Crystal Violet Staining Assay Research Articles

Anti-biofilm properties of eucalyptol in combination with antifungals against Candida albicans isolates in patients with hematological malignancy

Oral candidiasis is a fungal infection caused mainly by Candida albicans and it is a major problem among hematologic malignancy patients. Biofilm formation is an attributable factor to both virulence and drug resistance of Candida species. The aim of the study was to evaluate the biofilm-producing ability of oral C. albicans isolates and to evaluate the inhibitory activity of eucalyptol on Candida biofilm, alone and in combination with antifungal agents. Samples were collected from the oral cavity of 106 patients with hematologic malignancy. The isolated yeasts were identified by PCR-sequencing. Then C. albicans isolates were analyzed for their biofilm-producing ability by crystal violet staining and MTT assay. The minimum biofilm inhibition concentrations (MBIC) of eucalyptol, amphotericin B, itraconazole, and nystatin and the in vitro interaction of eucalyptol with these drugs were tested according to CLSI-M-27-A3 protocol and checkerboard methods, respectively. From 106 patients, 50 (47.2%) were confirmed for oral candidiasis [mean ± SD age 39 ± 14years; female 31 (62%) and male 19 (38%)]. C. albicans was isolated from 40 of 50 (80%) patients. From 40 C. albicans isolates, 24 (60%) and 16 (40%) were moderate and weak biofilm producer, respectively. The geometric mean MBIC of amphotericin B, itraconazole, nystatin and eucalyptol were 3.93µg/mL, 12.55µg/mL, 0.75µg/mL and 798µg/mL, respectively. Eucalyptol interacted synergistically with amphotericin B, itraconazole and nystatin against 12.5, 10, and 22.5% of isolates, respectively. Eucalyptol demonstrated promising activity against biofilm of C. albicans when tested alone or combined with antifungal drugs.

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice.

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 μg/mouse) of depolymerase intraperitoneal injection protected 90%–100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

First Human Cell Experiments With FLASH Carbon Ions.

This study aimed to establish a setup for ultra-high-dose-rate (FLASH) carbon-ion irradiation, and to conduct the first human cell experiments using FLASH carbon ions. A system for FLASH carbon-ion irradiation (1-3 Gy at 13 or 50 keV/μm) was developed. The growth and senescence of HFL1 lung fibroblasts were assessed by crystal violet staining assays and senescence-associated β-galactosidase staining, respectively. Survival of HSGc-C5 cancer cells was assessed by clonogenic assays. The dose rates of carbon ions ranged from 96-195 Gy/s, meeting the definition of FLASH. With both 13 and 50 keV/μm beams, no FLASH sparing effect was observed on the growth suppression and senescence of HFL1 cells, nor on the survival of HSGc-C5 cells. We successfully conducted the first human cell experiments with FLASH carbon ions. No FLASH effect was observed under the conditions examined.

Physicochemical, Antibacterial Properties, and Compatibility of ZnO-NP/Chitosan/β-Glycerophosphate Composite Hydrogels.

In this study we aimed to develop novel ZnO-NP/chitosan/β-glycerophosphate (ZnO-NP/CS/β-GP) antibacterial hydrogels for biomedical applications. According to the mass fraction ratio of ZnO-NPs to chitosan, mixtures of 1, 3, and 5% ZnO-NPs/CS/β-GP were prepared. Using the test-tube inversion method, scanning electron microscopy and Fourier-transform infrared spectroscopy, the influence of ZnO-NPs on gelation time, chemical composition, and cross-sectional microstructures were evaluated. Adding ZnO-NPs significantly improved the hydrogel's antibacterial activity as determined by bacteriostatic zone and colony counting. The hydrogel's bacteriostatic mechanism was investigated using live/dead fluorescent staining and scanning electron microscopy. In addition, crystal violet staining and MTT assay demonstrated that ZnO-NPs/CS/β-GP exhibited good antibacterial activity in inhibiting the formation of biofilms and eradicating existing biofilms. CCK-8 and live/dead cell staining methods revealed that the cell viability of gingival fibroblasts (L929) cocultured with hydrogel in each group was above 90% after 24, 48, and 72 h. These results suggest that ZnO-NPs improve the temperature sensitivity and bacteriostatic performance of chitosan/β-glycerophosphate (CS/β-GP), which could be injected into the periodontal pocket in solution form and quickly transformed into hydrogel adhesion on the gingiva, allowing for a straightforward and convenient procedure. In conclusion, ZnO-NP/CS/β-GP thermosensitive hydrogels could be expected to be utilized as adjuvant drugs for clinical prevention and treatment of peri-implant inflammation.

