r = 0.16, P = 0.34), the difference was not statistically significant. Conclusion: The clinical isolates of Candida glabrata in this study had aspartyl protease activity and low hemolytic activity, but no phospholipase, esterase and lipase activity. The activity of aspartyl aspartyl protease of Candida glabrata was positively correlated with the pathogenicity of Galleria mellonella larvae.
Animals ; Sheep ; Larva/microbiology* ; Virulence ; Candida glabrata ; Agar ; Moths/microbiology* ; Esterases ; Aspartic Acid Proteases ; Lipase

Glioblastoma is the most common primary cranial malignancy, and chemotherapy remains an important tool for its treatment. Sanggenon C(San C), a class of natural flavonoids extracted from Morus plants, is a potential antitumor herbal monomer. In this study, the effect of San C on the growth and proliferation of glioblastoma cells was examined by methyl thiazolyl tetrazolium(MTT) assay and 5-bromodeoxyuridinc(BrdU) labeling assay. The effect of San C on the tumor cell cycle was examined by flow cytometry, and the effect of San C on clone formation and self-renewal ability of tumor cells was examined by soft agar assay. Western blot and bioinformatics analysis were used to investigate the mechanism of the antitumor activity of San C. In the presence of San C, the MTT assay showed that San C significantly inhibited the growth and proliferation of tumor cells in a dose and time-dependent manner. BrdU labeling assay showed that San C significantly attenuated the DNA replication activity in the nucleus of tumor cells. Flow cytometry confirmed that San C blocked the cell cycle of tumor cells in G_0/G_1 phase. The soft agar clone formation assay revealed that San C significantly attenuated the clone formation and self-renewal ability of tumor cells. The gene set enrichment analysis(GSEA) implied that San C inhibited the tumor cell division cycle by affecting the myelocytomatosis viral oncogene(MYC) signaling pathway. Western blot assay revealed that San C inhibited the expression of cyclin through the regulation of the MYC signaling pathway by lysine demethylase 4B(KDM4B), which ultimately inhibited the growth and proliferation of glioblastoma cells and self-renewal. In conclusion, San C exhibits the potential antitumor activity by targeting the KDM4B-MYC axis to inhibit glioblastoma cell growth, proliferation, and self-renewal.
Humans ; Glioblastoma/genetics* ; Bromodeoxyuridine/therapeutic use* ; Signal Transduction ; Proto-Oncogene Proteins c-myc/metabolism* ; Agar ; Cell Proliferation ; Cell Line, Tumor ; Apoptosis ; Jumonji Domain-Containing Histone Demethylases/metabolism*

OBJECTIVE: Agar dilution method (ADM) was used as the golden standard to evaluate the consistency of Epsilometer test (E-test) in detecting the sensitivity of Helicobacter pylori (H. pylori) to metronidazole. METHODS: From August 2018 to July 2020, patients with H. pylori infection treated for the first time in Peking University Third Hospital for gastroscopy due to dyspepsia were included in this study. Gastric mucosas were taken from the patients with H. pylori infection. H. pylori culture was performed. Both the ADM and E-test were applied to the antibiotic susceptibility of H. pylori to metro-nidazole, and the consistency and correlation between the two methods were validated. RESULTS: In the study, 105 clinical isolates of H. pylori were successfully cultured, and the minimum inhibitory concentration ≥ 8 mg/L was defined as drug resistance. Both ADM and the E-test showed high resistance rates to metronidazole, 64.8% and 62.9%, respectively. Among them, 66 drug-resistant strains were detected by ADM and E-test, and 37 were sensitive strains, so the consistency rate was 98.1%. Two strains were evaluated as drug resistance by ADM, but sensitive by the E-test, with a very major error rate of 1.9%. There was zero strain sensitive according to ADM but assessed as resistant by the E-test, so the major error rate was 0%. Taking ADM as the gold standard, the sensitivity of E-test in the detection of metronidazole susceptibility was 97.1% (95%CI: 0.888-0.995), and the specificity was 100% (95%CI: 0.883-1.000). Cohen's kappa analysis showed substantial agreement, and kappa coefficient was 0.959 (95%CI: 0.902-1.016, P < 0.001). Spearmans correlation analysis confirmed this correlation was significant (r=0.807, P < 0.001). The consistency evaluation of Bland-Altman method indicated that it was good, and there was no measured value outside the consistency interval. In this study, cost analysis, including materials and labor, showed a 32.2% higher cost per analyte for ADM as compared with the E-test (356.6 yuan vs. 269.8 yuan). CONCLUSION The susceptibility test of H. pylori to metronidazole by E-test presents better agreement with ADM. Because it is less expensive, less labor intensive, and more rapid, it is an easy and reliable method for H. pylori susceptibility testing.
Humans ; Metronidazole/therapeutic use* ; Helicobacter pylori ; Agar/therapeutic use* ; Disk Diffusion Antimicrobial Tests ; Microbial Sensitivity Tests ; Helicobacter Infections/drug therapy* ; Anti-Bacterial Agents/therapeutic use*

