The role of quorum sensing and the effect of environmental conditions on biofilm formation by strains of Vibrio vulnificus

Abstract

It has been suggested that Vibrio vulnificus attaches to plankton and algae and is found in large numbers in the environment. Factors affecting attachment, biofilm formation and morphology of V. vulnificus have not been thoroughly investigated. This study evaluated the role of quorum sensing (QS) and environmental conditions on biofilm development of V. vulnificus. It was found that biofilm development by V. vulnificus was affected by nutrient and glucose concentration, but not by NaCl concentration or temperature under the conditions used here. Moreover, biofilm development of a QS mutant strain proceeded rapidly and sloughing occurred earlier than for the isogenic parent strain. There was a significant loss of viability for the QS mutant biofilm early in development. Hence, it is hypothesised that factors regulated by the QS system play a role in proper biofilm development and maintenance of V. vulnificus. Furthermore, it is shown that biofilm development varied among isolates.     

Characterization of two quaternary ammonium chloride-resistant bacteria isolated from papermaking processing water and the biocidal effect on their biofilm formation

Biofilm formation and growth on equipment surfaces is detrimental to papermaking processes. However, a fundamental understanding leading to an optimal control strategy is yet to be found. Quaternary ammonium compounds (QAC) are being increasingly applied in the papermaking processes. Among them, the most frequently applied, N-alkyl-benzyl-dimethyl ammonium chloride, was employed in this study. To foster fundamental understanding of QAC efficacy towards biofilm control, two of the highest QAC-resistant strains of bacteria were isolated from the papermaking processing water and employed as model organisms. By the 16S rRNA gene sequencing technique, two Gram-negative rods with QAC resistance were identified as Morganella morganii (HB22) and the biofilm-forming Pseudomonas putida (HB45). The minimal inhibition concentration (MIC) values were 8 mg L⁻¹ for HB22 and 16 mg L⁻¹ for HB45, respectively, against QAC in basal medium (BM). However, both strains could grow under more than 150 mg L⁻¹ QAC in basal medium at neutral pH. As observed by crystal violet assay and fluorescent confocal microscopy, HB45 formed biofilm more slowly on stainless steel coupon which is the prime material of papermachine than on the surface of polystyrene, the most common material for food packaging and semi-finished/finished products. HB45 formed biofilm more slowly on stainless steel coupons than on polystyrene Petri dish surfaces, as observed by crystal violet assay and fluorescent confocal microscopy. For HB45, there was a marginal increase of inhibition of biofilm formation by increasing QAC concentration from 50 to 75 mg L⁻¹. By comparison of inhibition concentration in liquid state and in biofilm formation, the results implicate that the current practice in papermaking processes of adding biocide to qualitatively control planktonic bacterial communities does not ensure control of biofilm formation.     

Potential effect of Allium sativum bulb for the treatment of biofilm forming clinical pathogens recovered from periodontal and dental caries

Biofilm producing clinical bacterial isolates were isolated from periodontal and dental caries samples and identified as, Lactobacillus acidophilus, Streptococcus sanguis, S. salivarius, S. mutansand Staphylococcus aureus. Among the identified bacterial species, S. aureus and S. mutansshowed strong biofilm producing capacity. The other isolated bacteria, Streptococcus sanguis, S. salivarius showed moderate biofilm formation. These pathogens were subjected for the production of extracellular polysaccharides (EPS) in nutrient broth medium and the strain S. aureus synthesized more amounts of EPS (610 ± 11.2 µg/ml) than S. sanguis (480 ± 5.8 µg/ml).EPS production was found to be less in S. salivarius (52 ± 3.8 µg/ml).The solvent extract of A. sativum bulb showed the phytochemicals such as, carbohydrate, total protein, alkaloids, saponins, flavonoids, tannins and sterioids. The solvent extract of A. sativum bulb showed wide ranges of activity against the selected dental pathogens. The difference in antibacterial activity of the solvent extract revealed differences in solubility of phytochemicals in organic solvents. Ethanol extract was highly active againstS. aureus (25 ± 2 mm). The Minimum Inhibitory Concentration (MIC) of crude garlic bulb varied widely and this clearly showed that bacteria exhibits different level of susceptibility to secondary metabolites. MIC value ranged between 20 ± 2 mg/ml and 120 ± 6 mg/ml and Minimum Bactericidal Concentration (MBC) value ranged from 60 ± 5 mg/l to 215 ± 7 mg/ml. To conclude, A. sativum bulb can be effectively used to treat periodontal and dental caries infections.     

