Biofilm-forming Species Research Articles

Evaluation of efficacy of new chalcone-based endodontic irrigant against dual biofilm Enterococcus faecalis and Candida albicans: a study in vitro.

The aim of this research was to develop a chalcone-based endodontic irrigant for cleaning and disinfecting the root canal. Minimal inhibitory concentration (MIC) experiments in C. albicans and E. faecalis strains with different aminochalcones (AM) were carried out, and the compound that presented the best activity against both pathogens was chosen. The formulation of an endodontic irrigant was elaborated, tested in mono and dual specie biofilms. Disks were sterilized and then incubated with E. faecalis, C. albicans and E. faecalis and C. albicans mixed for 72h for biofilm maturation. After contamination, samples were divided in 4 experimental groups and 2 positive control group as follows: Group1: Irrigant; Group2: Irrigant + AM-38; Group3: Chlorhexidine 2% (positive control) and, Group 4: 1.0% sodium hypochlorite (positive control). The samples were analyzed by CFU/ml count. The sample was taken to sonicador to remove the cells and then plated. The toxicity was determined in vitro with human gingival fibroblast cells (HGF) and in vivo using the Galleria mellonella model. Formulation showed antimicrobial activity, with MIC on C. albicans 15.6 and E. faecalis 7.8µg/ml. Treatment with formulation in concentration 156µg/ml significantly reduced mono or dual species biofilm formation and viability (p < 0.05). The results were significant against C. albicans and E. faecalis and did not show toxicity in cells and G. mellonella. In general, the formulation showed effective antibiofilm activity, significantly reducing microorganisms, opening paths in search of new endodontic irrigants.

Hesperetin-Between the Ability to Diminish Mono- and Polymicrobial Biofilms and Toxicity.

Hesperetin is the aglycone of citrus flavonoid hesperidin. Due to the limited information regarding hesperetin antimicrobial potential and emerging need for novel antimicrobials, we have studied its antimicrobial activity (microdilution assay), antibiofilm activity with different assays in two models (mono- and polymicrobial biofilm), and toxicity (MTT and brine shrimp lethality assays). Hesperetin inhibited growth of all Candida isolates (minimal inhibitory concentration, MIC, 0.165 mg/mL), while it’s inhibitory potential towards Staphylococcus aureus was lower (MIC 4 mg/mL). Hesperetin (0.165 mg/mL) reduced ability of Candida to form biofilms and moderately reduced exopolysaccharide levels in biofilm matrix. Effect on the eradication of 24 h old C. albicans biofilms was promising at 1.320 mg/mL. Inhibition of staphylococcal biofilm formation required higher concentrations of hesperetin (<50% inhibition with MIC 4 mg/mL). Establishment of polymicrobial C. albicans-S. aureus biofilm was significantly inhibited with the lowest examined hesperetin concentration (1 mg/mL) in crystal violet and CFU assays. Hesperetin toxicity was examined towards MRC-5 fibroblasts (IC50 0.340 mg/mL) and in brine shrimp lethality assay (LC50 > 1 mg/mL). Hesperetin is efficient in combating growth and biofilm formation of Candida species. However, its antibacterial application should be further examined due to the cytotoxic effects provoked in the antibacterial concentrations.

P068 Inhibition of Candida glabrata biofilm using the combination of a novel antifungal cyclic lipopeptide and azole: An in vitro synergistic approach

