Biofilm Formation In Microtiter Plates Research Articles

Incidence of Bacillus cereus, Bacillus sporothermodurans and Geobacillus stearothermophilus in ultra-high temperature milk and biofilm formation capacity of isolates

The presence of mesophilic and thermophilic spore-forming bacteria in UHT milk, as well as biofilm formation in dairy plants, are concerning. The current study explored the spore-forming bacilli diversity in 100 samples of UHT milk (skimmed and whole). Through this work, a total of 239 isolates from UHT milk samples were obtained. B. cereus s.s. was isolated from 7 samples, B. sporothermodurans from 19 and, G. stearothermophilus from 25 samples. Genes encoding hemolysin (HBL), and non-hemolytic (NHE) enterotoxins were detected in B. cereus s.s. isolates. All isolates of B. cereus s.s. (12) B. sporothermodurans (38), and G. stearothermophilus (47) were selected to verify the ability of biofilm formation in microtiter plates. The results showed all isolates could form biofilms. The OD595 values of biofilm formation varied between 0.14 and 1.04 for B. cereus, 0.20 to 1.87 for B. sporothermodurans, and 0.49 to 2.77 for G. stearothermophilus. The data highlights that the dairy industry needs to reinforce control in the initial quality of the raw material and in CIP cleaning procedures; avoiding biofilm formation and consequently a persistent microbiota in processing plants, which can shelter pathogenic species such as B. cereus s.s.

Biofilm Formation and Quantification Using the 96-Microtiter Plate.

Biofilm formation in microtiter plates is certainly the most commonly used method to grow and study biofilm. This simple design is very popular due to its high-throughput screening capacities, low cost, and easy handling. In the protocol described here, we focus on the use of 96-well optically clear, polystyrene flat-bottom plate to study biofilm formation by Leptospira spp. and quantify the biofilm formation by crystal violet (CV) staining. We also describe an alternative method, based on phase contrast image analysis that we believe is more suitable for accurately quantifying biofilm growth by reducing handling of this fragile structure.

Single-species (bacterial, fungal, or mycobacterial) biofilms or dual-species (mycobacterial-fungal) biofilms formed in dialysis fluids

Continuous hemodialysis system monitoring is necessary to prevent microorganism growth and health problems. This study evaluates single- and dual-species biofilm formation in microtiter plates by using dialysis solutions under aerobiosis or 5% CO2 atmosphere. Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida parapsilosis sensu lato, and Mycobacterium smegmatis produce single-species biofilms in all dialysis solutions in both oxygenation conditions. Dual-species biofilm cultures grown at 5% CO2 atmosphere and in dialysate containing glucose reveal that M. smegmatis benefits from its association with C. parapsilosis. The dialysate and its constituent solutions support the growth of all the mono-species and the inter-kingdom mycobacterial/yeast biofilms in both aerobiosis and microaerophilic conditions.

Coumarin Reduces Virulence and Biofilm Formation in Pseudomonas aeruginosa by Affecting Quorum Sensing, Type III Secretion and C-di-GMP Levels.

As one of the major pathogens in wound infections, Pseudomonas aeruginosa produces several virulence factors and forms biofilms; these processes are under the regulation of various quorum sensing (QS) systems. Therefore, QS has been regarded as a promising target to treat P. aeruginosa infections. In the present study, we evaluated the effect of the plant-derived QS inhibitor coumarin on P. aeruginosa biofilms and virulence. Coumarin inhibited QS in the P. aeruginosa QSIS2 biosensor strain, reduced protease and pyocyanin production, and inhibited biofilm formation in microtiter plates in different P. aeruginosa strains. The effects of coumarin in inhibiting biofilm formation in an in vitro wound model and reducing P. aeruginosa virulence in the Lucilia sericata infection model were strain-dependent. Transcriptome analysis revealed that several key genes involved in the las, rhl, Pseudomonas quinolone signal (PQS), and integrated QS (IQS) systems were downregulated in coumarin-treated biofilms of P. aeruginosa PAO1. Coumarin also changed the expression of genes related to type III secretion and cyclic diguanylate (c-di-GMP) metabolism. The cellular c-di-GMP level of P. aeruginosa PAO1 and recent clinical P. aeruginosa strains was significantly reduced by coumarin. These results provide new evidence for the possible application of coumarin as an anti-biofilm and anti-virulence agent against P. aeruginosa in wound infections.