Propolis ethanolic extract has double-face in vitro effect on the planktonic growth and biofilm formation of some commercial probiotics

This study investigated the in vitro effect of propolis ethanolic extract (PEE) on planktonic growth and biofilm forming abilities of five commercial probiotics (Enterol, Protexin, Normaflore, BioGaia and Linex). Broth microdilution method was used to investigate the susceptibility of the microbes of five commercial probiotics to PEE. Crystal violet assay was used for the quantitative assessment of biofilm formation and mature biofilm eradication tests. Effect of PEE on autoaggregation ability and swarming motility of Normaflore microbes was determined. Planktonic forms of probiotics showed varied susceptibilities with minimal inhibitory concentration values in the range of 100–800 µg/mL of PEE. However, low PEE concentrations significantly enhanced the planktonic growth of Linex and BioGaia microbes. Biofilm studies revealed that Enterol and Protexin were non-biofilm formers, while BioGaia, Linex and Normaflore showed weak biofilms, which were inhibited by 12.5, 25, and 800 µg/mL of PEE, respectively. PEE revealed double-face effect on the biofilms of Normaflore and Linex, which were enhanced at low concentrations of PEE and inhibited at higher concentrations. Interestingly, Normaflore biofilms were shifted from weak to strong biofilms at low PEE concentrations (12.5, 25, and 50 µg/mL). In conclusion, PEE has strain dependent controversial effects on the planktonic growth and biofilm forming ability of the tested probiotics, although high concentrations have inhibitory effect on all of them, low concentrations may have strain dependent prebiotic effect.     

A flow chamber assay for quantitative evaluation of bacterial surface colonization used to investigate the influence of temperature and surface hydrophilicity on the biofilm forming capacity of uropathogenic Escherichia coli

We have established a simple flow chamber-based procedure which provides an accurate and reproducible way to measure the amount of biofilm formed on an implantable biomaterial surface. The method enables the side-by-side evaluation of different materials under hydrodynamic flow conditions similar to those found on an implanted device. We have used the method to evaluate the biofilm forming capacity of clinically isolated Escherichia coli on silicone rubber and on silicone rubber containing a hydrophilic coating. It was found that the surface chemistry influenced the colonization of the isolates very differently. In addition, the temperature was found to have a considerable influence upon the adhesion and biofilm forming capacity of some of the isolates, and that the influence of surface chemistry depended on temperature. Our results suggest that the step from using E. coli laboratory strains to clinical isolates entails a significant rise in complexity and yields results that cannot be generalized. The results should be valuable information for researchers working with pre-clinical evaluation of device-associated E. coli infections.     

The effects of the Penicillium mycotoxins citrinin, cyclopiazonic acid, ochratoxin A, patulin, penicillic acid, and roquefortine C on in vitro proliferation of porcine lymphocytes

The in vitro effect of each of the Penicillium mycotoxins citrinin (CIT), cyclopiazonic acid (CPA), ochratoxin A (OTA), patulin (PAT), penicillic acid (PIA) and roquefortine C (RQC) on mitogen induced lymphocyte proliferation was determined using purified lymphocytes from 6 piglets. Dose response curves for each mycotoxin were generated and the concentrations producing 50% inhibition of cell proliferation (IC₅₀) were estimated. OTA and PAT were the most potent toxins with IC₅₀ of 1.3 and 1.2 μmol/l, respectively (0.52 and 0.18 mg/l, respectively). Based on molar concentrations, OTA was 15, 30, 40, and 65 times more potent as an inhibitor than PIA, CIT, CPA and RQC, respectively.     