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMThe general feature of the biofilm is a densely packed microbial community with an extracellular matrix composed of polysaccharides and proteins. Candida glabrata was reported as the most biofilm-forming species responsible for causing virulent Candida infections among Candida non-albicans (CNA). Candida glabrata is the most azole-resistant species of all Candida species. The early and maturing biofilms enable the Candida cells to overcome the effects of azoles and exhibit higher drug resistance. Since simple azole mono-therapy rarely eradicates recalcitrant Candida biofilms, removal of the infected device becomes necessary for curing the infection.ObjectiveThe objective was to assess the biofilm inhibition effect of antifungal cyclic lipopeptide which has been purified by a multi-step process from the cell-free supernatant of Bacillus subtilis, and to analyze the synergistic activity of the purified lipopeptide lead compound designated as AF4 with the standard antifungal fluconazole using different concentrations.MethodsBriefly, cell-free supernatant was extracted using a solvent mixture and silica gel-based adsorption chromatography. The potential antifungal cyclic lipopeptide variant AF4 was extracted from the soil isolate B. subtilis by a multi-step purification process that involves reversed-phase HPLC and exhibited a wide-spectrum of anti-Candida activity. The XTT reduction assay was used for metabolically active cells after treatment with a combination of drugs, and crystal violet (CV) assay was performed to quantify the cell biomass. Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were used to analyze the biofilm morphology and architecture of the AF4 lipopeptide at (2xMIC) and fluconazole (8x-16xMIC)-treated biofilms. For confocal microscopy-based biofilm visualization, a combination of two different dyes was used. Extracellular polymeric substances were stained by concanavalin A-Alexa Fluor 488 conjugate and, the FUN- 1 fluorescent dye was used for staining the live biofilm-forming Candida cells. After capturing the Z-stack images in CLSM, the biomass (μm3/μm2), average thickness (μm), and roughness of the biofilm were analyzed by Comstat 2.1 software.ResultsThe AFST analysis showed that the MIC of the lead antifungal lipopeptide was 8 μg/mL against the C. glabrata planktonic cells. Adding the antifungal compound at higher MICs such as by 2-folds exhibited strong antibiofilm effect that significantly enhanced the synergistic antifungal activity of the fluconazole against C. glabrata biofilm formation. Synergistic effect of fluconazole at 8x-16xMIC with 2xMIC of the lead compound AF4 on C. glabrata 24 h maturing biofilm had increased significantly as revealed by the XTT and CV assays. The results confirmed ˃ 50% inhibition of the biofilm in both 6 h developmental and 24 h maturing biofilms in 8xMIC fluconazole plus 2xMIC AF4 while compared with fluconazole alone at the same concentration. The results from the XTT and, CV assays and SEM and CLSM (Fig. 1) suggest that the combinatorial treatments of the lead antifungal lipopeptide and fluconazole at varying concentrations have the potential of eradicating or nearly eradicating the maturing biofilm of CAN.ConclusionsThe combinatorial study was found effective in combating the biofilm formation. A correlation between metabolic activity and antifungal resistance in maturing biofilms has been found.

Characterization of Cervus timorensis velvet antler and its effect on biofilm formation of Candida species.

Oral biofilms comprise extracellular polysaccharides and polymicrobial microorganisms. The objectives of the study were to characterize the deer velvet antler (DVA) compounds and their effect on Candida species biofilm formation with the hypothesis that DVA inhibits the biofilm of Candida spp. Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry (LC-QTOF-MS) was conducted to characterize the DVA compounds. To study the effect of DVA on biofilm, Candida albicans ATCC MYA-4901 (ALT5), AIDS isolate (ALC2), oral cancer isolate (ALC3), C. dubliniensis ATCC MYA-2975, C. glabrata ATCC 90030, C. krusei 14243, C. lusitaniae ATCC 34449, C. parapsilosis ATCC 22019, and C. tropicalis ATCC 13803 were inoculated with DVA in separate wells of a 96-well plate containing RPMI-1640 followed by 72h incubation. A total of 45 compounds were detected in the DVA extract. C. lusitaniae exhibited a higher percentage of biofilm biomass reduction when treated with DVA extract (66.10% ± 5.33), followed by ALC3 (44.12% ± 6.24). However, C. glabrata, C. krusei, and C. parapsilosis showed no reduction in biofilm biomass after being treated with DVA extract. Most Candida strains also exhibited decreased total cell count when treated with DVA extract, except for ALC3 and C. krusei. ALT5 had the lowest total cell count (0.17×105 cells/ml) when cultured with DVA extract. In conclusion, DVA extract inhibits Candida spp. biofilm formation except for C. glabrata, C. krusei, and C. parapsilosis.

