XTT Reduction Method Research Articles

Anti-Biofilm Activity of Cocultimycin A against Candida albicans

Candida albicans (C. albicans), the most common fungal pathogen, has the ability to form a biofilm, leading to enhanced virulence and antibiotic resistance. Cocultimycin A, a novel antifungal antibiotic isolated from the co-culture of two marine fungi, exhibited a potent inhibitory effect on planktonic C. albicans cells. This study aimed to evaluate the anti-biofilm activity of cocultimycin A against C. albicans and explore its underlying mechanism. Crystal violet staining showed that cocultimycin A remarkably inhibited biofilm formation in a dose-dependent manner and disrupted mature biofilms at higher concentrations. However, the metabolic activity of mature biofilms treated with lower concentrations of cocultimycin A significantly decreased when using the XTT reduction method. Cocultimycin A could inhibit yeast-to-hypha transition and mycelium formation of C. albicans colonies, which was observed through the use of a light microscope. Scanning electron microscopy revealed that biofilms treated with cocultimycin A were disrupted, yeast cells increased, and hypha cells decreased and significantly shortened. The adhesive ability of C. albicans cells treated with cocultimycin A to the medium and HOEC cells significantly decreased. Through the use of a qRT-PCR assay, the expression of multiple genes related to adhesion, hyphal formation and cell membrane changes in relation to biofilm cells treated with cocultimycin A. All these results suggested that cocultimycin A may be considered a potential novel molecule for treating and preventing biofilm-related C. albicans infections.

The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state.

Listeria monocytogenes is a typical foodborne pathogen that causes hard-to-treat bacterial infections, mainly due to its ability to form biofilm and enter into a viable but non-culturable state (VBNC). In this study, we investigated the removal effects of four antimicrobial agents on L. monocytogenes biofilms formed at 32°C and 10°C, analysed the resistances of the mature biofilms to antimicrobial agents, and explored the VBNC state of cells in mature biofilms induced by lavender essential oil (LEO). The results showed that the growth of L. monocytogenes was completely inhibited when 1·6% (v/v) of the LEO was added. Meanwhile, the results of the crystal violet staining and XTT reduction method indicated that different concentrations of LEO significantly reduced L. monocytogenes biofilms biomass and metabolic activities, followed by sodium hypochlorite, lactic acid, and hydrogen peroxide. Moreover, the confocal laser scanning microscopy (CLSM) images confirmed that the treated biofilms became thinner, the structure was sparse, and the appearance was blurry. More interestingly, L. monocytogenes biofilms developed at 10°C were less susceptible to the sanitizers than those formed at 32°C. In addition, LEO presented a more significant dispersing effect on the biofilm cells, and 1/2 MIC to 4 MIC of LEO could induce fewer VBNC state cells in biofilm and plankton compared with sodium hypochlorite. This study indicated that the LEO could be considered as an ideal antibiofilm agent for controlling L. monocytogenes. But we should pay attention to the resistance of the biofilms developed at low temperatures.

Development of novel isatin thiazolyl-pyrazoline hybrids as promising antimicrobials in MDR pathogens.

Microbial Multidrug Resistance (MDR) is an emerging global crisis. Derivatization of natural or synthetic scaffolds is among the most reliable strategies to search for and obtain novel antimicrobial agents for the treatment of MDR infections. Here, we successfully manipulated the synthetically flexible isatin moieties to synthesize 22 thiazolyl-pyrazolines hybrids, and assessed their potential antimicrobial activities in vitro against various MDR pathogens, using the broth microdilution calorimetric XTT reduction method. We chose 5 strains to represent the major MDR microorganisms, viz: Methicillin-resistant S. aureus (MRSA), and Vancomycin-resistant E. faecalis (VRE) as Gram-positive bacteria; Carbapenem-resistant K. pneumonia (CRKP), and Extended-spectrum beta-lactamase E. coli (ESBL-E), as Gram-negative bacteria; and Fluconazole-resistant C. albicans (FRCA), as a yeast-like unicellular fungus. The cytotoxicity of compounds 9f and 10h towards mammalian lung fibroblast (MRC-5) cells demonstrated their potential satisfactory safety margin as represented by their relatively high IC50 values. The target compounds showed promising anti-MDR activities, suggesting they are potential leads for further development and in vivo studies.

Exploring Photoactivated Disinfection-Induced Bystander Effects on Microbial Biofilms of Aggregatibacter actinomycetemcomitans.