Inhibitory effect of different chicken-derived lactic acid bacteria isolates on drug resistant Salmonella SE47 isolated from eggs

Lactic Acid Bacteria (LAB) regulate and maintain the stability of healthy microbial flora, inhibit the adhesion of pathogenic bacteria and promote the colonization of beneficial micro-organisms. The drug resistance and pathogenicity of Salmonella enteritis SE47 isolated from retail eggs were investigated. Meanwhile, Enterococcus faecalis L76 and Lactobacillus salivarius LAB35 were isolated from intestine of chicken. With SE47 as indicator bacteria, the diameters of L76 and LAB35 inhibition zones were 12 mm and 8·5 mm, respectively, by agar inhibition circle method, which indicated that both of them had inhibitory effect on Salmonella, and L76 had better antibacterial effect; two chicken-derived lactic acid bacteria isolates and Salmonella SE47 were incubated with Caco-2. The adhesion index of L76 was 17·5%, which was much higher than that of LAB35 (10·21%) and SE47 (4·89%), this experiment shows that the higher the bacteriostatic effect of potential probiotics, the stronger the adhesion ability; then Caco-2 cells were incubated with different bacteria, and the survival of Caco-2 cells was observed by flow cytometry. Compared with Salmonella SE47, the results showed that lactic acid bacteria isolates could effectively protect Caco-2 cells; finally, after different bacteria incubated Caco-2 cells, according to the cytokine detection kit, the RNA of Caco-2 cells was extracted and transcribed into cDNA, then detected by fluorescence quantitative PCR, the results showed that L76 could protect Caco-2 cells from the invasion of Salmonella SE47, with less cell membrane rupture and lower expression of MIF and TNF genes. Therefore, the lactic acid bacteria isolates can effectively inhibit the adhesion of Salmonella and protect the integrity of intestinal barrier.     

Characterization of two quaternary ammonium chloride-resistant bacteria isolated from papermaking processing water and the biocidal effect on their biofilm formation

Biofilm formation and growth on equipment surfaces is detrimental to papermaking processes. However, a fundamental understanding leading to an optimal control strategy is yet to be found. Quaternary ammonium compounds (QAC) are being increasingly applied in the papermaking processes. Among them, the most frequently applied, N-alkyl-benzyl-dimethyl ammonium chloride, was employed in this study. To foster fundamental understanding of QAC efficacy towards biofilm control, two of the highest QAC-resistant strains of bacteria were isolated from the papermaking processing water and employed as model organisms. By the 16S rRNA gene sequencing technique, two Gram-negative rods with QAC resistance were identified as Morganella morganii (HB22) and the biofilm-forming Pseudomonas putida (HB45). The minimal inhibition concentration (MIC) values were 8 mg L⁻¹ for HB22 and 16 mg L⁻¹ for HB45, respectively, against QAC in basal medium (BM). However, both strains could grow under more than 150 mg L⁻¹ QAC in basal medium at neutral pH. As observed by crystal violet assay and fluorescent confocal microscopy, HB45 formed biofilm more slowly on stainless steel coupon which is the prime material of papermachine than on the surface of polystyrene, the most common material for food packaging and semi-finished/finished products. HB45 formed biofilm more slowly on stainless steel coupons than on polystyrene Petri dish surfaces, as observed by crystal violet assay and fluorescent confocal microscopy. For HB45, there was a marginal increase of inhibition of biofilm formation by increasing QAC concentration from 50 to 75 mg L⁻¹. By comparison of inhibition concentration in liquid state and in biofilm formation, the results implicate that the current practice in papermaking processes of adding biocide to qualitatively control planktonic bacterial communities does not ensure control of biofilm formation.     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

Inhibitory effect of 2,4‐dichlorophenoxyacetic acid on ROS,autophagy formation,and mRNA replication for influenza virus infection