Biofilm Formation by Coagulase-Positive Staphylococcus aureus Isolated from Mozzarella Cheese Elaborated with Buffalo Milk and its Effect on Sensitivity to Sanitizers

Background: The buffalo milk mozzarella cheese is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the proliferation of microorganisms that can cause food-borne diseases in the consumer. Staphylococcus aureus can cause gastro-enteritis in humans by the production of enterotoxins in food. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by Staphylococcus aureus isolated from buffalo mozzarella cheese on sensitivity to sanitizers.Materials, Methods & Results: Fifty samples of buffalo mozzarella cheese were analyzed to investigate the presence of S. aureus. The isolates were obtained through microbiological analysis and identified by PCR. The similarity of the strains was compared through rep-PCR. The distinct strains were tested for biofilm formation in microtiter plates. Soy Tripticase Broth (TSB) was placed in each well of the microtiter plate and overnight cultures of each strain was added. Wells without bacterial culture were used as controls. A villous cap was then placed on the plate and incubated for 48 h at 37°C. During incubation, the biofilms formed on the surface of the villi of the caps. For quantification of biofilm formation, material that remained attached to the cap was stained with crystal violet, the stained biofilm was extracted and the OD570 of each well was measured. Each strain was classified as non-biofilm forming, weak forming, moderately formed or formative strong. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. Plates of 4 cm² of the different materials were placed in TSB where the culture of each isolate was inoculated separately. At each 48 h incubation the plates were washed to remove unbound cells and re-inserted into TSB without the inoculum. After five replicates of the procedure, sterile swabs were passed over the entire surface of each plate for counting in Baird-Parker agar. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. The biofilm plates were immersed in flasks containing sanitizers, where they remained for 10 min. At the established contact time, the plates were immersed in neutralizing solution for 30 s. After washing with PBS, a sterile swab was passed on the surface of each plate and counts on Baird-Parker agar were performed. The bands profiles obtained on rep-PCR were identical when compared to isolates from the same sample, indicating that each sample was contaminated with only one S. aureus strain. From the twenty S. aureus strain identified, two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains.Discussion: Most strains of S. aureus isolated from buffalo milk mozzarella cheese have the ability to form biofilm on the surfaces of equipment and utensils that have stainless steel, glass or high density polyethylene components. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers. These results support the recommendation that the good hygienic practices adopted by industries processing buffalo milk mozzarella cheese should include specific measures to control the Staphylococcus aureus contamination.

Biofilm formation and antibiotic susceptibility in dispersed cells versus planktonic cells from clinical, industry and environmental origins.

We examined the cell-surface physicochemical properties, the biofilm formation capability and the antibiotic susceptibility in dispersed cells (from an artificial biofilm of alginate beads) and compared with their planktonic (free-swimming) counterparts. The strains used were from different origins, such as clinical (Acinetobacter baumannii AB4), cosmetic industry (Klebsiella oxytoca EU213, Pseudomonas aeruginosa EU190), and environmental (Halomonas venusta MAT28). In general, dispersed cells adhered better to surfaces (measured as the "biofilm index") and had a greater hydrophobicity [measured as the microbial affinity to solvents (MATS)] than planktonic cells. The susceptibility to two antibiotics (ciprofloxacin and tetracycline) of dispersed cells was higher compared with that of their planktonic counterparts (tested by the "bactericidal index"). Dispersed and planktonic cells exhibited differences in cell permeability, especially in efflux pump activity, which could be related to the differences observed in susceptibility to antibiotics. At 1h of biofilm formation in microtiter plates, dispersed cells treated with therapeutic concentration of ciprofloxacin yielded a lower biofilm index than the control dispersed cells without ciprofloxacin. With respect to the planktonic cells, the biofilm index was similar with and without the ciprofloxacin treatment. In both cases there were a reduction of the number of bacteria measured as viable count of the supernatant. The lower biofilm formation in dispersed cells with ciprofloxacin treatment may be due to a significant increase of biofilm disruption with respect to the biofilm from planktonic cells. From a clinical point of view, biofilms formed on medical devices such as catheters, cells that can be related to an infection were the dispersed cells. Our results showed that early treatment with ciprofloxacin of dispersed cells could diminishe bacterial dispersion and facilitate the partial elimination of the new biofilm formed.

