Crystal Violet Staining Method Research Articles

Listeria monocytogenes in Fruits and Vegetables: Antimicrobial Resistance, Biofilm, and Genomic Insights

Background/Objectives: Listeria monocytogenes is a foodborne pathogen that can infect both humans and animals and cause noninvasive gastrointestinal listeriosis or invasive listeriosis. The objectives of this study were to determine the genetic diversity of L. monocytogenes; the genes associated with its resistance to antibiotics, benzalkonium chloride (BC), and cadmium chloride (CdCl2); and its biofilm formation. Methods: A total of 132 fresh fruits (44 samples) and vegetables (88 samples) were selected for this study. The genetic diversity of the isolates and the genes associated with their antibiotic resistance were determined using PCR amplification; meanwhile, their levels of susceptibility to antibiotics were determined using the agar diffusion method. Their levels of resistance to BC and CdCl2 were determined using the minimum inhibitory concentration method, and their capacity for biofilm formation was evaluated using the crystal violet staining method. Results: A total of 17 L. monocytogenes strains were collected: 12.8% (17/132) from fresh fruits and vegetables in this study. The isolates of L. monocytogenes belonged to phylogenetic groups I.1 (29.4% (5/17); serotype 1/2a) and II.2 (70.5% (12/17); serotype 1/2b); strains containing Listeria pathogenicity islands (LIPIs) were also identified at prevalence rates of 100% for LIPI-1 and LIPI-2 (17/17), 29.4% for LIPI-3 (5/17), and 11.7% for LIPI-4 (2/17). The antibiotic susceptibility tests showed that the L. monocytogenes isolates exhibited six different multiresistant patterns, with multiple antibiotic resistance (MAR) index of ≥0.46 (70.5%; 12/17); additionally, the genes Ide, tetM, and msrA, associated with efflux pump Lde, tetracycline, and ciprofloxacin resistance, were detected at 52.9% (9/17), 29.4% (5/17), and 17.6% (3/17), respectively. The phenotypic tests showed that 58.8% (10/17) of cadmium-resistant L. monocytogenes isolates had a co-resistance of 23.5% (4/17) to BC. Finally, all strains of L. monocytogenes exhibited moderate biofilm production. Conclusions: The results of this study contribute to our understanding of the persistence and genetic diversity of L. monocytogenes strains isolated from fresh fruits and vegetables; in addition, their resistance to CdCl2, which is correlated with co-resistance to BC disinfectant, is helpful for the food industry.

Anti-staphylococcal, antibiofilm and trypanocidal activities of CrataBL encapsulated into liposomes: Lectin with potential against infectious diseases

The present study aimed to evaluate the anti-staphylococcal, antibiofilm, cytotoxicity and trypanocidal activity, mechanisms of parasite death and immunomodulatory effect of CrataBL encapsulated into liposomes (CrataBL-Lipo). CrataBL-Lipo were prepared by the freeze-thaw technique and characterized. Anti-staphylococcal and antibiofilm activities of CrataBL and CrataBL-Lipo were evaluated against standard and clinical strains of Staphylococcus aureus susceptible and resistant. Thus, broth microdilution method was performed to determine the Minimum Inhibitory Concentration (MIC). Antibiofilm activity at subinhibitory concentrations was evaluated using the crystal violet staining method. Cytotoxicity of CrataBL-Lipo was verified in L929 fibroblasts and J774A.1 macrophages by determining the inhibitory concentration necessary to kill 50 % of cells (IC50). Trypanocidal activities of CrataBL-Lipo was evaluated in Trypanosoma cruzi and the efficacy was expressed as the concentration necessary to kill 50 % of parasites (EC50). The mechanisms of parasite death and immunomodulatory effect of CrataBL-Lipo were evaluated using flow cytometry analysis. CrataBL-Lipo presented Ø of 101.9 ± 1.3 nm (PDI = 0.245), ζ of +33.8 ± 1.3 mV and %EE = 80 ± 0.84 %. CrataBL-Lipo presented anti-staphylococcal activity (MIC = 0.56 mg/mL to 0.72 mg/mL). CrataBL-Lipo inhibited 45.4 %–75.6 % of biofilm formation. No cytotoxicity of CrataBL-Lipo was found (IC50 > 100 mg/L). CrataBL-Lipo presented EC50 of 1.1 mg/L, presenting autophagy, apoptosis and necrosis as death profile. In addition, CrataBL-Lipo reduced the production of IL-10 and TNF-α levels, causing an immunomodulatory effect. CrataBL-Lipo has a therapeutic potential for the treatment of staphylococcal infections and Chagas disease exhibiting a high degree of selectivity for the microorganism, and immunomodulatory properties.

