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

Candida albicans (C. albicans) is the most prevalent opportunistic human pathogenic fungus and can cause mucosal membrane infections and invade the blood. In the oral cavity, it can ferment dietary sugars, produce organic acids and therefore has a role in caries development. In this study, we examined whether the polyphenol rich extractions Polyphenon from green tea (PPFGT) and Padma Hepaten (PH) can inhibit the caries-inducing properties of C. albicans. Biofilms of C. albicans were grown in the presence of PPFGT and PH. Formation of biofilms was tested spectrophotometrically after crystal violet staining. Exopolysaccharides (EPS) secretion was quantified using confocal scanning laser microscopy (CSLM). Treated C. albicans morphology was demonstrated using scanning electron microscopy (SEM). Expression of virulence-related genes was tested using qRT-PCR. Development of biofilm was also tested on an orthodontic surface (Essix) to assess biofilm inhibition ability on such appliances. Both PPFGT and PH dose-dependently inhibited biofilm formation, with no inhibition on planktonic growth. The strongest inhibition was obtained using the combination of the substances. Crystal violet staining showed a significant reduction of 45% in biofilm formation using a concentration of 2.5mg/ml PPFGT and 0.16mg/ml PH. A concentration of 1.25 mg/ml PPFGT and 0.16 mg/ml PH inhibited candidal growth by 88% and EPS secretion by 74% according to CSLM. A reduction in biofilm formation and in the transition from yeast to hyphal morphotype was observed using SEM. A strong reduction was found in the expression of hwp1, eap1, and als3 virulence associated genes. These results demonstrate the inhibitory effect of natural PPFGT polyphenolic extraction on C. albicans biofilm formation and EPS secretion, alone and together with PH. In an era of increased drug resistance, the use of phytomedicine to constrain biofilm development, without killing host cells, may pave the way to a novel therapeutic concept, especially in children as orthodontic patients.

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Temperature Effect on Listeria Monocytogenes Planktonic Growth and Biofilm-Forming Ability

Journal of Veterinary Medicine and Animal Sciences ◽

10.33582/2640-1223/1044 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Joana Catarina Andrade ◽

◽

Rita Bernardo ◽

António Salvador Barreto ◽

Telmo Nunes ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Temperature Effect ◽

Optical Density ◽

Crystal Violet ◽

Biofilm Formation ◽

Viable Cell ◽

Crystal Violet Staining ◽

Cell Counts ◽

Viable Cells ◽

Density Results

Listeria Monocytogenes is an important foodborne pathogen with the capacity to grow at low temperatures and the ability to form biofilms. These features are particularly significant to food business operators producing readyto-eat foods with a long refrigerated shelf-life not undergoing any listericidal treatment before consumption. Objectives: This work aims to assess the temperature effect on L. monocytogenes growth in planktonic suspension and in mono-species biofilms. Methods and results: Isothermal planktonic growth at 12o C and 37o C was assayed using viable cell counts and optical density measurements that revealed a strong positive correlation, confirming the reliability of combining both methods to estimate L. monocytogenes concentration. Experimental data were then fitted to Baranyi and Roberts primary predictive model and the estimated growth parameters confirmed that μmax at 37o C (0.375 ± 0.072 log Cfu/ ml/h) was higher than at 120 C (0.054 ± 0.001 log Cfu/ml/h), with identical L. monocytogenes final concentrations which emphasizes its ability to grow at refrigerated temperatures. Experimental results from the isothermal growth assay and ComBase Predictor growth model were similar, with slightly higher estimated μmax (37o C: 0.480 log Cfu/ml/h; 12o C: 0.068 log Cfu/ml/h) in the predictor growth model. The studied strains demonstrated biofilm-forming ability at 12o C, 20o C and 300 C after 5 days of growth. No significant differences in biofilm formation at different temperatures were detected considering viable cell counts values, but when using crystal violet staining optical density results significant differences were found, with the highest formation occurring at 30ºC. A positive strong correlation was found between viable cell counts and crystal violet staining optical density results. In fact, both methods complement each other, because while viable cell counts measures viable cells, crystal violet staining optical density considers total biomass (viable and non-viable cells and extracellular matrix components). Nevertheless, in this work all L. monocytogenes strains revealed to be weak biofilm producers. Conclusion: Overall, this studys results contribute with important initial information on L. monocytogenes growth and biofilm formation to further assist predictive growth modeling in food matrices and environments, also enabling subsequent quantitative microbial risk assessment, to improve pathogen’s control.