Influenza virus has had a high rate of antigenic shift and drift that causes significant morbidity and mortality in humans and animals. The lack of excellent pharmacological treatment underlines the importance of the development of the novel antiviral drugs. We investigated the anti‐influenza A and B viruses of 2,4‐dichlorophenoxyacetic acid (2,4‐D), which is the synthetic analog to auxin and is used as a popular herbicide in the agricultural practices. 2,4‐D was evaluated using a cytopathic effect reduction method; assay results showed that 2,4‐D possessed strong anti‐influenza A and B viruses inhibiting the formation of a visible cytopathic effect. Influenza viral RNA expression was performed by quantitative real‐time polymerase chain reaction. 2,4‐D also inhibited virus replication in the early stage of influenza virus infection without direct interaction with virus particles. Additionally, 2,4‐D significantly inhibited various factors occur during influenza virus infection as the acidic vesicular formation and reactive oxygen species production. Moreover, 2,4‐D represented no cytotoxicity in normal kidney cell. Therefore, these findings provide an understanding of the mechanism and efficient use of 2,4‐D in pharmacological applications against influenza virus infection.     

Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: the inhibitory effect of linoleic acid on the DNA-binding step

c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90(+/-0.54)x10(-7)M for Max85/Max85 homodimer, 6.85(+/-0.25)x10(-3)M for Myc87/Myc87 homodimer, and 2.55(+/-0.29)x10(-8)M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33(+/-0.21)x10(-9)M for Max85/Max85/DNA, 2.27(+/-0.08)x10(-12)M for Myc87/Myc87/DNA, and 4.43(+/-0.37)x10(-10)M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.     

Phytoextraction of copper from contaminated soil by Elsholtzia splendens as affected by EDTA, citric acid, and compost

Phytoextraction of copper (Cu) from contaminated soils greatly depends on the metal bioavailability in the soils and metal uptake ability of the plant. In this study, the effects of chelators [ethylenediamine tetraacetic acid (EDTA), citric acid (CA)] and compost amendments on Cu phytoextraction potential by a tolerant and accumulating plant species (E. splendens) were examined in two types of contaminated soils, ie., the mined soil from Cu-mined area (MS) and a paddy soil polluted by Cu refining (PS). The results showed that EDTA application at 2.5-5.0 mmol kg(-1) increased phytoextraction of Cu by four- and eight-fold from both MS and PS, respectively, which is mainly attributed to increased H2O extractable Cu in the soil. The Cu amount extracted by the shoots of E. splendens reached 800-1000 microg Cu plant(-1) from the MS and 400-700 microg Cu plant(-1) from the PS at EDTA application rates of 2.5-5.0 mmol kg(-1). The application of CA at 5.0 mmol kg(-1) had minimal effects on Cu extractability in both soils and slightly decreased Cu extraction efficiency by E. splendens. Plant biomass production was enhanced by CA at 0.25 mmol L(-1) in nutrient solution, but inhibited by CA at 5.0 mmol kg(-1) in both MS and PS. Increasing the compost rate significantly decreased H2O extractable Cu in the MS, but raised H2O-extractable Cu in the PS, which resulted mainly front the reduced exchangeable Cu in the MS and the increased exchangeable and organic fractions of Cu in the PS by compost. At high compost rate (5%), the shoots of E. splendens extracted 3.6-fold higher Cu from the PS than from the MS. These results indicate that, among the soil amendments, efficiency of Cu phytoextraction is enhanced mostly by 2.5-5.0 mmol kg(-1) EDTA, followed by 5% (w:w) compost, whereas < 5.0 mmol kg(-1) CA has minimal effects on Cu phytoextraction by E. splendens in the PS. As for the MS, only 2.5-5.0 mmol kg(-1) EDTA can elevate the efficiency of Cu, while 5% compost amendment and < 5.0 mmol kg(-1) CA application have no marked effects on Cu phytoextraction by E. splendens.     