Antimicrobial Sub-MIC induces Staphylococcus aureus biofilm formation without affecting the bacterial count

BackgroundBiofilm formation is an essential virulence factor that creates a highly protected growth mode for Staphylococcus aureus (S. aureus) to survive in any hostile environment. Antibiotic sub-minimal inhibitory concentration (sub-MIC) may modulate the biofilm formation ability of bacterial pathogens, thereby affecting bacterial pathogenesis and infection outcomes. Intense antimicrobial therapy to treat biofilm-associated infections can control the pathogenic infection aggravation but cannot guarantee its complete eradication.ObjectiveThis study aimed to assess the sub-MICs effect of 5 different antimicrobial classes on biofilm-forming capacity among Staphylococcus aureus clinical isolates using three different biofilm quantitation techniques.MethodsIn this study, the effects of 5 different antimicrobial agents, namely, azithromycin, gentamicin, ciprofloxacin, doxycycline, and imipenem, at sub-MICs of 12.5%, 25%, and 50% were tested on 5 different clinical isolates of S. aureus. The biofilms formed in the absence and presence of different antimicrobial sub-MICs were then assessed using the following three different techniques: the crystal violet (CV) staining method, the quantitative PCR (qPCR) method, and the spread plate method (SPM).ResultsBiofilm formation was significantly induced in 64% of the tested conditions using the CV technique. On the other hand, the qPCR quantifying the total bacterial count and the SPM quantifying the viable bacterial count showed significant induction only in 24% and 17.3%, respectively (Fig. 1). The difference between CV and the other techniques indicates an increase in biofilm biomass without an increase in bacterial growth. As expected, sub-MICs did not reduce the viable cell count, as shown by the SPM. The CV staining method revealed that sub-MICs of imipenem and ciprofloxacin had the highest significance rate (80%) showing an inductive effect on the biofilm development. On the other hand, doxycycline, azithromycin, and gentamicin displayed lower significance rates of 73%, 53%, and 47%, respectively.ConclusionExposure to sub-MIC doses of antimicrobial agents induces the biofilm-forming capacity of S. aureus via increasing the total biomass without significantly affecting the bacterial growth of viable count.

Reduction of Multispecies Biofilms on an Acrylic Denture Base Model by Antimicrobial Photodynamic Therapy Mediated by Natural Photosensitizers.

The aim of this study is to investigate the antimicrobial efficacy of antimicrobial photodynamic therapy (PDT) using natural photosensitizers (curcumin, riboflavin, and phycocyanin) and light-emitting diode (LED) irradiation against multispecies biofilms in an acrylic denture base model. Forty-five acrylic specimens were fabricated using heat-curing acrylic resin. The specimens were then infected with a mixed culture of bacterial and fungal species (including Streptococcus mutans, Streptococcus sanguinis, Candida albicans, and Candida glabrata) for 4 days. The acrylic discs were divided into nine groups, with each group containing five discs: control, 0.2% chlorhexidine, 5.25% sodium hypochlorite, curcumin, riboflavin, phycocyanin alone or along with LED. After treatment, the number of colony-forming units (CFUs) per milliliter was counted. In addition, the extent of biofilm degradation was assessed using the crystal violet staining method and scanning electron microscopy. All experimental groups exhibited a significant reduction in colony numbers for both bacterial and fungal species compared to the control (p < 0.001). The PDT groups exhibited a statistically significant reduction in colony counts for both bacteria and fungi compared to the photosensitizer-only groups. The results of this in vitro study show that PDT with natural photosensitizers and LED devices can effectively reduce the viability and eradicate the biofilm of microorganisms responsible for causing denture infections.