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.
Agar ; Bacteria ; Bacteriophages ; Caudovirales ; Classification ; DNA ; Electrophoresis ; Eukaryotic Cells ; Head ; Humans ; Livestock ; Membranes ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Microscopy, Electron, Scanning Transmission ; Microscopy, Electron, Transmission ; Sewage ; Siphoviridae ; Staphylococcal Infections ; Tail

PURPOSE: We investigated the clinical features and epidemiology of staphylococcal scalded skin syndrome (SSSS) from year 2006 to 2015 in Changwon city, Korea. METHODS: We reviewed medical records of 69 patients diagnosed with SSSS from year 2006 to 2015. Antibiotic susceptibility testing was performed by agar dilution method. Methicillin-resistant Staphylococcus aureus (MRSA) was phenotypically identified by oxacillin susceptibility testing and genotypically confirmed by the existence of the mecA gene. RESULTS: The median age of patients was 2.0 years (range 0.2–6 years). Three (4.3%), 53 (76.8%), and 13 (18.9%) patients showed the generalized type, the intermediate type, and the abortive type, respectively. Patients occurred throughout the year, but most patients occurred between July and October. MRSA was isolated from 54 of the 60 patients regardless of the clinical types. All patients recovered without any complications. CONCLUSIONS: There was a constant occurrence of SSSS patients caused by MRSA in Changwon area during 2006 and 2015. It is needed to constantly monitor the occurrence of patients with SSSS.
Agar ; Epidemiology ; Gyeongsangnam-do ; Humans ; Korea ; Medical Records ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Oxacillin ; Staphylococcal Scalded Skin Syndrome ; Staphylococcus aureus

OBJECTIVES: Irrigants are imperative in endodontic therapy for the elimination of pathogens from the infected root canal. The present study compared the antimicrobial efficacy of octenidine dihydrochloride (OCT) with chlorhexidine (CHX) and sodium hypochlorite (NaOCl) against Staphylococcus epidermidis (S. epidermidis) for root canal disinfection. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) was obtained using serial dilution method. The agar diffusion method was then used to determine the zones of inhibition for each irrigant. Lastly, forty 6-mm dentin blocks were prepared from human mandibular premolars and inoculated with S. epidermidis. Samples were randomly divided into 4 groups of 10 blocks and irrigated for 3 minutes with saline (control), 2% CHX, 3% NaOCl, or 0.1% OCT. Dentin samples were then collected immediately for microbial analysis, including an analysis of colony-forming units (CFUs). RESULTS: The MICs of each tested irrigant were 0.05% for CHX, 0.25% for NaOCl, and 0.0125% for OCT. All tested irrigants showed concentration-dependent increase in zones of inhibition, and 3% NaOCl showed the largest zone of inhibition amongst all tested irrigants (p < 0.05). There were no significant differences among the CFU measurements of 2% CHX, 3% NaOCl, and 0.1% OCT showing complete elimination of S. epidermidis in all samples. CONCLUSIONS: This study showed that OCT was comparable to or even more effective than CHX and NaOCl, demonstrating antimicrobial activity at low concentrations against S. epidermidis.
Agar ; Anti-Bacterial Agents ; Bicuspid ; Chlorhexidine ; Dental Pulp Cavity ; Dentin ; Diffusion ; Disinfection ; Humans ; In Vitro Techniques ; Methods ; Microbial Sensitivity Tests ; Sodium Hypochlorite ; Staphylococcus epidermidis ; Staphylococcus ; Stem Cells

PURPOSE: The purpose of this study was to compare the diagnostic yield of five systematic randomized protocols using 12–20 biopsy cores with variably-sized phantoms. METHODS: A total of 100 prostate phantom models were produced by casting liquid devil's tongue jelly using silicone molds. Sets of 20 phantoms were created with the following volumes: 20 mL, 40 mL, 60 mL, 80 mL, and 100 mL. Three focal lesions were created by injecting 0.5 mL of warm agar solution stained with red, blue, and green ink into each phantom model. The focal lesions were verified by ultrasonography. The systematic randomized biopsy protocols consisted of 12, 14, 16, 18, and 20 biopsy cores. The diagnostic yield of the multiple systematic biopsy protocols was compared. RESULTS: The overall detection rates of each model set were 93.3% for 20 mL, 88.3% for 40 mL, 71.7% for 60 mL, 43.3% for 80 mL, and 30.0% for 100 mL. Statistically significant differences in the detection rate were found between 40 mL and 60 mL and between 60 mL and 80 mL. No statistically significant increase in the detection rate was observed within a given volume set even when the number of core biopsies increased from 12 to 20. CONCLUSION: The diagnostic yield of systematic randomized biopsies is inversely proportional to the phantom volume.
Agar ; Amorphophallus ; Biopsy* ; Fungi ; Ink ; Prostate* ; Silicon ; Silicones ; Tongue* ; Ultrasonography