In Vitro Activity of Water Extracts of Olive Oil against Planktonic Cells and Biofilm Formation of Arcobacter-like Species.

Extra-virgin olive oils contain many bioactive substances that are phenolic compounds. The survival of Arcobacter-like strains in non-buffered (WEOO) and buffered (BEOO) extracts of olive oils were studied. Time kill curves of different strains were measured in the environment of olive oil extracts of different grades. The activity of the extracts was also monitored for biofilm formation using the Christensen method. In vitro results revealed that extra-virgin olive oil extracts exhibited the strongest antimicrobial effects, especially non-buffered extracts, which exhibited strain inhibition after only 5 min of exposure. The weakest inhibitory effects were observed for olive oil extracts. A decrease in biofilm formation was observed in the environment of higher WEOO concentrations, although at lower concentrations of extracts, increased biofilm formation occurred due to stress conditions. The dialdehydic forms of oleuropein derivatives, hydroxytyrosol, and tyrosol were the main compounds detected by HPLC-CoulArray. The results indicate that not all olive oils had a similar bactericidal effect, and that bioactivity primarily depended on the content of certain phenolic compounds.

Synbiotic Musa acuminata skin extract and Streptococcus salivarius K12 inhibit candida species biofilm formation

This study aimed to determine the effect of synbiotic Musa acuminata skin extract (MASE) and Streptococcus salivarius K12 (K12) on Candida species biofilm formation. Liquid chromatography quadrupole time-of-flight (LC-Q-TOF-MS) was conducted to characterize MASE. To determine the effect of synbiotic on Candida biofilm, 200 µL of RPMI-1640 containing Candida, K12, and MASE were pipetted into the same well and incubated at 37 °C for 72 h. A similar protocol was repeated with K12 or MASE to determine the probiotic and prebiotic effects, respectively. Dimorphism, biofilm biomass, and Candida total cell count (TCC) were determined. A total of 60 compounds were detected in MASE. C. albicans (ALT5) and Candida lusitaniae exhibited the highest reduction in biofilm biomass when co-cultured with prebiotic (77.70 ± 7.67%) and synbiotic (97.73 ± 0.28%), respectively. All Candida spp. had decreased TCC and hyphae when co-cultured with synbiotic. In conclusion, MASE and K12 inhibit Candida biofilm formation.

Prevalence, virulence-related genes and antimicrobial resistance of Aeromonas spp. from loach Misgurnus anguillicaudatus with skin ulcer and healthy controls in Southern China

Aeromonas are primarily inhabitants of the aquatic environment, but they can also be isolated from soil, vertebrates and invertebrates. They have been identified as important pathogens in fish and are considered to be emerging pathogens in humans, causing enteric and extraintestinal diseases. The pathogenic factors associated with Aeromonas are multifactorial, involving structural components, secretion systems, quorum sensing, metal ions, extracellular enzymes and exotoxins. Aeromonas pathogenic mechanisms are complex and not well understood. In this study, we investigated the prevalence and diversity of Aeromonas species from loach with skin ulcers and healthy controls in southern China and characterized their virulence profile, antibiotic resistance and biofilm formation. A total of 104 Aeromonas strains were isolated and confirmed at the species level using housekeeping genes gyrA and rpoD and all 104 isolates were identified as: 83% of Aeromonas veronii (n = 87), 10% of Aeromonas jandaei (n = 10), 4% of Aeromonas hydrophila (n = 4), 3% of Aeromonas allosaccharophila (n = 3). Only one isolate carried all 10 virulence genes tested, while at least 3 of these genes were present in all 104 isolates. The 10 virulence genes are present in the following proportions: 64% of hlyA, 67% of ascV, 74% of aexT, 100% of aer, 98% of act, 19% of ast, 41% of alt, 56% of lip,65% of ela, 89% of fla. There are multiple combinations of virulence genes in different strains of the same species. The high prevalence of these virulence factors, even in healthy loach isolates, suggests potential pathogenicity. Furthermore, Aeromonas isolates exhibited high levels of resistance to lincomycin, nalidixic acid, ampicillin-sulbactam, erythromycin and imipenem; however, none were resistant to aztreonam. The incidence of multiple antibiotic resistance (MAR) in Aeromonas isolates was 89.4%. These isolates have a variety of resistance gene profiles, among which CphA, qnrS, sul1 and tem are the most common. Our data suggest that antimicrobial resistance in Aeromonas species of loach origin is mainly due to a wide range of acquired genes. Additionally, all the isolates formed weak biofilm or moderate biofilms in microtiter plates. The capability of such isolates to form biofilms may lead to difficulties in the treatment and control of Aeromonas-related infections. In summary, this study provides valuable data on the species prevalence, virus-related genes, antibiotic resistance, and biofilm formation of Aeromonas in diseased and healthy loaches. It will contribute to the understanding of the interaction between Aeromonas and animals and disease control.