Inhibitory effects of clove and oregano essential oils on biofilm formation of Salmonella Derby isolated from beef processing plant

The biofilm of Salmonella is a major source of meat contamination and disease transmission. This study aimed to investigate biofilm inhibitory effects and mechanisms of clove essential oil (CEO) and oregano essential oil (OEO) at different concentrations on Salmonella Derby (S. Derby). Crystal violet staining assay was employed to investigate the anti-biofilm effects of the essential oils (EOs), and the initial adhesion, bacterial metabolism, extracellular polymer (EPS) and microscopic examination were used to explore the inhibition mechanism. The results showed that EOs at minimum inhibitory concentrations (MIC) significantly inhibited the biofilm formation of S. Derby. At 1/8 MIC, the two EOs had no significant inhibitory effect on the growth of S. Derby planktonic bacteria, but the biofilm inhibition rates of CEO and OEO were 90.29% and 48.79%, respectively. Scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) analysis also confirmed distinguishable anti-biofilm effects of CEO and OEO. In addition, our results showed that CEO and OEO inhibited S. Derby biofilm formation by inhibiting metabolic activity and EPS production. This work provides a theoretical basis for the future mechanism research of EOs in the control of biofilm formation of S. Derby and supports the potential application of CEO and OEO in the food industry.

Antibiofilm potential of lactobionic acid against Salmonella Typhimurium

This study aimed to explore the antibiofilm activity and possible mechanism of lactobionic acid (LBA) against Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium). The minimum inhibitory concentration (MIC) of LBA against S. Typhimurium was 8 mg/mL. At sub-inhibitory concentrations (SICs), LBA inhibited the biofilm formation of S. Typhimurium on glass surface, as evidenced by the results of microscope observations. Crystal violet staining assay and cell enumeration assay revealed that LBA was also effective in inhibiting the biofilm formation on stainless steel surface. Moreover, LBA repressed the swimming and swarming motility of S. Typhimurium and also reduced the production of extracellular polysaccharide and protein during biofilm formation. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis revealed that LBA down-regulated the expression of genes involved in S. Typhimurium biofilm formation. These data suggested that LBA exerted an inhibitory effect on S. Typhimurium biofilm formation through interfering with cell motility, extracellular polysaccharide and protein production as well as expression of biofilm-related genes. Additionally, LBA at 4MIC and 8MIC had the efficacy to inactivate S. Typhimurium biofilms formed on stainless steel and chicken meat.

In vitro physicochemical characterization of five root canal sealers and their influence on an ex vivo oral multi-species biofilm community.

AimTo evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque‐derived polymicrobial community.MethodologyDental plaque‐derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio‐C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy‐dispersive spectroscopy. Data normality was assessed using the Shapiro–Wilk test. One‐way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two‐way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray–Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05).ResultsThe sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity.ConclusionDespite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