To diagnose colibacillosis, detection of O-serogroups and virulence genes has been recommended worldwide. The prevalence of virulence factors can fluctuate over time. The objectives of this study were to determine the prevalence of O-serogroups, virulence genes, and F18 subtypes among pathogenic Escherichia coli isolated from weaned piglets with diarrhea in Korea. Between 2008 and 2016, 362 E. coli were isolated from weaned piglets with diarrhea. Hemolysis was determined in blood agar, and O-serogroups were identified using the slide agglutination technique. The genes for the toxins and fimbriae were amplified by polymerase chain reaction (PCR). Real-time PCR was conducted to discriminate between F18 subtypes. Although the most prevalent serogroup was O149 (11.3%) in the last 9 years, O139 (19.1%) became the most prevalent in recent years (2015–2016). The most predominant pathotype was enterotoxigenic E. coli (61.3%). The frequencies of Shiga-like toxin-producing E. coli (STEC) (23.4%), O139 (19.1%), Stx2e (35.1%), and F18ab (48.7%) increased over the most recent years. Although enterotoxigenic E. coli was the most predominant pathotype, the frequencies of O139, Stx2e, STEC, and F18ab have increased in recent years. These results demonstrate that there have been temporal changes in the predominant O-serogroups and virulence genes over the last decade in Korea. These findings can be practicable for use in epidemiology and control measures for enteric colibacillosis in Korean piggeries.
Agar ; Agglutination ; Diarrhea ; Enterotoxigenic Escherichia coli ; Epidemiology ; Escherichia coli ; Escherichia ; Hemolysis ; Korea ; Polymerase Chain Reaction ; Prevalence ; Real-Time Polymerase Chain Reaction ; Serogroup ; Shiga-Toxigenic Escherichia coli ; Virulence Factors ; Virulence

PURPOSE: The purpose of this study was to evaluate the antimicrobial effects of a toothbrush with light-emitting diodes (LEDs) on periodontitis-associated dental biofilm attached to a zirconia surface by static and dynamic methods. MATERIALS AND METHODS: Zirconia disks (12 mm diameter, 2.5 mm thickness) were inserted into a 24-well plate (static method) or inside a Center for Disease Control and Prevention (CDC) biofilm reactor (dynamic method) to form dental biofilms using Streptococcus gordonii and Fusobacterium nucleatum. The disks with biofilm were subdivided into five treatment groups-control, commercial photodynamic therapy (PDT), toothbrush alone (B), brush with LED (BL), and brush with LED+erythrosine (BLE). After treatment, the disks were agitated to detach the bacteria, and the resulting solutions were spread directly on selective agar. The number of viable bacteria and percentage of bacterial reduction were determined from colony counts. Scanning electron microscopy (SEM) was performed to visualize alterations in bacterial morphology. RESULTS: No significant difference in biofilm formation was observed between dynamic and static methods. A significant difference was observed in the number of viable bacteria between the control and all experimental groups (P < 0.05). The percentage of bacterial reduction in the BLE group was significantly higher than in the other treated groups (P < 0.05). SEM revealed damaged bacterial cell walls in the PDT, BL, and BLE groups, but intact cell walls in the control and B groups. CONCLUSION: The findings suggest that an LED toothbrush with erythrosine is more effective than other treatments in reducing the viability of periodontitis-associated bacteria attached to zirconia in vitro.
Agar ; Bacteria ; Biofilms ; Cell Wall ; Centers for Disease Control and Prevention (U.S.) ; Dihydroergotamine ; Erythrosine ; Fusobacterium nucleatum ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Photochemotherapy ; Streptococcus gordonii ; Toothbrushing

OBJECTIVES: The purpose of this study is to determine methods of dental caries prevention by investigating the use of compounds of Diospyros kaki (D. kaki) peel, Momordica charantia (M. charantia), and Canavalia gladiata (C. gladiata) extracts to limit the cariogenic traits of Streptococcus mutans (S. mutans), such as their ability to proliferate and adhere to the tooth surface. METHODS: Broth microdilution and the agar spreading assay were used to determine the antimicrobial effect and minimum inhibitory concentration (MIC) of S. mutans extracts. In order to identify the adhesive ability of S. mutans at varying concentrations, culture plates were first stained with 1 ml of 0.01% crystal violet for 15 minutes at room temperature, and then eluted with 1 ml of EtOH:Acetone (8:2) solution for 15 minutes in a 37℃ incubator. Eluted solutions were then evaluated by use of a spectrophotometer at 575 nm. RESULTS: Experiments were conducted in order to investigate the effectiveness of D. kaki peel, M. charantia, and C. gladiata extracts on limiting the proliferation of S. mutans. The MIC was measured as an indication of whether the antibacterial activity of D. kaki peel, M. charantia, and C. gladiata extracts had a significant bacteriostatic effect on S. mutans. M. charantia extract was effective for growth inhibition on S. mutans at a minimum concentration of 0.25%. From the adhesion ability assay, M. charantia extract had an anti-adhesive effect. CONCLUSIONS: These results indicate that M. charantia extract demonstrates antibacterial activity and has an anti-adhesive effect on S. mutans. Due to these properties, M. charantia extract may be used to prevent dental caries.
Adhesives ; Agar ; Canavalia ; Dental Caries ; Diospyros ; Gentian Violet ; Incubators ; Microbial Sensitivity Tests ; Momordica charantia ; Momordica ; Streptococcus mutans ; Streptococcus ; Thiram ; Tooth