Phenotypic and genotypic determination of resistance to common disinfectants among strains of Acinetobacter baumannii producing and non-producing biofilm isolated from Iran

BackgroundNosocomial infections are a global problem in hospitals all around the world. It is considered a major health problem, especially in developing countries. The increase in the patient’s stay in hospitals has increased the mortality rate, and consequently, the costs drastically increase. The main purpose of using disinfectants in the hospital environment is to reduce the risk of nosocomial infections. Ethylene diamine tetra acetic acid (EDTA) causes lysis and increases susceptibility to antimicrobial agents in the planktonic form of bacteria. This substance affects the permeability of the outer membrane of bacteria. It also prevents the formation of biofilms by bacteria.Materials and methodsIn the current study, 120 isolates of Acinetobacter baumannii (A. baumannii) were confirmed by phenotypic and genotypic methods. Antibiogram was performed and then the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of isolates against 5% sodium hypochlorite, ethanol %70, sayasept-HP 2%, chlorhexidine 2%, dettol 4/8% were evaluated. In addition, the disinfectant effect was re-evaluated with the mixture of EDTA solution. All isolates were examined for biofilm presence by crystal violet staining method in triplicates and repeated three times for each strain. Also for all isolates detection of efflux pump genes (Qac-E, qacE-Δ1, SUG-E) by PCR technique was done.ResultsAntibiogram results of A. baumannii showed that 6.7% were Multi-drug-resistant (MDR), and 89.2% were Extensively drug-resistant (XDR) isolates. The highest effect of disinfectants was related to 5% sodium hypochlorite, and the least effect was 70% ethanol. EDTA increases the efficacy of selected disinfectants significantly. The highest prevalence of the efflux pump genes was related to SUG-E (95%) and Qac-E (91.7%), and, the qacE-Δ1 gene with 12.5%. The biofilm production rate was 91.3% among all isolates.ConclusionThe best and safest way to disinfect hospital floors and surfaces is to choose the right disinfectants, and learn how to use them properly. In this study, a mixture of disinfectants and EDTA had a significant effect on bactericidal activity. it was found that improper use of disinfectants, especially the use of sub-inhibitory dilutions, increases the resistance of bacteria to disinfectants.

Evaluation of In Vitro Biofilm Formation of Helicobacter pylori in Different Culture Media

Objective: Biofilms are surface-attached cell communities that play a role in the survival of bacteria. Helicobacter pylori, a gram-negative pathogen that colonizes the human gastric mucosa, forms biofilms, causing treatment failure and the risk of developing peptic ulcers, gastritis and gastric cancer in infected individuals. The aim of the study is to evaluate the biofilm formation abilities of H. pylori ATCC 26695 and three clinical strains in different culture media. Material and Method: Biofilm formation characteristics of H. pylori strains using different culture media were evaluated, and the crystal violet (CV) staining method (measured at OD 595 ) was used. Various media were used for incubating H. pylori strains: Brucella broth (BB), TSB with 10% FBS, BHI with 10% FBS, BB with 10% FBS, BB with 10% FBS + 0.25% glucose, and BB with 10% FBS + 1% glucose (incubated for 3 days). Additionally, BB with 1% FBS, BB with 10% FBS, and BB with 5% inactivated human serum were incubated for 2 and 4 days at 37°C under microaerophilic conditions. Results: It was observed that 5% inactived human serum was more effective in biofilm formation of H. pylori ATCC 26695 and three clinical strains. However, there was no biofilm production in the strains cultured with Brucella broth alone and that the strains cultured with TSB + 10% FBS could not form a strong biofilm compared to other media. Conclusion: Different culture media used for H. pylori ATCC 26695 and three clinical strains affect biofilm formation. It is thought that in vitro experiments to prevent biofilm formation may provide a solution to the prevention of H. pylori infection.

Antibiofilm Metabolites from Marine-Derived Streptomyces sp. ZS-A31.