BIOFILM INHIBITION POTENTIAL OF AQUEOUS LEAF EXTRACT OF SARACA ASOCA LINN. AGAINST PSEUDOMONAS AERUGINOSA

About 95-99% of microorganisms exist in nature as biofilms. The homogenous or heterogeneous communities of pathogenic bacteria constituting microbial biofilms exert anti-microbial resistance. Several natural anti-biofilm agents have been recognized, like phytochemicals, biosurfactants, anti-microbial peptides, and microbial enzymes. The present study investigates the effect of aqueous extract of Saraca asoca Linn. on the in vitro formation of pseudomonas aeruginosa biofilm. The quantitative assessment of biofilm formation was done by crystal violet staining assay. Various factors such as temperature, pH, and concentration were optimized for enhanced effect in inhibiting the biofilm by aq. Saraca asoca leaves extract. Aq. Saraca asoca leaves extract was characterized using GC-MS analysis. Brine shrimp lethality bioassay was also carried out to investigate the cytotoxicity of aq. extract. Saraca asoca was observed as a potential source of anti-biofilm molecules and can be further harnessed to maximum potential.

Does ibuprofen affect the expression of alginate genes in pathogenic Pseudomonas aeruginosa strains?

Conversion to mucoid form is a crucial step in the pathogenesis of P. aeruginosa in burns and cystic fibrosis (CF) patients. Alginate is considered the major component of biofilm and is highly associated with the formation of mucoid biofilm in this species. Nonsteroid anti-inflammatory drugs (NSAIDs), including ibuprofen, have shown promising antibacterial and antibiofilm potential for bacterial pathogens. In this study, we aimed to evaluate the effect of ibuprofen on the expression of alginate synthetase (alg8), GDP-mannose dehydrogenase (algD), and alginate lyase (algL) genes in multiple drug-resistant (MDR) P. aeruginosa strains. The biofilm formation potential and the expression of alg8, algD, and algL among the bacteria treated with ibuprofen (at sub-inhibitory concentration) were investigated using the crystal violet staining and real-time PCR assays, respectively. The minimum inhibitory concentration of ibuprofen for the studied strains was determined 1024-2048µg/mL. We observed that ibuprofen was able to reduce bacterial biofilm by 51-77%. Also, the expression of alg8, algD, and algL decreased by 32, 52, and 48%, respectively. The reduction of the genes responsible for alginate synthesis indicates promising antivirulece potential of ibuprofen to combat P. aeruginosa infection, especially in burns and CF patients. Our findings suggest that ibuprofen could be used to reduce the pathogenicity of P. aeruginosa that could be used in combination with antibiotics to treat drug-resistant infections.

Involvement of PhoP/PhoQ two-component system in biofilm formation in Cronobacter sakazakii

The aim of this study was to investigated the effect of PhoP/PhoQ two component system on the biofilm formation of Cronobacter sakazakii BAA-894. Crystal violet staining assay and XTT assay showed that the biofilm biomass and the viability of biofilm cells were significantly reduced in ΔphoPQ strain, while these defects were reversed in the complementation strain ΔphoPQC. Moreover, the phoPQ gene deletion weakened the ability of BAA-894 strain to form biofilms on glass and silicon wafers, and affected the content of extracellular polymeric substance (EPS) in biofilms formed on stainless-steel plates. In addition, qRT-PCR results showed that the PhoP/PhoQ system of C. sakazakii played roles in regulating the expression of several genes associated with flagella and cellulose biosynthesis, which are both involved in biofilm formation. These finding suggest that the PhoP/PhoQ system plays important roles in biofilm formation in C. sakazakii and the system could be considered as a potential target for developing novel alternative strategies for controlling biofilms.

Evaluation of pathogenesis and biofilm formation ability of Yersinia pestis after 40-day exposure to simulated microgravity