Bio-inspired gold nanoparticles synthesis and their anti-biofilm efficacy

Gold nanoparticles (AuNPs) are ‘smart nanomaterials’ with a variety of applications in the different fields. The conventional methods used for AuNPs have some drawbacks and therefore, search of an alternative ‘green method’ is of high importance. In this study, Acinetobacter baumannii AB01 isolated from an infected apple sample was used as a green route for AuNPs synthesis. Effect of various physicochemical parameters such as pH, temperature, cell density and gold chloride salt concentrations on AuNPs synthesis was studied. These AuNPs were further characterized by UV–Vis spectrophotometer, transmission electron microscopy (TEM), energy dispersive spectroscopy, X-ray diffraction and dynamic light scattering. TEM images study showed presence of an extracellular polymeric substance around cells of A. baumannii AB01. The synthesized biogenic AuNPs were tested for their antibacterial activity by well diffusion method. Anti-biofilm efficacy of AuNPs was tested on the glass slide surfaces against the biofilms of Pseudomonas aeruginosa, Vibrio cholera and Brevibacterium linens and visualised by using a fluorescence microscopy. Biofilm formation was reduced about 60–80 % when compared with control biofilm (without AuNPs). Effect of different AuNPs concentrations on biofilm formation in microtiter plates was studied by 96-well plate assay. The bacterial biofilm inhibition (%) was increased with increasing concentrations of AuNPs.

In Vitro Evaluation of Bacteriocins Activity Against Listeria monocytogenes Biofilm Formation.

The present study aimed to assess the activity of cell-free supernatant (CFS) containing bacteriocins on the formation and maintenance of biofilms developed by Listeria monocytogenes, and the associated effect of bacteriocins and ethylene-diamine-tetra-acetic acid (EDTA) on the formed biofilm. CFS from 9 lactic acid bacteria (LAB) strains was tested for inhibitory activity against 85 L. monocytogenes isolates and 21 LAB strains. Then, 12 L. monocytogenes strains were selected based on genetic profiles and sensitivity to CFS and were subjected to an in vitro assay to assess biofilm formation in microtiter plates, considering different culture media and incubation conditions. Based on these results, 6 L. monocytogenes strains were subjected to the same in vitro procedure to assess biofilm formation, being co-inoculated with CFS. In addition, these strains were subjected to the same in vitro procedure, modified by adding the CFS after biofilm formation. Relevant decrease in biofilm formation was observed in the first experiment, but CFS added after biofilm formation did not eliminate them. CFS from Lactobacillus curvatus ET31 were selected due to its anti-biofilm activity, being associated to EDTA at different concentrations and tested for biofilm control of three strains of L. monocytogenes, using the same in vitro procedure described previously. Concentrated bacteriocin presented poor performance in eliminating formed biofilms, and EDTA concentration presented no evident interference on biofilm elimination. Twelve selected L. monocytogenes strains were positive for investigated virulence makers and negative for luxS gene, recognized as being involved in biofilm formation. Selected L. monocytogenes strains were able to produce biofilms under different conditions. CFSs have the potential to prevent biofilm formation, but they were not able to destroy already formed biofilms. Nevertheless, low concentrations of CFS combined with EDTA caused a relevant reduction in already formed biofilms, but this association was not able to eliminate them. The activity of selected CFS was demonstrated against L. monocytogenes-formed biofilms, being more effective when associated to EDTA at different concentrations.