Biofilm-formation capability depends on environmental oxygen concentrations in Candida species

BackgroundAlthough oxygen concentrations inside of the human body vary depending on organs or tissues, few reports describe the relationships between biofilm formation of Candida species and oxygen concentrations. In this study, we investigated the biofilm-forming capabilities of Candida species under various oxygen conditions. MethodsWe evaluated the adhesion and biofilm formation of Candida albicans and C. tropicalis under aerobic, microaerobic (oxygen concentration 5%), or anaerobic conditions. We also examined how oxygen concentration affects adhesion/maturation by changing adhesion/maturation phase conditions. We used crystal violet assay to estimate the approximate biofilm size, performed microscopic observation of biofilm morphology, and evaluated adhesion-associated gene expression. ResultsThe adhered amount was relatively small except for a clinical strain of C. tropicalis. Our biofilm-formation analysis showed that C. albicans formed a higher-size biofilm under aerobic conditions, while C. tropicalis favored microaerobic conditions to form mature biofilms. Our microscopic observations were consistent with these biofilm-formation analysis results. In particular, C. tropicalis exhibited more hyphal formation under microaerobic conditions. By changing the adhesion/maturation phase conditions, we represented that C. albicans had favorable biofilm-formation capability under aerobic conditions, while C. tropicalis showed enhanced biofilm formation under microaerobic adhesion conditions. In good agreement with these results, the C. tropicalis adhesion-associated gene expression tended to be higher under microaerobic or anaerobic conditions. ConclusionsC. albicans favored aerobic conditions to form biofilms, whereas C. tropicalis showed higher biofilm-formation ability and promoted hyphal growth under microaerobic conditions. These results indicate that favorable oxygen conditions significantly differ for each Candida species.

Phototherapy and optical waveguides for the treatment of infection.

With rapid emergence of multi-drug resistant microbes, it is imperative to seek alternative means for infection control. Optical waveguides are an auspicious delivery method for precise administration of phototherapy. Studies have shown that phototherapy is promising in fighting against a myriad of infectious pathogens (i.e. viruses, bacteria, fungi, and protozoa) including biofilm-forming species and drug-resistant strains while evading treatment resistance. When administered via optical waveguides, phototherapy can treat both superficial and deep-tissue infections while minimizing off-site effects that afflict conventional phototherapy and pharmacotherapy. Despite great therapeutic potential, exact mechanisms, materials, and fabrication designs to optimize this promising treatment option are underexplored. This review outlines principles and applications of phototherapy and optical waveguides for infection control. Research advances, challenges, and outlook regarding this delivery system are rigorously discussed in a hope to inspire future developments of optical waveguide-mediated phototherapy for the management of infection and beyond.