A novel compound streptothiomycin F (1), and a new natural product, N-(5-nitropentyl)acetamide (2), were discovered alongside ten previously identified compounds (3-12) through solid fermentation of marine-derived Streptomyces sp. ZS-A31 based on rice. The chemical structures of compounds 1-2 were elucidated using 1D and 2D NMR, as well as HRESIMS data analysis. Evaluation of all isolated compounds for their antibiofilm and antibacterial activities against P. aeruginosa was carried out using microdilution and crystal violet staining methods. Results highlighted the weak potency of the known compounds lumichrome (3) and vanillic acid (7) in inhibiting biofilm formation.

Detection of SpaP and vicR Genes in Non-biofilm Producing Streptococcus mutans Isolated from Dental Caries and Plaque

Evidence accumulated over many decades has clearly shown that Streptococcus mutans is a major agent in dental caries. S. mutans SpaP gene (Cell Surface Antigen) mediates the binding of these cariogenic bacteria to tooth surfaces. The aim of this study was to detect the presence of SpaP and vicR genes in non-biofilm forming Streptococcus mutant isolated from dental plaques. In this study, 120 clinical samples were obtained from different dental clinics and hospitals in Baghdad. 26 Bacterial isolates identified as Streptococcus mutans by using selective media (Mitis-salivarius agar) and microscopic examination after gram staining then confirmed S. mutans by molecular detection using SpaP genes. Interestingly all 26 isolates were non-biofilm forming after microtiter plate assay based on the crystal violet staining method, and negatively resulted for the detection of vicR gene which responsible for biofilm formation. 10 antibiotics (Doxycycline, Ofloxacin, Tetracycline, Erythromycin, Vancomycin, Clindamycin, Rifampicin, Imipenem, Amikacin and Cefepime) were used to examine sensitivity of S. mutans isolates, all isolates were multi-drugs resist (MDR) for all 10 antibiotics used. We can conclude that absence of biofilm forming ability does not affecting antibiotic resistance and virulence of Streptococcus mutance.

Marine Microalgae Schizochytrium sp. S31: Potential Source for New Antimicrobial and Antibiofilm Agent.

The rise of antibiotic-resistant bacteria necessitates the discovery of new, safe, and bioactive antimicrobial compounds. The antibacterial and antibiofilm activity of microalgae makes them a potential candidate for developing natural antibiotics to limit microbial infection in various fields. This study aimed to analyze the antibacterial effect of the methanolic extract of Schizochytrium sp. S31 microalgae by broth microdilution and spot plate assays. The antibacterial effects of Schizochytrium sp. S31 extract was studied on gramnegative pathogens, Pseudomonas aeruginosa, Escherichia coli 35218, Klebsiella pneumonia, which cause many different human infections, and the gram-positive pathogen Streptococcus mutans. At the same time, the antibiofilm activity of the Schizochytrium sp. S31 extract on Pseudomonas aeruginosa and Escherichia coli 35218 bacteria were investigated by crystal violet staining method. Schizochytrium sp. S31 extract at a 60% concentration for 8 hours displayed the highest antibacterial activity against P. aeruginosa, E. coli 35218, and K. pneumonia, with a decrease of 87%, 92%, and 98% in cell viability, respectively. The experiment with Streptococcus mutans revealed a remarkable antibacterial effect at a 60% extract concentration for 24 hours, leading to a notable 93% reduction in cell viability. Furthermore, the extract exhibited a dose-dependent inhibition of biofilm formation in P. aeruginosa and E. coli 35218. The concentration of 60% extract was identified as the most effective dosage in terms of inhibition. This research emphasizes the potential of Schizochytrium sp. S31 as a natural antibacterial and antibiofilm agent with promising applications in the pharmaceutical sectors. This is the first study to examine the antibacterial activity of Schizochytrium sp. S31 microalgae using broth microdilution, spot plate assays, and the antibiofilm activity by a crystal staining method. The findings of this study show that Schizochytrium sp. S31 has antibacterial and antibiofilm activities against critical bacterial pathogens.

Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells.