Photoactivated disinfection (PAD) has been indicated as one of the potential methods for the initial periodontal therapy in periodontitis and peri-implantitis, which may have bystander effects on the viability of off-target cells. In this article, we evaluated the effects of PAD-induced bystander on biofilm formation ability, metabolic activity, and gene expression level of Aggregatibacter actinomycetemcomitans as one of the bacteria involved in causing periodontitis and peri-implantitis. Bystander effects induced by bacterial cell suspension (BCST) obtained from treated A. actinomycetemcomitans culture with indocyanine green (ICG)-PAD on biofilm formation of A. actinomycetemcomitans were determined using microbial viability assay. Additionally, metabolic activity and changes in gene expression of fimA were assessed using the XTT reduction method and quantitative real-time PCR (qRT-PCR) assay, respectively. The results revealed that 1000 and 125 μg/mL of ICG combined with diode laser irradiation for 1 min were significant and sub-significant reduction doses of ICG-PAD, respectively, versus A. actinomycetemcomitans. The biofilm formation ability, metabolic activity, as well as, expression level of fimA gene in A. actinomycetemcomitans were significantly reduced after treatment by a significant reduction dose of ICG-PAD-induced bystander effects (P<0.05). ICG-PAD could significantly reduce the microbial population, cell metabolic activity, and gene expression through the bystander effects and could enhance the efficiency of PAD as an adjunct approach for the nonsurgical treatment of periodontitis and peri-implantitis.

Changes of microbial cell survival, metabolic activity, efflux capacity, and quorum sensing ability of Aggregatibacter actinomycetemcomitans due to antimicrobial photodynamic therapy-induced bystander effects.

The bystander effects, whereby naive (bystander) microbial cells near microbial cells directly exposed to certain treatment show responses that would not have happened in the absence of the directly targeted microbial cells, is recently documented in the field of microbiology. In this article, we discuss that substantial bystander responses are also observed after antimicrobial photodynamic therapy (aPDT) using curcumin (Cur). Bystander effects induced by whole bacterial cell suspension (WBCST), cell-free supernatants fluid (CFSFT), and bacterial cell pellet (BCPT) obtained from A. actinomycetemcomitans culture treated with Cur-aPDT on cell survival, quorum sensing (QS) ability, metabolic activity and efflux capacity of A. actinomycetemcomitans were determined using microbial viability assay, Escherichia coli-based bioassay, XTT reduction method, and ethidium bromide (EtBr) accumulation assay, respectively. A. actinomycetemcomitans cell survival reduced by 82.7% (P = 0.001) and 76.2% (P = 0.01) after exposure to WBCST and CFSFT, respectively. The A. actinomycetemcomitans population increased by 5.5% (P = 0.7) after exposure to BCPT. Bacterial metabolic activity decreased by 42.6% (P = 0.02), 35.3% (P = 0.03), and 9.4% (P = 0.5) after exposure to WBCST, CFSFT, and BCPT, respectively. A. actinomycetemcomitans exposed to WBCST, CFSFT, and BCPT showed a reduction of 83.2% (P = 0.001), 77.2% (P = 0.01) and 21.9% (P = 0.09) in the QS mediator compared to the WBCSU, CFSFU, and BCPU of untreated A. actinomycetemcomitans, respectively. No significant change of the EtBr accumulation was observed in the three preparations of the Cur-aPDT-treated culture (i.e. WBCST, CFSFT, and BCPT) compared to their respective controls. The results of the current study revealed that Cur-aPDT could significantly reduce microbial cell survival, cell metabolic activity, efflux capacity, and QS ability through the bystander effects. As a result, the bystander effects of Cur-aPDT along with the direct effect of Cur-aPDT can enhance the efficiency of aPDT as an adjunct therapeutic strategy for treatment of local infections.