AbstractWith the increase of crewed space missions and the rise of space microbiology, the research of microbes grown under microgravity environment has been attracting more attention. The research scope in space microbiology has been extended beyond pathogens directly related to spaceflight. Y. pestis, the causative agent of plague, is also of interest to researchers. After being cultivated for 40 consecutive passages in either simulated microgravity (SMG) or normal gravity (NG) conditions, the Y. pestis strain 201 cultures were analysed regarding their phenotypic features. By using crystal violet staining assays, increased biofilm amount was detected in Y. pestis grown under SMG condition. Besides that, the damage degrees of Hela cell caused by SMG-grown Y. pestis were found diminished in comparison to those under NG condition. Consistent with this observation, the death course was delayed in mice infected with SMG-grown Y. pestis, suggesting that microgravity condition can contribute the attenuated virulence. RNA-seq-based transcriptomics analysis showed that a total of 218 genes were differentially regulated, of which 91 upregulated and 127 downregulated. We found that dozens of virulence-associated genes were downregulated, which partially explained the reduced virulence of Y. pestis under SMG condition. Our study demonstrated that long-term exposure to SMG influences the pathogenesis and biofilm formation ability of Y. pestis, which provides a novel avenue to study the mechanism of physiology and virulence of this pathogen. Microgravity enhanced the ability of biofilm formation and reduced the virulence and cytotoxicity of Y. pestis. Many virulence-associated genes of Y. pestis were differentially regulated in response to the stimulated microgravity. However, there is no molecular evidence to explain the enhanced biofilm formation ability, which requires further research. Taken together, the phenotype changes of Y. pestis under SMG conditions can provide us a new research direction of its potential pathogenesis.

Antibacterial Effect of Chitosan-Modified Fe3O4 Nanozymes on Acinetobacter baumannii.

The aim of this study was to determine whether the antibacterial activity of chitosan-modified Fe3O4 (CS@Fe3O4) nanomaterials against Acinetobacter baumannii (A. baumannii) is mediated through changes in biofilm formation and reactive oxygen species (ROS) production. For this purpose, the broth dilution method was used to examine the effect of CS@Fe3O4 nanoparticles on bacterial growth. The effects of CS@Fe3O4 nanoparticles on biofilm formation were measured using a semi-quantitative crystal violet staining assay. In addition, a bacterial ROS detection kit was used to detect the production of ROS in bacteria. The results showed that CS@Fe3O4 nanoparticles had a significant inhibitory effect on the colony growth and biofilm formation of drug-resistant A. baumannii (p < 0.05). The ROS stress assay revealed significantly higher ROS levels in A. baumannii subjected to CS@Fe3O4 nanoparticle treatment than the control group (p < 0.05). Thus, we demonstrated for the first time that CS@Fe3O4 nanoparticles had an inhibitory effect on A. baumannii in vitro, and that the antibacterial effect of CS@Fe3O4 nanoparticles on drug-resistant A. baumannii was more significant than on drug-sensitive bacteria. Our findings suggest that the antibacterial mechanism of CS@Fe3O4 nanoparticles is mediated through inhibition of biofilm formation in drug-resistant bacteria, as well as stimulation of A. baumannii to produce ROS. In summary, our data indicate that CS@Fe3O4 nanoparticles could be used to treat infections caused by drug-resistant A. baumannii.

Effect of paeonol against bacterial growth, biofilm formation and dispersal of Staphylococcus aureus and Listeria monocytogenes in vitro

Previous studies have demonstrated the antibacterial activity of paeonol against bacterial pathogens, but its anti-biofilm activities against Staphylococcus aureus and Listeria monocytogenes remain largely unexplored. Here, the antibacterial and anti-biofilm activities of paeonol against S. aureus and L. monocytogenes were examined using the crystal violet staining assay (CVSA), field emission scanning electron microscopy (FESEM), and confocal laser scanning microscopy (CLSM) analysis. Paeonol effectively inhibited the growth of S. aureus and L. monocytogenes with a minimum inhibitory concentration (MIC) of 500 and 125 μg ml−1, respectively, and disrupted the integrity of cell membranes. Moreover, sub-MIC paeonol exhibited an inhibitory effect on the attachment of S. aureus and L. monocytogenes to the abiotic surface and biofilm formation. Further, paeonol effectively destroyed cell membranes within biofilms, and dispersed mature biofilms of both strains. The results indicate that paeonol might be a promising antibacterial and anti-biofilm agent for combating infections caused by S. aureus and L. monocytogenes.