Frankincense is produced by Boswellia trees, which can be found throughout the Middle East and parts of Africa and Asia. Boswellia serrata extract has been shown to have anti-cancer, anti-inflammatory, and antimicrobial effects. Periodontitis is an oral chronic inflammatory disease that affects nearly half of the US population. We investigated the antimicrobial effects of B. serrata extract on two oral pathogens associated with periodontitis. Using the minimum inhibitory concentration and crystal violet staining methods, we demonstrated that Porphyromonas gingivalis growth and biofilm formation were impaired by treatment with B. serrata extracts. However, the effects on Fusobacterium nucleatum growth and biofilm formation were not significant. Using quantification of colony-forming units and microscopy techniques, we also showed that concentrations of B. serrata that were not toxic for host cells decreased intracellular P. gingivalis infection in human gingival epithelial cells. Our results show antimicrobial activity of a natural product extracted from Boswellia trees (B. serrata) against periodontopathogens. Thus, B. serrata has the potential for preventing and/or treating periodontal diseases. Future studies will identify the molecular components of B. serrata extracts responsible for the beneficial effects.

Prevalence of Extensively and Pan-drug-resistant Acinetobacter spp. in Nosocomial Infections in Western Saudi Arabia

Multidrug resistance patterns of Acinetobacter spp. have led to their emergence as an important source of nosocomial infections. This study investigated the prevalence and clinical characteristics of Acinetobacter spp. in hospital-acquired wound and urinary tract infections. A total of 432 samples [wound swabs (210) and urine samples (222)] were analyzed for the presence of Acinetobacter spp. through selective culturing on MacConkey and Leeds Acinetobacter medium followed by identification with API 20E strips and Vitek 2 compact system. Antimicrobial susceptibility was assessed by adopting the disk diffusion method on Muller-Hinton agar, whereas the minimum inhibitory concentration procedure was carried out by using a ComASP™ Colistin test kit. Biofilm formation was examined using microtiter plates and following the crystal violet staining method. PCR was performed to amplify virulence (lasB, bap, and plcN) and antimicrobial resistance (blaOXA-51like) genes. The results revealed a low prevalence of Acinetobacter spp. (1.85 %) where Acinetobacter baumannii was the predominant species. Acinetobacter baumannii isolates harbored blaOXA-51-like gene to exert extensive or pan-drug resistance. Most Acinetobacter baumannii isolates demonstrated weaker to moderate biofilm-forming capabilities and carried the bap gene. Acinetobacter baumannii isolates lacked the combination of virulence factors encoding lasB and plcN genes. Acinetobacter baumannii infections are rising in Saudi Arabia. The results of this study highlight the epidemiology of virulent and antibiotic-resistant Acinetobacter spp., particularly A. baumannii, in Saudi Arabia. The detailed elaboration on the diversity, virulence, and antimicrobial susceptibility patterns of Acinetobacter spp. in Saudi Arabia requires further in-depth molecular investigations.

Antibacterial and antibiofilm potentials of Rumex dentatus root extract characterized by HPLC-ESI-Q-TOF-MS

The control of infections is one of the key strategies to treat cuts, wounds, lung, and skin infections. In this study the folkloric use of Rumex dentatus (R. dentatus) roots in the mentioned conditions was scientifically investigated. The methanolic (MeOH) crude extract of R. dentatus root was fractionated (n-hexane, ethyl acetate and water) via bioassay-guided method, and its antibacterial activity was evaluated using the agar well diffusion and Minimum inhibitory concentration (MIC) assays against clinical isolate of Pseudomonas aeruginosa (P. aeruginosa). The antibiofilm activity was measured using the crystal violet staining method. The crude extract, fractions and sub-fractions tested showed the MICs values ranging from 200 to 1000 μg/mL respectively. Among the fractions, notably, the water fraction exhibited the highest activity against P. aeruginosa. The water fraction was then subjected to thin layer chromatography (TLC). Following spectrometric analysis using HPLC-ESI-Q-TOF-MS, gallic acid and emodin were identified as the primary components within the same fraction, responsible for eliciting antibacterial and antibiofilm effects. The in-silico studies conducted with AutoDock Vina on the LasR protein, using both isolated gallic acid and emodin, confirm the binding affinity of these molecules to the active sites of the LasR protein that has regulatory role in building of biofilm formation and its pathogenicity. By scientifically validating the infection-controlling properties of R. dentatus, this research provides compelling evidence that supports its traditional use as reported in folklore. Moreover, this study contributes to our understanding of the plant's potential in managing infections, thereby substantiating its traditional therapeutic application in a scientific context.