Biofilm formation and antifungal susceptibility of Trichosporon asahii isolates from Mexican patients

BackgroundTrichosporon asahii is a yeast-like fungus that has recently gained importance as a cause of opportunistic systemic infections. The pathogenicity and virulence factors of T. asahii remain largely unknown. Because of the association between invasive infections and the use of catheters and related devices, the ability of the microorganism to adhere and form biofilms may play an important role in the pathogenicity during a trichosporonosis. AimsThe aim of this study is to identify an association between biofilm formation by T. asahii isolates and their genotype and/or clinical source. MethodsThe biofilm production of 49 T. asahii strains isolated from Mexican patients was measured using the crystal violet stain method, and a comparison made with different adhesion phase incubation times. Antifungal susceptibility testing was performed using a modified CLSI protocol coupled with the quantification of the viable cells with the XTT reduction method. ResultsAll the T. asahii isolates assayed were able to produce biofilm in vitro, with an intraspecific variability being observed. Overall, increased biofilm production was found when extending the adhesion phase incubation time from 2 to 4h. No association could be established between the biofilm-producing phenotype and either the genotype or clinical source. Higher antifungal resistance to amphotericin B and fluconazole was linked to increased biofilm production by T. asahii. ConclusionsAll clinical isolates tested were able to produce biofilm. No association could be established between biofilm formation and genotype or clinical source.

New Triterpenoid Saponins from the Herb Hylomecon japonica.

Background: Hylomecon japonica, a plant of the Papaveraceae family which is well-known for the alkaloids they produce, is a perennial plant widely distributed in the northeast, central and east regions of China. Although a variety of chemical constituents, including alkaloids, flavonoids, and megastigmoids, have been isolated from H. japonica, the investigation of saponins in H. japonica has not been reported until now. Methods: Various separation techniques, including polyporous resin column chromatography, silica gel column chromatography and hemi-preparative HPLC were applied to the isolation of triterpenoid saponins, and chemical methods such as acid hydrolysis and spectroscopic methods including HRESIMS and NMR were applied to their structure elucidation, and the XTT reduction method was used to assay cytotoxicity. Results: Two new triterpenoid saponins, named hylomeconoside A (1) and B (2) which were identified as 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-quinovopyranoside (1) and 3-O-β-d-galactopyranosyl-(1→2)-β-d-glucuronopyranosyl-gypsogenin-28-O-β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranoside (2), and two known triterpenoid saponins identified as dubioside C (3) and lucyoside P (4) on the basis of spectroscopic and chemical evidence, were isolated from H. japonica. Compound 1 exhibited moderate cytotoxicity on MGC-803 and HL-60 cells, with IC50 values of 43.8 and 32.4 μg·mL−1, respectively. Conclusions: Compounds 1 and 2 are new saponins, and 1 is considered to be one of the antitumor principles in this plant. This is the first time that triterpenoid saponins have been isolated from plants of the Papaveraceae family.

Investigation of the correlation between biofilm forming ability of urinary Candida isolates with the use of urinary catheters and change of antifungal susceptibility in the presence of biofilm

Frequency of Candida species causing urinary tract infections is increasing, and this increase is outstanding in nosocomial urinary tract infections especially in intensive care units. The ability of biofilm formation that is contributed to the virulence of the yeast, plays a role in the pathogenesis of biomaterial-related infections and also constitutes a risk for treatment failure. The aims of this study were to compare biofilm forming abilities of Candida strains isolated from urine cultures of patients with and without urinary catheters, and to investigate the change of antifungal susceptibility in the presence of biofilm. A total of 50 Candida strains isolated from urine cultures of 25 patients with urinary catheters (10 C.tropicalis, 6 C.glabrata, 4 C.albicans, 4 C.parapsilosis, 1 C.krusei) and 25 without urinary catheters (8 C.tropicalis, 6 C.albicans, 4 C.krusei, 3 C.parapsilosis, 2 C.kefyr, 1 C.glabrata, 1 C.lusitaniae) were included in the study. Biofilm forming ability was tested by Congo red agar (CRA) and microplate XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction methods. Fluconazole (FLU) and amphotericin B (AMP-B) susceptibilities of the isolates were determined by reference microdilution method recommended by Clinical and Laboratory Standards Institute for planktonic cells and by XTT reduction assay in case of biofilm presence. Biofilm formation was detected in 12 (24%) by CRA and 50 (100%) of the isolates by XTT reduction method. None of the C.albicans (n= 10) and C.tropicalis (n= 18) strains were detected as biofilm positive by CRA, however, these strains were strongly positive by XTT reduction method. No statistically significant correlation was detected between the presence of urinary catheter and biofilm forming ability of the isolate (p> 0.05). This might be caused by the advantage of biofilm forming strains in adhesion to bladder mucosa at the initial stages of infection. For all of the isolates in planktonic form, minimum inhibitory concentration (MIC) values were below the clinical resistance breakpoints or epidemiological cut-off values. When tested in presence of biofilms, all the isolates became resistant aganist FLU. Similarly, 100% inhibition, which is recommended in the standard method to determine AMP-B MIC, could not be obtained in any of the isolates with the highest dilution 8 µg/ml AMP-B tested. When evaluation was performed according to 80% inhibition, only 14 (28%) of the isolates had an AMP-B MIC below species-specific epidemiological cut-off values in the presence of biofilm. As a result, no correlation between urinary catheters and biofilm formation ability of Candida isolates were detected. XTT reduction method was considered as more reliable than CRA for investigating biofilm formation of Candida species. In addition, CRA failed to detect biofilm formation in frequently isolated species such as C.albicans and C.tropicalis. Fluconazole activity was lost, while AMP-B could not provide 100% inhibition in presence of biofilm for all isolates tested. Even if 80% inhibition was taken into account, AMP-B activity was still variable according to strain.