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Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

Applied and Environmental Microbiology ◽

10.1128/aem.02544-14 ◽

2014 ◽

Vol 80 (22) ◽

pp. 7088-7095 ◽

Cited By ~ 22

Author(s):

Carmen Puig ◽

Arnau Domenech ◽

Junkal Garmendia ◽

Jeroen D. Langereis ◽

Pascal Mayer ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Respiratory Disease ◽

Otitis Media ◽

Crystal Violet ◽

Solid Surface ◽

Biofilm Formation ◽

Invasive Disease ◽

Crystal Violet Staining ◽

Content Type ◽

Initial Adhesion

ABSTRACTBiofilm formation by nontypeable (NT)Haemophilus influenzaeremains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NTH. influenzaeby comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NTH. influenzaeisolation, with significantly increased biofilm formation for NTH. influenzaeisolates collected from patients with invasive disease and OM compared with NTH. influenzaeisolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.

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Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates

BMC Research Notes ◽

10.1186/s13104-021-05567-y ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Narjess Bostanghadiri ◽

Abdollah Ardebili ◽

Zohreh Ghalavand ◽

Samane Teymouri ◽

Mahsa Mirzarazi ◽

...

Keyword(s):

Crystal Violet ◽

Biofilm Formation ◽

Staining Method ◽

Clinical Isolates ◽

Diffusion Method ◽

Crystal Violet Staining ◽

Disc Diffusion Method ◽

Antimicrobial Susceptibility Pattern ◽

Combination Strategies ◽

Biofilm Production

Abstract Objective The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. Results All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.

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Synergy between Indoloquinolines and Ciprofloxacin: An Antibiofilm Strategy against Pseudomonas aeruginosa

Antibiotics ◽

10.3390/antibiotics10101205 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1205

Author(s):

Emilie Charpentier ◽

Ludovic Doudet ◽

Ingrid Allart-Simon ◽

Marius Colin ◽

Sophie C. Gangloff ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Antibiotic Resistance ◽

Drug Discovery ◽

Crystal Violet ◽

Biofilm Formation ◽

Bacterial Biofilm ◽

Quinoline Derivatives ◽

Crystal Violet Staining ◽

Radical Process

Antibiotic treatments can participate in the formation of bacterial biofilm in case of under dosage. The interest of indoloquinoline scaffold for drug discovery incited us to study the preparation of new indolo [2,3-b]quinoline derivatives by a domino radical process. We tested the effect of two different “indoloquinoline” molecules (Indol-1 and Indol-2) without antimicrobial activity, in addition to ciprofloxacin, on biofilm formation thanks to crystal violet staining and enumeration of adhered bacteria. This association of ciprofloxacin and Indol-1 or Indol-2 attenuated the formation of biofilm up to almost 80% compared to ciprofloxacin alone, or even prevented the presence of adhered bacteria. In conclusion, these data prove that the association of non-antimicrobial molecules with an antibiotic can be a solution to fight against biofilm and antibiotic resistance emergence.

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In vitro Streptococcus mutans adhesion and biofilm formation on different esthetic orthodontic archwires

The Angle Orthodontist ◽

10.2319/121220-998.1 ◽

2021 ◽

Author(s):

Deise C. Oliveira ◽

Joshua J. Thomson ◽

Jamal A. Alhabeil ◽

Jonathan M. Toma ◽

Sarah C. Plecha ◽

...