Prevalence of biofilms in Candida spp. bloodstream infections: A meta-analysis.

Candida-related infections are nowadays a serious Public Health Problem emerging multidrug-resistant strains. Candida biofilm also leads bloodstream infections to invasive systemic infections. The present meta-analysis aimed to analyze Candida biofilm rate, type, and antifungal resistance among hospitalized patients between 1995 and 2020. Web of Science, Scopus, PubMed, and Google Scholar databases were searched for English papers using the following medical subject heading terms (MESH): "invasive candidiasis"; "bloodstream infections"; "biofilm formation"; "biofilm-related infections"; "mortality"; and "prevalence". The major inclusion criteria included reporting the rate of biofilm formation and the prevalence of biofilm-related to Candida species, including observational studies (more exactly, cohort, retrospective, and case-control studies). Furthermore, data regarding the mortality rate, the geographical location of the study set, and the use of anti-fungal agents in clinical isolates were also extracted from the studies. Independent extraction of articles by 2 authors using predefined data fields, including study quality indicators. A total of 31 studies from publicly available databases met our inclusion criteria. The biofilm formation in the data set varied greatly from 16 to 100% in blood samples. Most of the studies belonged to Europe (17/31) and Asia (9/31). Forest plot showed a pooled rate of biofilm formation of 80.0% (CI: 67-90), with high heterogeneity (Q = 2567.45, I2 = 98.83, τ2 = 0.150) in random effects model (p < 0.001). The funnel plot and Egger's linear regression test failed to find publication bias (p = 0.896). The mortality rate in Candida-related bloodstream infections was 37.9% of which 70.0% were from biofilm-associated infections. Furthermore, Candida isolates were also characterized in low, intermediate, or high biofilm formers through their level of biofilm mass (crystal violet staining or XTT assays) after a 24h growth. When comparing between countries, statistical differences were obtained (p = 0.0074), showing the lower and higher biofilm prevalence values in Italy and Spain, respectively. The prevalence of low, intermediate, and high biofilms were 36.2, 18.9, and 35.0% (p < 0.0001), respectively. C. tropicalis was the prevalent species in high biofilm formation (67.5%) showing statistically significant differences when compared to other Candida species, except for C. krusei and C. glabrata. Finally, the rates of antifungal resistance to fluconazole, voriconazole, and caspofungin related to biofilm were 70.5, 67.9 and 72.8% (p < 0.001), respectively. Early detection of biofilms and a better characterization of Candida spp. bloodstream infections should be considered, which eventually will help preserve public health resources and ultimately diminish mortality among patients.

Antiproliferative pharmacophore azo-hydrazone analogue BT-1F exerts death signalling pathway targeting STAT3 in solid tumour.

Anomalous activation of intra-cellular signalling cascades confers neoplastic properties on malignant cells. The JAK2/STAT3 proteins play a pivotal role in the pathogenesis of most of the solid malignancies. The over expression of STAT3 in these tumours results in an evasion of apoptosis and thereby pathogenesis. Hence, strategy to target STAT3 to regress tumour development is an emerging new concept. As an approach, anti-neoplastic drug, Azo-hydrozone analogue, BT-1F with potential anti-proliferative effect was evaluated to demonstrate its capacity to counteract STAT3 signal with mechanistic approach. Cell based screening for cytotoxicity was performed through MTT, LDH and Trypan blue. The BT-1F induced anti-clonogenic property by clonogenic assay. The apoptotic capacity was examined by crystal violet staining, flow cytometry, Annexin-FITC, DAPI and TUNEL assay. The altered signalling events were studied using immunoblot. The drug-induced anti-tumour effect was evaluated in an in-vivo solid tumour model and molecular interaction was further validated by in-silico studies. The BT-1F exerts chemo-sensitivity specifically against EAC and A549 cells without altering its normal counterpart. The anti-proliferative/anti-clonogenic effect was due to the induction of apoptosisthrough inhibition of STAT3Tyr705 signal. Eventuallydownstream signalling proteins p53, Bax, Bad and Bcl-xL were significantly altered. Further in-vivo experimental results validated in-vitro findings.Thecomputational approaches assuresthe BT-1F efficiencyin binding with STAT3. Systemic validation of STAT3 target drug, BT-1F in in-vitro, in-silico and in-vivo models has promising strategy for solid cancer treatment.