Study on how nanosilver-based inorganic antibacterial agent functions on biofilm formation of Candida albicans, inside the oral cavity

Background: Candida albicans is a common symbiotic fungus in the oral cavity, which can easily adhere to the surface of implanted materials. Highlighted by a broad antibacterial spectrum and potent antibacterial effects, nanosilver-based inorganic antibacterial agents (NSBIAA) are currently being hotly discussed with regard to their influences on biofilm formation of Candida albicans. Purpose: This paper aims to explore the influence of NSBIAA on biofilm formation of Candida albicans. Method: The XTT reduction method and the method of crystal violet determination were applied in measuring the influence of NSBIAA on biofilm formation of Candida albicans. In addition, biofilm morphology was determined by crystal violet staining. Result: It was observed that with the application of liquid antibacterial agent, at a concentration of 0.62 mg/ml, the biofilm activity of Candida albicans reduced (96.1 ± 3.0) %, along with a reduction in the biomass (95.4 ± 2.7) %, and biofilm formation was not observed under an inverted microscope. Conclusion: NSBIAA are able to inhibit biofilm formation.

Eradication of a Mature Methicillin-Resistant Staphylococcus aureus (MRSA) Biofilm From Acrylic Surfaces

Methicillin-resistant Staphylococcus aureus (MRSA) can grow as structured biofilm in different surfaces, including oral mucosa and denture surfaces. Such biofilms can be released into the oral fluids and aspirated, causing systemic infections such as aspiration pneumonia. This study evaluated the efficacy of two disinfectant solutions and microwave irradiation in disinfecting acrylic specimens contaminated with MRSA biofilm. Thirty-six acrylic specimens were made, sterilized and contaminated with MRSA (107 cfu/mL). After incubation (37 °C/48 h), the specimens were divided into 4 groups: not disinfected (positive control); soaking in 1% sodium hypochlorite for 10 min; soaking in 2% chlorhexidine gluconate for 10 min; and irradiating by microwave for 3 min at 650 W. The viability of cells was evaluated by XTT reduction method. All specimens from the positive control group showed biofilm formation after 48 h incubation. The mean absorbance value of the control specimens was 1.58 (OD at 492 nm). No evidence of biofilm formation was observed on specimens after the disinfection methods. Disinfection by soaking in 1% sodium hypochlorite and 2% chlorhexidine gluconate and irradiating by microwaves resulted in 100% reduction of MRSA biofilm metabolism. The use of chemical solutions and microwave irradiation was shown to be effective for eradicating mature MRSA biofilms on acrylic resin specimens.

Effectiveness of mechanical brushing with different denture cleansing agents in reducing in vitro Candida albicans biofilm viability

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis, a common disease diagnosed among denture wearers. A routine of denture cleansing is essential to prevent biofilm formation and the onset of this infection. The aim of this study was to investigate the effectiveness of combining brushing and cleansing agents in killing C. albicans biofilm. Disks of acrylic resin were made, sterilized, and inoculated with C. albicans (10(7) cfu/mL). After incubation (37°C/48 h), specimens were randomly assigned to 10 experimental groups (n=9): 5 subjected to brushing with distilled water or cleansing agents - dentifrice slurry, 2% chlorhexidine gluconate (CHX), 1% sodium hypochlorite (NaOCl), and Polident fresh cleanse(®) (combined method) - and 4 exposed to the cleansing agents without brushing (immersion). Non-cleansed specimens were used as positive controls. The viability of cells was evaluated by XTT reduction method. Results were analyzed by Mann-Whitney and Kruskal-Wallis tests (α=0.05). The combined method was significantly more effective (p<0.0001) in reducing biofilm viability than the immersion. Brushing with CHX and NaOCl resulted in 100% removal of the biofilm. Immersion in the agents reduced significantly (p<0.0001) the biofilm viability, with CHX being the most effective (p<0.0001). The use of the combined method of brushing with cleansing agents is an effective method to reduce C. albicans biofilm, being CHX and NaOCl the most effective solutions.