The effect of glucose concentration and shaking conditions on Escherichia coli biofilm formation in microtiter plates

Microtiter plates are one of the most widely used platforms for biofilm studies. However, these systems have been used rather blindly without knowledge of the flow characteristics inside them. Plates with 96-wells were used to study flow dynamics and its influence on Escherichia coli JM109(DE3) biofilm formation. Two different glucose concentrations (1gL−1 and 0.25gL−1) and two different shaking conditions were tested using incubators with orbital diameters of 50 and 25mm. Biofilm growth was monitored for 60h and the hydrodynamics were simulated by computational fluid dynamics. These simulations have shown that wall shear stress was higher near the air/liquid interface and average values of 0.070 and 0.034Pa were obtained for the largest and smallest orbital diameter. For the high glucose concentration, the maximum biofilm amount was attained at 24h and similar values were obtained in both incubators. For the low glucose concentration, lower values were attained. Numerical simulations indicated that microtiter plates can adequately model biofilm formation in relevant biomedical systems.

Increased production of biofilms by Escherichia coli in the presence of enrofloxacin

The literature has demonstrated that subinhibitory concentrations of some antimicrobials are able to induce biofilm formation by certain bacterial species. Biofilms present in the mammary glands of cattle contribute to antimicrobial resistance, resulting in the appearance of persistent mastitis and consequent great losses to the dairy sector worldwide. The present study aimed to investigate the induction of biofilm formation by enrofloxacin in Escherichia coli isolates obtained from bovine mastitis. Twenty-seven isolates were reactivated in brain heart infusion (BHI) broth supplemented with different subinhibitory concentrations of enrofloxacin. Biofilm formation in microtiter plates was measured and confirmed by scanning electron microscopy (SEM). Isolates submitted to the concentration 0.0125mg/mL of enrofloxacin showed greater biofilm formation compared to the control (p<0.001). Biofilm formation results obtained for the other concentrations did not differ from those obtained for the control (p>0.05). Using SEM it was possible to visualize the typical architecture of biofilms. These results represent the first report of inducing the production of biofilms in the presence of enrofloxacin, a quinolone antibiotic used to treat clinical mastitis.

Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci

The details of all steps involved in the quantification of biofilm formation in microtiter plates are described. The presented protocol incorporates information on assessment of biofilm production by staphylococci, gained both by direct experience as well as by analysis of methods for assaying biofilm production. The obtained results should simplify quantification of biofilm formation in microtiter plates, and make it more reliable and comparable among different laboratories.

Establishment and Early Succession of a Multispecies Biofilm Composed of Soil Bacteria

Most soil bacteria are likely to be organized in biofilms on roots, litter, or soil particles. Studies of such biofilms are complicated by the many nonculturable species present in soil, as well as the interspecific bacterial interactions affecting biofilm biology. We in this study describe the development of a biofilm flow model and use this system to establish an early (days 1-7) flow biofilm of soil bacteria from agricultural soil. It was possible to follow the succession in the early flow biofilm by denaturing gradient gel electrophoresis (DGGE) analysis, and it was demonstrated that the majority of strains present in the biofilm were culturable. We isolated and identified nine strains, all associated with unique DGGE profiles, and related their intrinsic phenotypes regarding monospecies biofilm formation in microtiter plates and planktonic growth characteristics to the appearance of the strains in the flow biofilm. The ability of the strains to attach to and establish biofilm in microtiter plates was reflected in their flow biofilm appearance, whereas no such reflection of the planktonic growth characteristics in the flow biofilm appearance was observed. One strain-specific synergistic interaction, strongly promoting biofilm formation of two strains when cultured together in a dual-species biofilm, was observed, indicating that some strains promote biofilm formation of others. Thus, the biofilm flow model proved useful for investigations of how intrinsic phenotypic traits of individual species affect the succession in an early soil biofilm consortium.