3Y-TZP/Ta Biocermet as a Dental Material: An Analysis of the In Vitro Adherence of Streptococcus Oralis Biofilm and an In Vivo Pilot Study in Dogs.

The surface adhesion of bacterial cells and the in vivo biocompatibility of a new ceramic-metal composite made of zirconium dioxide and tantalum were evaluated. Within the framework of an in vitro study using the crystal violet staining and colony counting methods, a relatively similar adhesion of Streptococcus oralis to the 3Y-TZP/Ta biocermet (roughness Ra = 0.12 ± 0.04 µm) and Ti-Al6-V4 titanium alloy (Ra = 0.04 ± 0.01 µm) was found. In addition, in an in vivo preliminary study focused on the histological analysis of a series of rods implanted in the jaws of beagle dogs for a six-month period, the absence of any fibrous tissue or inflammatory reaction at the interface between the implanted 3Y-TZP/Ta biocermets and the new bone was found. Thus, it can be concluded that the developed ceramic-metal biocomposite may be a promising new material for use in dentistry.

Role and related mechanisms of LiaSR two-component system in acid tolerance and biofilm formation of Streptococcus mutans

Objective: To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans (Sm) 593. Methods: The growth curves of various Sm strains in pH=5.5 brian heart infusion (BHI) medium were analyzed. And colony forming unit (CFU) was also performed to evaluate the acid tolerance of Sm. Laurdan probe, H+-K+adenosine triphosphate (ATP)ase activity analysis kit, proton permeability assay and real-time fluorescence quantitative PCR (RT-qPCR) were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm. Crystal violet staining, CFU, SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms. RT-qPCR was conducted to detect the expression levels of underlying regulated genes. Results: The growth of mutants in acidic BHI were inhibited (P<0.05). The acid tolerance of mutants significantly decreased compared to the wild-type strain (P<0.05). In mutants, the activity of H+-ATPase (917.06±59.53 and 469.53±47.65) were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain (127.00±50.71) (P<0.001, P<0.001) and the encoded gene atpD (3.39±0.21 and 1.94±0.17) were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain (1.00±0.15) (P<0.001, P=0.001). The Laurdan generalized polarization of mutants (0.18±0.04 and 0.18±0.05) increased significantly compared to the wild-type strain (0.08±0.05) (P=0.006, P=0.003) and the expression levels of fabM gene were decreased in mutants (0.52±0.11 and 0.57±0.05) by 1/2 (P=0.014, P=0.022). In liaR deletion mutant, the reduced terminal pH (4.76±0.01) can also be observed (P<0.001). The total amount of the biofilms of three Sm didn't show significant differences (P>0.05). But the number of viable bacteria of mutants' biofilms were decreased [Sm 593: (12.00±2.80)×107 CFU/ml; Sm ΔliaS: (2.95±1.13)×107 CFU/ml; Sm ΔliaR: (7.25±1.60)×107 CFU/ml] (P=0.001, P=0.024). The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants' biofilms (128.73±15.65 and 46.38±5.52) compared to the wild-type strain (7.16±3.62) (P<0.001, P=0.003). Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant [(138.73±10.12) μg/ml] (P=0.003) along with the expression level of gtfC gene (1.65±0.39) (P=0.014). The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants (P<0.001, P=0.010). Conclusions: The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance. The LiaR can also decrease the proton permeability and restrict the entrance of H+. The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.

The combination of allicin with domiphen is effective against microbial biofilm formation.

Microorganisms in biofilms are particularly difficult to control because of their increased survival and antibiotic resistance. Allicin and domiphen were employed to inhibit the microbial growth and biofilm formation of Staphylococcus aureus, Escherichia coli, and Candida albicans strains. Broth microdilution method and checkerboard assay were conducted to determine the efficacy of allicin combined with domiphen against S. aureus, E. coli, and C. albicans. Microbial biofilm formation was measured using the crystal violet staining method and fluorescence microscopy. And the total viable count of the biofilm cells on material surface after the treatment with antimicrobial reagents was calculated with the plate count technique. The two drugs showed synergistic effects against the pathogens with a fractional bactericidal concentration of less than 0.38. The combination of 64 μg/mL allicin with 1 μg/mL domiphen dispersed over 50% of the biofilm mass of S. aureus, E. coli, and C. albicans. In addition, the drug combination reduced the total viable counts of E. coli and C. albicans biofilm cells on stainless steel and polyethylene surfaces by more than 102 CFU/mL. The combination of allicin and domiphen is an effective strategy for efficiently decreasing biofilms formation on various industrial materials surfaces.