Keyword(s):

Statistical Analysis ◽

Crystal Violet ◽

Biofilm Formation ◽

Clinical Use ◽

Crystal Violet Staining ◽

Orthodontic Wires ◽

Colony Forming Units ◽

Oral Biofilms ◽

Orthodontic Archwires

ABSTRACT Objectives To evaluate the ability of different esthetic archwires to retain oral biofilms in vitro. Materials and Methods Seven different brands of coated orthodontic archwires were tested: two epoxy coated, two polytetrafluoroethylene coated, two rhodium coated, and one silver plus polymer coated. Conventional uncoated metallic archwires were used as controls. Streptococus mutans adherence to archwires was quantified by colony count following 24 hours of biolfilm growth, and total wire-associated biofilm was measured using a crystal violet staining assay. For both tests, two conditions were used: 0% sucrose and 3% sucrose. For statistical analysis, P < .05 was considered as statistically significant. Results For S. mutans colony forming units per biofilm, there were no statistically significant differences among the various archwires (P = .795 for 0% sucrose; P = .905 for 3% sucrose). Regarding total biofilm formed on archwires in the 3% sucrose condition, there were statistically significant differences in crystal violet staining only for the comparison between Niti Micro Dental White and Copper Ni-Ti wires (P < .05). Conclusions The clinical use of esthetic-coated orthodontic wires may be considered to have similar risks as uncoated archwires for biofilm retention.

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Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

Journal of Medical Microbiology ◽

10.1099/jmm.0.001451 ◽

2021 ◽

Vol 70 (11) ◽

Author(s):

Letícia Roloff Stallbaum ◽

Beatriz Bohns Pruski ◽

Suelen Cavalheiro Amaral ◽

Stella Buchhorn de Freitas ◽

Daniela Rodriguero Wozeak ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Crystal Violet ◽

Biofilm Formation ◽

Staining Method ◽

Multidrug Resistant ◽

Crystal Violet Staining ◽

Content Type ◽

Klebsiella Pneumoniae Carbapenemase

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

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Biofilm-Formation-Related Genes csgD and bcsA Promote the Vertical Transmission of Salmonella Enteritidis in Chicken

Frontiers in Veterinary Science ◽

10.3389/fvets.2020.625049 ◽

2021 ◽

Vol 7 ◽

Author(s):

Sujuan Chen ◽

Zheng Feng ◽

Hualu Sun ◽

Ruonan Zhang ◽

Tao Qin ◽

...

Keyword(s):

Vertical Transmission ◽

Crystal Violet ◽

Biofilm Formation ◽

Salmonella Enteritidis ◽

Wild Type Strain ◽

Laying Hens ◽

Bacterial Load ◽

Lethal Dose ◽

Health Concern ◽

Crystal Violet Staining

The contamination of Salmonella Enteritidis in eggs and chicken meat via vertical transmission has become a worldwide public health concern. Biofilm formation by S. Enteritidis further enhances its antibacterial resistance. However, whether genes related to biofilm formation affect the level of vertical transmission is still unclear. Here, S. Enteritidis mutants ΔcsgD, ΔcsgA, ΔbcsA, and ΔadrA were constructed from wild type strain C50041 (WT), and their biofilm-forming ability was determined by Crystal violet staining assay. Then the median lethal dose (LD50) assay was performed to determine the effects of the selected genes on virulence. The bacterial load in eggs produced by infected laying hens via the intraperitoneal pathway or crop gavage was determined for evaluation of the vertical transmission. Crystal violet staining assay revealed that S. Enteritidis mutants ΔcsgD, ΔcsgA, and ΔbcsA, but not ΔadrA, impaired biofilm formation compared with WT strain. Furthermore, the LD50 in SPF chickens showed that both the ΔcsgD and ΔbcsA mutants were less virulent compared with WT strain. Among the intraperitoneally infected laying hens, the WT strain-infected group had the highest percentage of bacteria-positive eggs (24.7%), followed by the ΔadrA group (16%), ΔcsgA group (9.9%), ΔbcsA group (4.5%), and ΔcsgD group (2.1%). Similarly, among the crop gavage chickens, the WT strain group also had the highest infection percentage in eggs (10.4%), followed by the ΔcsgA group (8.5%), ΔadrA group (7.5%), ΔbcsA group (1.9%), and ΔcsgD group (1.0%). Our results indicate that the genes csgD and bcsA help vertical transmission of S. Enteritidis in chickens.