Gibberellic acid and kinetin partially reverse the effect of water stress on germination and seedling growth in chickpea

The percent germination and seedling growth of chickpea (Cicer arietinum L. cv. PBG-1) decreased with increasing concentrations of exogenous polyethylene glycol 6000 (PEG). With 15% PEG in the growth medium germination was only 33% while with 10% PEG it was 58% as compared to 93% in control. Addition of gibberellic acid (GA₃) and kinetin to medium containing 10% PEG increased the germination and seedling growth and the effect was maximum with 6 µM GA₃ which was a better inducer of growth and germination under reduced water potential than kinetin. However, indole acetic acid (IAA) inhibited germination and growth of stressed seedlings. The activity of amylase in cotyledons under stress was significantly increased with GA₃ while kinetin and IAA were less effective. Gibberellic acid also enhanced the mobilization of starch from cotyledons of stressed seedlings resulting in low starch levels in cotyledons compared with stressed seedlings.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Factors affecting the inhibitory effect of indolylacetic acid on root formation in pea cuttings

Indol-3yl-acetic acid (IAA) present in the rooting solution for 1–4 days appreciably decreased the number of roots subsequently formed in pea stem cuttings. Removal of the lowest 10 mm of the cutting base after IAA treatment abolished the inhibitory effect of IAA almost completely. The number of roots formed from the remaining part of the base internode after excision of the basal centimeter of the internode 4 days after the cuttings were taken was similar to that of the control. It is concluded that the first root primordia, which develop near the cut surface, have an inhibitory influence on development of further roots. The inhibitory effect of IAA may be explained as a strengthening of this dominance phenomenon.
Addition of charcoal to the rooting solution delayed the appearance of roots but increased the number of roots developed during a standard rooting period. Adsorption of stimulatory and inhibitory compounds at the surface of the cutting base is believed to be the reason for these results as no evidence of accumulation of inhibitory compounds in the solution was obtained. Charcoal reversed the inhibitory effect of IAA if added after the IAA treatment. This reversal was almost complete if the IAA treatment lasted for one day only but decreased drastically if the IAA treatment was extended over 4 days.     

Determination of isonicotinic acid in the presence of isoniazid and acetylisoniazid Studies on isonicotinic acid formation from isoniazid in isolated rat hepatocytes

In comparison with the hepatocytes obtained from intact rats and rats pretreated with phenobarbital or 3-methylchoranthrene, the amount of isonicotinic acid (INA) formed from isoniazid (INH) increased substantially after incubation at 37°C using the pretreated hepatocytes. This suggests an oxidative pathway for INA formation from INH, apart from hydrolysis. In order to explore the exact mechanism of INA formation in the hepatocytes, an HPLC assay for INA in the presence of INH and acetylisoniazid was developed. In this assay, INA was extracted after the preparation of an ion pair with tetra-n-butylammonium hydroxide, and analysed using an ODS column and a mobile phase consisting of 0.067 M potassium dihydrogenphosphate solution-methanol (96:4 v/v). The method is simple, accurate and especially suitable for INA determination after incubation of INH in isolated rat hepatocytes.     

CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响

目的 研究CRK1基因缺失对白念珠菌形态、黏附、生物被膜的影响.方法 显微镜下观察,计算菌丝形成率,比较CRK1基因缺失菌（Δcrk1菌）及标准菌SC5314形成菌丝的能力;建立肠黏膜模型,计算黏附率,评价CRK1基因缺失对白念珠菌黏附的影响;MTT法及结晶紫法（CV）评价CRK1基因缺失对白念珠菌生物被膜形成的影响.结果 与SC5314相比,Δcrk1菌分别在10％胎牛血清和RPMI-1640培养条件下形成菌丝能力均较弱,两者之间有统计学差异;Δcrk1菌在60、90、120 min时对肠黏膜的黏附数明显少于标准菌SC5314,两者之间有统计学差异;通过MTT法、结晶紫法两种方法证实了,在经48 h培养后,Δcrk1菌与其标准菌SC5314相比,形成生物膜的能力弱,两者之间差异有统计学差异.结论 CRK 1基因缺失影响白念珠菌菌丝二态性的转化,进而影响黏附力和生物被膜的形成.