HSP70 alleviates sepsis-induced cardiomyopathy by attenuating mitochondrial dysfunction-initiated NLRP3 inflammasome-mediated pyroptosis in cardiomyocytes.

Sepsis-induced cardiomyopathy (SIC) is an identified serious complication of sepsis that is associated with adverse outcomes and high mortality. Heat shock proteins (HSPs) have been implicated in suppressing septic inflammation. The aim of this study was to investigate whether HSP70 can attenuate cellular mitochondrial dysfunction, exuberated inflammation and inflammasome-mediated pyroptosis for SIC intervention. Mice with cecal ligation plus perforation (CLP) and lipopolysaccharide (LPS)-treated H9C2 cardiomyocytes were used as models of SIC. The mouse survival rate, gross profile, cardiac function, pathological changes and mitochondrial function were observed by photography, echocardiography, hematoxylin-eosin staining and transmission electron microscopy. In addition, cell proliferation and the levels of cardiac troponin I (cTnI), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by Cell Counting Kit-8, crystal violet staining and enzyme-linked immunosorbent assay. Moreover, mitochondrial membrane potential was assessed by immunofluorescence staining, and dynamin-related protein 1 and pyroptosis-related molecules [nucleotide-binding domain, leucine-rich-repeat containing family pyrin domain-containing 3 (NLRP3), caspase-1, gasdermin-D (GSDMD), gasdermin-D N-terminal (GSDMD-N)] were measured by western blotting, immunoprecipitation and immunoblotting. Finally, hsp70.1 knockout mice with CLP were used to verify the effects of HSP70 on SIC and the underlying mechanism. Models of SIC were successfully established, as reduced consciousness and activity with liparotrichia in CLP mice were observed, and the survival rate and cardiac ejection fraction (EF) were decreased; conversely, the levels of cTnI, TNF-α and IL-1β and myocardial tissue damage were increased in CLP mice. In addition, LPS stimulation resulted in a reduction in cell viability, mitochondrial destabilization and activation of NLRP3-mediated pyroptosis molecules in vitro. HSP70 treatment improved myocardial tissue damage, survival rate and cardiac dysfunction caused by CLP. Additionally, HSP70 intervention reversed LPS-induced mitochondrial destabilization, inhibited activation of the NLRP3 inflammasome, caspase-1, GSDMD and GSDMD-N, and decreased pyroptosis. Finally, knockout of hsp70.1 mice with CLP aggravated cardiac dysfunction and upregulated NLRP3 inflammasome activity, and exogenous HSP70 significantly rescued these changes. It was further confirmed that HSP70 plays a protective role in SIC by attenuating mitochondrial dysfunction and inactivating pyroptotic molecules. Our study demonstrated that mitochondrial destabilization and NLRP3 inflammasome activation-mediated pyroptosis are attributed to SIC. Interestingly, HSP70 ameliorates sepsis-induced myocardial dysfunction by improving mitochondrial dysfunction and inhibiting the activation of NLRP3 inflammasome-mediated pyroptosis, and such a result may provide approaches for novel therapies for SIC.

In vitro Antifungal Activity of a Novel Antimicrobial Peptide AMP-17 Against Planktonic Cells and Biofilms of Cryptococcus neoformans.