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Copper(II) and Zinc(II) Complexes with the Clinically Used Fluconazole: Comparison of Antifungal Activity and Therapeutic Potential

Pharmaceuticals ◽

10.3390/ph14010024 ◽

2020 ◽

Vol 14 (1) ◽

pp. 24

Author(s):

Nevena Lj. Stevanović ◽

Ivana Aleksic ◽

Jakob Kljun ◽

Sanja Skaro Bogojevic ◽

Aleksandar Veselinovic ◽

...

Keyword(s):

Antifungal Activity ◽

Biofilm Formation ◽

Candida Parapsilosis ◽

Therapeutic Potential ◽

A549 Cells ◽

Candida Krusei ◽

Strong Inhibition ◽

Subinhibitory Concentrations ◽

Pyocyanin Production

Copper(II) and zinc(II) complexes with clinically used antifungal drug fluconazole (fcz), {[CuCl2(fcz)2].5H2O}n, 1, and {[ZnCl2(fcz)2]·2C2H5OH}n, 2, were prepared and characterized by spectroscopic and crystallographic methods. The polymeric structure of the complexes comprises four fluconazole molecules monodentately coordinated via the triazole nitrogen and two chlorido ligands. With respect to fluconazole, complex 2 showed significantly higher antifungal activity against Candida krusei and Candida parapsilosis. All tested compounds reduced the total amount of ergosterol at subinhibitory concentrations, indicating that the mode of activity of fluconazole was retained within the complexes, which was corroborated via molecular docking with cytochrome P450 sterol 14α-demethylase (CYP51) as a target. Electrostatic, steric and internal energy interactions between the complexes and enzyme showed that 2 has higher binding potency to this target. Both complexes showed strong inhibition of C. albicans filamentation and biofilm formation at subinhibitory concentrations, with 2 being able to reduce the adherence of C. albicans to A549 cells in vitro. Complex 2 was able to reduce pyocyanin production in Pseudomonas aeruginosa between 10% and 25% and to inhibit its biofilm formation by 20% in comparison to the untreated control. These results suggest that complex 2 may be further examined in the mixed Candida-P. aeruginosa infections.

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Prevention of postoperative intrapericardial adhesion by dextrin hydrogel

General Thoracic and Cardiovascular Surgery ◽

10.1007/s11748-020-01581-2 ◽

2021 ◽

Author(s):

Satoshi Kikusaki ◽

Kazuyoshi Takagi ◽

Takahiro Shojima ◽

Kosuke Saku ◽

Tomofumi Fukuda ◽

...

Keyword(s):

Crystal Violet ◽

Proliferative Response ◽

Sham Group ◽

Cardiac Tissue ◽

Crystal Violet Staining ◽

Pericardial Cavity ◽

Uptake Assay ◽

Surgically Induced ◽

Observation Period

Abstract Objective Postoperative intrapericardial adhesion increases the risk of complications in patients undergoing reoperation. We investigated the effect of a bioabsorbable dextrin hydrogel (DHG) on the formation of intrapericardial adhesions. Methods Intrapericardial adhesion was surgically induced in Japanese white rabbits with DHG treatment (Adh + DHG) or without DHG treatment (Adh). The sham group was not treated with DHG and intrapericardial adhesion was not induced. The extent of intrapericardial adhesion was assessed by adhesion scoring and crystal violet staining of the pericardial cavity. Bromodeoxyuridine (BrdU) uptake assay was performed to assess the proliferative response to the injury in the tissue beneath the intrapericardial adhesion. Results The Adh + DHG group showed looser intrapericardial adhesions compared to the Adh group. The adhesion area of the Adh + DHG group was 4.6 ± 2.2%, whereas that of the Adh group was 32.6 ± 6.4% at the end of the 28-day observation period (p < 0.01). The induction of intrapericardial adhesion resulted in a proliferative response mainly in the cardiac tissue just beneath the adhesion. There were 48.6 ± 10.7 cells/0.1 mm2 BrdU-positive cells in the Adh + DHG group and 135.7 ± 23.8 cells/0.1 mm2 BrdU-positive cells in the Adh group on day 28 (p < 0.05). Conclusion These findings indicate that DHG effectively prevented intrapericardial adhesion in this model.

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