The Synthesis and Antibacterial Properties of Pillar[5]arene with Streptocide Fragments.

The growing problem of bacterial resistance to antimicrobials actualizes the development of new approaches to solve this challenge. Supramolecular chemistry tools can overcome the limited bacterial resistance and side effects of classical sulfonamides that hinder their use in therapy. Here, we synthesized a number of pillar[5]arenes functionalized with different substituents, determined their ability to self-association using DLS, and characterized antimicrobial properties against S. typhimurium, K. pneumoniae, P. aeruginosa, S. epidermidis, S. aureus via a resazurin test. Biofilm prevention concentration was calculated for an agent with established antimicrobial activity by the crystal-violet staining method. We evaluated the mutagenicity of the macrocycle using the Ames test and its ability to affect the viability of A549 and LEK cells in the MTT-test. It was shown that macrocycle functionalized with sulfonamide residues exhibited antimicrobial activity an order higher than pure streptocide and also revealed the ability to prevent biofilm formation of S. aureus and P. aeruginosa. The compound did not show mutagenic activity and exhibited low toxicity to eukaryotic cells. The obtained results allow considering modification of the macrocyclic platforms with classic antimicrobials as an opportunity to give them a "second life" and return to practice with improved properties.

Chitin scaffolds derived from the marine demosponge Aplysina fistularis stimulate the differentiation of dental pulp stem cells.

The use of stem cells for tissue regeneration is a prominent trend in regenerative medicine and tissue engineering. In particular, dental pulp stem cells (DPSCs) have garnered considerable attention. When exposed to specific conditions, DPSCs have the ability to differentiate into osteoblasts and odontoblasts. Scaffolds are critical for cell differentiation because they replicate the 3D microenvironment of the niche and enhance cell adhesion, migration, and differentiation. The purpose of this study is to present the biological responses of human DPSCs to a purified 3D chitin scaffold derived from the marine demosponge Aplysina fistularis and modified with hydroxyapatite (HAp). Responses examined included proliferation, adhesion, and differentiation. The control culture consisted of the human osteoblast cell line, hFOB 1.19. Electron microscopy was used to examine the ultrastructure of the cells (transmission electron microscopy) and the surface of the scaffold (scanning electron microscopy). Cell adhesion to the scaffolds was determined by neutral red and crystal violet staining methods. An alkaline phosphatase (ALP) assay was used for assessing osteoblast/odontoblast differentiation. We evaluated the expression of osteogenic marker genes by performing ddPCR for ALP, RUNX2, and SPP1 mRNA expression levels. The results show that the chitin biomaterial provides a favorable environment for DPSC and hFOB 1.19 cell adhesion and supports both cell proliferation and differentiation. The chitin scaffold, especially with HAp modification, isolated from A. fistularis can make a significant contribution to tissue engineering and regenerative medicine.

Molecular characteristics of Staphylococcus aureus strains isolated from subclinical mastitis of water buffaloes in Guangdong Province, China.