Biofilm mediated strategies to mitigate heavy metal pollution: A critical review in metal bioremediation

Mining and various industrial activities, along with generation and accumulation of heavy metals due to agricultural and domestic activities are languishing human health and the environment as a whole. Employment of conventional strategies in eradicating these toxic loads, are way expensive and concerned with the production of secondary wastes. Biofilm being a syntropic microbial consortium, through the mechanism of cell communication and signaling, quorum sensing and modulating strategies accomplish the removal of contaminant metals from environment. Such biofilm mediated bioremediation may be explicitly explained by implementing synthetic biology models and through unique mathematical modelling of mechanisms of multi species biofilm formation and survival strategies in toxic environments. Microbial metabolism for extracellular electron transfer (EET) and development of niche-specific microbial electrochemistry-driven biotechnologies, exploit the bio electrochemical system of electroactive biofilms (EABs) for removal of metal ions from environment. The multi omics platform predicts the pathways of modified metal-biofilm interaction and sequestration strategies as crucial mechanism to accelerate biofilm mediated bioremediation processes.

Chemical composition and antifungal activities of aromatic water of Zataria multiflora Boiss.

Background and Purpose: In Iranian culture, aromatic waters harboring a slight amount of essential oil have been popularly used for many years as a pleasant non-alcoholic drink with various medicinal properties.In this study, chemical composition of Zataria multiflora Boiss. (ZM) aromatic water was determined and its in vitro and in vivo antifungal properties were investigated. Materials and Methods: Chemical composition of the essential oil extracted from aromatic water (AW) of ZM was analyzed by Gas chromatography and gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity of the AW against Candida species was determined by broth micro-dilution methods. Additionally, biofilm formation inhibition and antioxidant activity of the AW were measured using XTT reduction and DPPH methods, respectively. Antifungal activities of the AW in the prevention and/or treatment of gastrointestinal (GI) candidiasis in animal models were also evaluated.Results: The GC-MS analysis revealed that the major constituents of ZM AW were Carvacrol (46.56%) and Thymol (40.67%). The ZM AW inhibited the growth and biofilm formation of Candida species in the range of 0.25-0.5 V/V. Moreover, ZM AW significantly decreased Candida colonization in therapeutic groups (P<0.05).Conclusion: Given the wide therapeutic potential of ZM AW, including antifungal and antioxidant activities, it might be possible to use it in the management of mucocutaneous or alimentary candidiasis.

Fabrication of Microstructured Surface Topologies for the Promotion of Marine Bacteria Biofilm.

Several marine bacteria of the Roseobacter group can inhibit other microorganisms and are especially antagonistic when growing in biofilms. This aptitude to naturally compete with other bacteria can reduce the need for antibiotics in large-scale aquaculture units, provided that their culture can be promoted and controlled. Micropatterned surfaces may facilitate and promote the biofilm formation of species from the Roseobacter group, due to the increased contact between the cells and the surface material. Our research goal is to fabricate biofilm-optimal micropatterned surfaces and investigate the relevant length scales for surface topographies that can promote the growth and biofilm formation of the Roseobacter group of bacteria. In a preliminary study, silicon surfaces comprising arrays of pillars and pits with different periodicities, diameters, and depths were produced by UV lithography and deep reactive ion etching (DRIE) on polished silicon wafers. The resulting surface microscale topologies were characterized via optical profilometry and scanning electron microscopy (SEM). Screening of the bacterial biofilm on the patterned surfaces was performed using green fluorescent staining (SYBR green I) and confocal laser scanning microscopy (CLSM). Our results indicate that there is a correlation between the surface morphology and the spatial organization of the bacterial biofilm.

Morphogenesis and cell ordering in confined bacterial biofilms

Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm-environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

Biosynthesized selenium nanoparticles: characterization, antimicrobial, and antibiofilm activity against Enterococcus faecalis.