BackgroundCryptococcus neoformans is a common human fungal pathogen in immunocompromised people, as well as a prevalent cause of meningitis in HIV-infected individuals. With the emergence of clinical fungal resistance and the shortage of antifungal drugs, it is urgent to discover novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica, has antifungal activity against C. neoformans. However, its antifungal and anti-biofilm activities remain unclear. Thus, this study aimed to evaluate the antifungal activity of AMP-17 against planktonic cells and biofilms of C. neoformans.MethodsThe minimum inhibitory concentration (MIC), the biofilm inhibitory and eradicating concentration (BIC and BEC) were determined by the broth microdilution assay or the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, respectively. The inhibitory and killing activities of AMP-17 against C. neoformans were investigated through the time-inhibition/killing kinetic curves. The potential antifungal mechanism of AMP-17 was detected by flow cytometry, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The efficiency of AMP-17 against biofilm formation or preformed biofilm was evaluated by crystal violet staining and XTT reduction assays. The morphology of pre-biofilms was tested by optical microscopy (OM) and CLSM.ResultsAMP-17 exhibited in vitro antifungal activity against C. neoformans planktonic cells and biofilms, with MICs of 4~16 μg/ml, BIC80 and BEC80 of 16~32 μg/ml, 64~128 μg/ml, respectively. In addition, the 2× and 4× MIC of AMP-17 exhibited similar inhibition levels compared to the 2× and 4× MIC of the clinical drugs FLC and AMB in C. neoformans growth. Moreover, the time-kill results showed that AMP-17 (8× MIC) did not significantly eliminate colony forming units (CFU) after 6 h of treatment; however, there was 2.9-log reduction in CFU of C. neoformans. Furthermore, increasing of the permeability of the fungal cell membrane was observed with the treatment of AMP-17, since the vast change as fungal leakage and cell membrane disruption. However, the DNA binding assay of AMP-17 indicated that the peptide did not target DNA. Besides, AMP-17 was superior in inhibiting and eradicating biofilms of C. neoformans compared with FLC.ConclusionAMP-17 exhibited potential in vitro antifungal activity against the planktonic cells and biofilms of C. neoformans, and it may disrupt fungal cell membranes through multi-target interactions, which provides a promising therapeutic strategy and experimental basis for Cryptococcus-associated infections.

Comparison of Crystal Violet Staining Assay and XTT Methods in the Evaluation of Biofilm Formation in Candida parapsilosis Candidemia Isolates

Biofilm formation is one of the most important virulence factors for Candida species. Biofilm can be defined as a whole microbial community formed by microorganisms producing extracellular polymers and clustering in the matrix (...)

Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 × 10-11 to 2.80 × 10-3 *s-1 per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Attenuation of Quorum Sensing Controlled Virulence Factors and Biofilm Formation by Edible Fruit Extract of Coccinia indica against Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that mainly infects immunocompromised individuals and patients with urinary tract infection and chronic infections of the respiratory pathways, including cystic fibrosis. Many quorum sensing (QS) controlled components such as bio surfactants and swarming motilities play an important role in the establishment of biofilms. Targeting these factors through anti-QS strategies prevent biofilm formation and treating infections. Coccinia indica commonly called little gourd is used to treat diabetes, wound, burn infections and has antioxidant, antibacterial and antitussive properties.&#x0D; Methods: The methanolic fruit extract of C. indica was prepared and screened for anti-QS and anti-biofilm formation activity. Pyocyanin inhibition, rhamnolipid, crystal violet staining assay tests was performed and the extract was observed under fluorescent microscope.&#x0D; Results: The results obtained are as follows - the fruit extract inhibits the pyocyanin at 58.13% and 42.27% at 0.5 mg/ml and 1.0 mg/ml, biofilm at 69.86% and 49.06% at 0.5 mg/ml and 1.0 mg/ml, inhibits rhamnolipid assay and under fluorescent microscope it is seen scattered whereas control produce biofilm matrix like appearance.&#x0D; Conclusion: Since less study has been made on the quorum sensing and biofilm activity of C.indica our study aimed to fulfil it and it was found that it exhibits good biofilm formation and thus can be used for treating infections.