Adherence in vitro of Candida albicans to plasma treated acrylic resin. Effect of plasma parameters, surface roughness and salivary pellicle

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis. Objective Hence, this study investigated if surface modifications with plasma treatments could reduce the adherence of C. albicans to a denture base resin. Methods Specimens ( n = 180) with roughened and smooth surfaces were made and divided into five groups: control—specimens were left untreated; experimental groups—specimens were submitted to plasma treatments to obtain surfaces with different hydrophobicity (Ar/50 W; ArO 2/70 W; AAt/130 W) or incorporation of fluorine (Ar/SF 670 W). Contact angle measurements were performed immediately after the treatments and after immersion in water for 48 h. For each group, half of the specimens were incubated with saliva prior to the adhesion assay. The number of adherent yeasts was evaluated by XTT reduction method. Results For the experimental groups, there was significant change in the mean contact angle after 48 h of immersion in water. Groups ArO 2/70 W and ArSF 6/70 W showed significantly lower absorbance readings than the other groups, regardless the presence or absence of saliva and surface roughness. Conclusions Results demonstrated that ArO 2/70 W and ArSF 6/70 W plasma treatments showed promising potential for reducing the adherence of C. albicans to denture base resins.

Biofilm formation of the pathogens of fatal bacterial granuloma after trauma: Potential mechanism underlying the failure of traditional antibiotic treatments

The pathogen of a new type of disease - fatal bacterial granuloma after trauma (FBGT) - was found to be Propionibacterium acnes (P. acnes). Although in vitro studies showed that the pathogenic P. acnes are sensitive to conventional antibiotics, treatments of FBGT patients with these antibiotics were ineffective. The underlying mechanisms were not clear. Since P. acnes are able to form biofilm on orthopaedic biomaterials in vitro, and pathogenic P. acnes of acnes vulgaris was known to form biofilm in vivo, we hypothesize that the pathogens of FBGT are also able to form biofilm during the pathogenesis, which may be 1 of the reasons for antibiotics tolerance of FBGT. Biofilm forming capacity of the pathogens of FBGT were examined with XTT reduction method, as well as with scanning electron microscope. The effect of long-term subminimal inhibitory concentration (MIC) lincomycin on the biofilm forming ability of the pathogens was also tested. Our results show that both the type strain (NCTC737) and the pathogenic P. acnes of FBGT can form biofilm in vitro. These data demonstrated the biofilm formation of the FBGT pathogens in vitro, and its acceleration by lincomycin, which may be 1 of the major mechanisms for the failure of antibiotic treatment.

Direct enumeration of injured Escherichia coli cells harvested onto membrane filters

Four methods testing either biosynthesis capacity (Direct Viable Counts), respiratory activity (CTC and XTT reduction assays), or membrane integrity (LIVE/DEAD® BacLight™ VIABILITY kit) were performed to enumerate E. coli injured cells previously harvested onto 0.2- μm black membranes. Some of them remained viable but were no longer culturable when exposed to hyperosmotic or oxidative stresses. The damaged cellular functions were dependent on the stress applied, and the results provided by these methods (except the XTT reduction method which is not sensitive enough), gave information on cell behaviour to one stress, and on the different cellular targets of this stress. The osmotic shock in artificial seawater led to moderate plasma membrane damage (0.8 log reduction in counts determined with the LIVE/DEAD® BacLight™ VIABILITY kit after 5 days in seawater), and to similar reductions of respiration and elongation capacities (2.7 log reduction of CTC counts, and 2.6 log reduction of DVC counts, respectively). After a 30-min oxidative stress induced by peracetic acid (8 mg l −1), the plasma membranes remained intact (no reduction of LIVE/DEAD® BacLight™ VIABILITY counts), and respiration was less impaired than elongation capacity (2.1 log reduction of CTC counts and 3.1 log reduction of DVC counts, respectively).