BackgroundControl over microbial growth is a crucial factor in determining the success of endodontic therapy. Enterococcus faecalis is the most resistant biofilm-forming species leading to endodontic failure. Hence, the current researches are directed towards discovering materials with superior disinfection properties and lesser cytotoxicity. This study aimed to synthesize and characterize biogenically produced Selenium Nanoparticles, and to evaluate the antimicrobial and antibiofilm efficacy, against Enterococcus Faecalis, for the following test groups: Group I: Distilled water (control), Group II: SeNPs (1 mg/ml), Group III: Calcium hydroxide (1 mg/ml), Group IV: 2% Chlorhexidine gluconate (CHX), Group V: 5.25% Sodium hypochlorite (NaOCl).Materials and MethodsSelenium nanoparticles were derived using fresh guava leaves (Psidium guajava) and were characterized. The antibacterial efficacy against E. faecalis was evaluated by agar well diffusion method. The antibiofilm efficacy of the test groups was observed by viable cell count, antibiofilm assay, and Anthrone and Bradford’s tests. The morphology of the biofilms was analysed using the Scanning Electron Microscope and Fourier Transform Infrared spectroscopy.ResultsAntibacterial and antibiofilm efficacy of all tested solutions showed superior antibacterial and antibiofilm efficacy when compared to the control group. Overall, SeNPs (Group II) was the most effective against E. faecalis biofilm, followed by NaOCl (Group V), CHX (Group IV), and Ca(OH)2 (Group III).ConclusionBiogenically produced SeNPs emerged as a novel antibacterial and antibiofilm agent against E. faecalis. This nano-formulation demonstrates the potential to be developed as a root canal disinfectant combating bacterial biofilm in endodontics after the results have been clinically extrapolated.

Whole slide imaging is a high-throughput method to assess Candida biofilm formation

New strategies that enable fast and accurate visualization of Candida biofilms are necessary to better study their structure and response to antifungals agents. Here, we applied whole slide imaging (WSI) to study biofilm formation of Candida species. Three relevant biofilm-forming Candida species (C. albicans ATCC 10231, C. glabrata ATCC 2001, and C. tropicalis ATCC 750) were cultivated on glass coverslips both in presence and absence of widely used antifungals. Accumulated biofilms were stained with fluorescent markers and scanned in both bright-field and fluorescence modes using a WSI digital scanner. WSI enabled clear assessment of both size and structural features of Candida biofilms. Quantitative analyses readily detected reductions in biofilm-covered surface area upon antifungal exposure. Furthermore, we show that the overall biofilm growth can be adequately assessed across both bright-field and fluorescence modes. At the single-cell level, WSI proved adequate, as morphometric parameters evaluated with WSI did not differ significantly from those obtained with scanning electron microscopy, considered as golden standard at single-cell resolution. Thus, WSI allows for reliable visualization of Candida biofilms enabling both large-scale growth assessment and morphometric characterization of single-cell features, making it an important addition to the available microscopic toolset to image and analyse fungal biofilm growth.

Analysis of Biofilm-Related Genes and Antifungal Susceptibility Pattern of Vaginal Candida albicans and Non-Candida albicans Species.

Background Vulvovaginal candidiasis caused by Candida species is a prevalent fungal infection among women. It is believed that the pathogenesis of Candida species is linked with the production of biofilm which is considered a virulence factor for this organism. The aim of this study was molecular identification, antifungal susceptibility, biomass quantification of biofilm, and detection of virulence markers of Candida species. Methods We investigated the molecular identification of 70 vaginal isolates of Candida species, antifungal resistance to amphotericin B, fluconazole, itraconazole, and voriconazole according to CLSI M27-A3 and M27-S4, biofilm formation, and frequency analysis of biofilm-related ALS1, ALS3, and HWP1 genes. Results Our findings showed that the most common yeast isolated from vaginal discharge was C. albicans (67%), followed by the non-Candida albicans species (33%). All C. albicans complex isolates were confirmed as C. albicans by HWP-PCR, and all isolates of the C. glabrata complex were revealed to be C. glabrata sensu stricto using the multiplex PCR method. FLC resistance was observed in 23.4% of C. albicans and 7.7% of C. glabrata. The resistance rate to ITC was found in 10.6% of C. albicans. The frequency of ALS1, ALS3, and HWP1 genes among Candida species was 67.1%, 80%, and 81.4%, respectively. Biofilm formation was observed in 54.3% of Candida species, and the highest frequency detected as a virulence factor was for the ALS3 gene (97.3%) in biofilm-forming species. Discussion. Our results showed the importance of molecular epidemiology studies, investigating antifungal susceptibility profiles, and understanding the role of biofilm-related virulence markers in the pathogenesis of Candida strains.