Intramammary infections (IMI) in animals reared for milk production can result in large economic losses and distress to the animals. Staphylococcus aureus is an important causative agent of IMI in dairy cows, but its prevalence in water buffaloes has not been determined. Therefore, the current study was conducted to investigate the prevalence of subclinical mastitis in water buffaloes and the antimicrobial susceptibility, virulence genes and biofilm formation abilities of Staphylococcus aureus isolates recovered from water buffaloes in Guangdong, China. Staphylococcus aureus strains were isolated from milk samples of water buffaloes with subclinical mastitis, and twofold microdilution, PCR and crystal violet staining methods were used to determine antimicrobial susceptibility, distributions of virulence and antimicrobial resistance genes and biofilm formation ability, respectively. Our results indicated that 29.44% of water buffaloes were diagnosed with subclinical mastitis, and the most prevalent pathogens were Escherichia coli (96.17%), coagulase-negative staphylococci (CoNS) (67.60%) and S. aureus (28.57%). Most S. aureus isolates showed resistance to bacitracin, doxycycline, penicillin, florfenicol, and tetracycline but were susceptible to ciprofloxacin, ceftizoxime, cefoquinoxime, and ofloxacin. Moreover, 63.72% of S. aureus isolates were positive for tetM, and the prevalence of msrB, blaZ, mecA, fexA, and tetK ranged from 21.24 to 6.19%. All S. aureus isolates harbored clfB and icaA genes, and the virulence genes hla (93.8%), hld (91.15%), clfA (90.27%), fnbA (86.73%), and hlb (83.19%), and tsst, icaD, sec, see, fnbB, and sea showed a varied prevalence ranging from 3.5 to 65.49%. All S. aureus isolates possessed the ability to form biofilms, and 30.09% of isolates showed strong biofilm formation abilities, while 19.47% of isolates were weak biofilm producers. Our results indicated that subclinical mastitis is prevalent in water buffaloes in Guangdong, China, and S. aureus is prevalent in samples from water buffaloes with subclinical mastitis. Most S. aureus isolates were susceptible to cephalosporins and fluoroquinolones; thus, ceftizoxime and cefoquinoxime can be used to treat subclinical mastitis in water buffaloes.

Detection of virulence genes among Staphylococcus saprophyticus isolated from women with urinary tract infections: first report from Iran

ObjectiveThe purpose of the present study was to investigate the biofilm production, and the presence of virulence genes and biochemical characteristics among the S. saprophyticus clinical isolates. A total of 35 clinical isolates of S. saprophyticus were collected from patients referred to several hospitals. By the crystal violet staining method, the capability of biofilm formation was performed. The genes associated with surface of S. saprophyticus were investigated by the PCR-sequencing techniques. Hemagglutination and lipase activity assays were also performed.ResultsThe results of crystal violet staining assay showed that 32 isolates (91%) form biofilm. Moreover, seven (20%), 13 (37%), and 12(34%) isolates were categorized as weak, moderate, and strong biofilm producers, respectively. virulence genes including UafA, Aas and Ssp had an overall prevalence of 88%, 91% and 80%, respectively. None of the isolates exhibited lipolytic activities. Regarding hemagglutination properties, only 11 (31%) isolates demonstrated hemagglutination of sheep erythrocytes. The results of this study indicate a high prevalence of UafA and Aas genes that can enhance the pathogenicity of S. saprophyticus, and Identification and better understanding of the functions of these genes can be used for therapeutic purposes. Maybe in the future we will be switch to anti-adhesion therapy because of drug resistance.

The effect of carbon source and temperature on the formation and growth of Bacillus licheniformis and Bacillus cereus biofilms

This study focused on biofilm characteristics of Bacillus licheniformis and Bacillus cereus which were the residual dominant strains in milk powder production system. The effects of different carbon sources (glucose, maltose, lactose and skimmed milk) and temperatures (37 and 55 °C) on biofilm formation and metabolic activity were investigated by crystal violet staining and CCK-8 method. The results showed that the biofilm formation enhanced with the increase of the concentration of carbon source in nutrient broth. The images of the scan electron microscope and light microscope showed that glucose and maltose could significantly increase（p < 0.05） the biomass and metabolic activity of biofilm. Skimmed milk can significantly improve（p < 0.05） the biomass of Bacillus licheniformis and Bacillus cereus biofilm and the metabolic activity of Bacillus licheniformis biofilm. While lactose can only significantly improve （p < 0.05） the metabolic activity of Bacillus licheniformis biofilm, which meant that lactose was not the main component in skimmed milk that affected the biofilm forming ability of Bacillus licheniformis and Bacillus cereus. This study estimated the effect of carbon source and temperature on the formation characteristics of biofilms, and was conductive to the prevention and control of Bacillus pollution in food industry.