Physiological, biochemical and proteomic insight into the response system of Streptomyces hygroscopicus OsiSh-2 to rice blast fungus toxins

Magnaporthe oryzae is a major hazard to the growth and yield of rice and other graminaceous crops. The rice endophyte Streptomyces hygroscopicus OsiSh-2 has been reported to have a strong antagonistic effect on M. oryzae. However, the defence mechanism of OsiSh-2 against pathogens remains to be established. In this study, a combination of physiological, biochemical and label-free proteomics analyses were used to investigate the global response of OsiSh-2 to the toxins of M. oryzae (Mo-toxins). When exposed to Mo-toxins, OsiSh-2 regulated its growth rate to get rapid transition to the stationary growth phase where stronger antibiotic activity was achieved. Additionally, OsiSh-2 triggered reactive oxygen species (ROS) generation, hydrolase activity and biofilm formation in response to Mo-toxin stress. Finally, proteomic profiling confirmed that OsiSh-2 possesses the following sophisticated coping strategies against Mo-toxins: (1) sensing and transmittance of Mo-toxin signals by signal-transmitting proteins; (2) damaging the toxicity of M. oryzae by hydrolytic enzymes and other secondary products; and (3) guaranteeing the secretion and function of proteins by proteases and protein isomerases. Our research may promote the understanding of the interaction between antagonistic microbes and further broaden the scope of utilizing endophytic Streptomyces for biological control.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Association of different Candida species with catheter-related candidemia, and the potential antifungal treatments against their adhesion properties and biofilm-forming capabilities.

BackgroundTo compare the adhesion properties and biofilm‐forming capabilities of 27 Candida isolates obtained from catheter‐related candidemia patients and to evaluate the inhibitory effects of antifungal agents on different Candida species.Material and MethodsSeven C. albicans, six C. parapsilosis, five C. guilliermondii, five C. tropicalis, and four C. glabrata clinical isolates were investigated. We quantified the adherence of these Candida species by flow cytometric method and evaluated the formation of biofilms by XTT reduction and crystal violet methods. Actions of micafungin (MF), fluconazole (FZ), and N‐acetylcysteine (NAC) on the adhesion and biofilm formation of different Candida species were determined.ResultsNon‐albicans Candida species were demonstrated to have stronger adhesion abilities compared with C. albicans. The biofilm‐forming capabilities of different Candida species were varied considerably, and the degree of biofilm formation might be affected by different assay approaches. Interestingly, C. parapsilosis displayed the highest biofilm formation abilities, while C. glabrata exhibited the lowest total biomass and metabolic activity. Furthermore, the inhibitory activities of MF, FZ, and NAC on fungal adhesion and biofilm formation were evaluated, and the results indicated that MF could reduce the adhesion ability and biofilm metabolism more significantly (p < 0.05), and its antifungal activity was elevated in a dose‐dependent manner.ConclusionNon‐albicans Candida species, especially C. guilliermondii, C. tropicalis, and C. parapsilosis, exhibited higher adhesion ability in catheter‐related candidemia patients. However, these Candida species had varied biofilm‐forming capabilities. MF tended to have stronger inhibitory effects against both adhesion and biofilm formation of different Candida species.

The Lack of SNARE Protein Homolog Syn8 Influences Biofilm Formation of Candida glabrata.

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.