Integration and decontamination of Bacillus cereus in Pseudomonas fluorescens biofilms

Aims:  The hypothesis that surrogate planktonic pathogens ( Bacillus cereus and polystyrene microspheres) could be integrated in biofilms and protected from decontamination was tested.
Methods and Results:  Pseudomonas fluorescens biofilms were grown on polyvinyl chloride coupons in annular reactors under low nutrient conditions. After biofilm growth, B. cereus spores and polystyrene microspheres (an abiotic control) were introduced separately. Shear stress at the biofilm surface was varied between 0·15 and 1·5 N m⁻². The amount of surrogate pathogens introduced ranged from approximately 10⁵ CFU ml⁻¹ to 10¹⁰ spheres ml⁻¹. The quantity of surrogate pathogens integrated in the biofilm was proportional to the amount introduced. In 14 of the 16 cases, 0·4–3·0% of the spores or spheres introduced were measured in the biofilms. The other two cases had 10% and 21% of the spores detected. Data suggested that the spores germinated in the system. The amount of surrogate pathogens detected in the biofilms was higher in the mid-shear range. Chlorine treatment reduced the quantity of both surrogate pathogens and biofilm organisms. In one experiment, the biofilms and B. cereus recovered when the chlorine treatment was terminated.
Conclusions:  Planktonic surrogate pathogens can be integrated in biofilms and protected from chlorination decontamination.
Significance and Impact of the Study:  This knowledge assists in understanding the impact of biofilms on harbouring potential pathogens in drinking-water systems and protecting the pathogens from decontamination.     

The structure of Pseudomonas fluorescens biofilms in contact with flowing systems

Using a specially designed flow system Pseudomonas fluorescens has been grown on the inside of glass tubes under carefully controlled conditions. Results show that films developed from water flowing at 0–5 m.s^‐1 are less compact and thicker than with a water velocity of 2–5 m.s^‐1. In the latter all the cells are aligned in the direction of flow whereas in the former the individual cells directly attached to the surface, are randomly distributed with groups of cells lying parallel to the tube axis.     

Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus

Despite the recent enthusiasm for using bacteriophages as bacterial control agents, there are only limited studies concerning phage interaction with their respective hosts residing in mixed biofilm consortia and especially in biofilms where the host species is a minor constituent. In the present work, a study was made of mono and dual species biofilms formed by Pseudomonas fluorescens (Gram-negative) and/or Staphylococcus lentus (Gram-positive) and their fate after infection with phages. The dual species biofilms consisted predominantly of S. lentus. The exposure of these biofilms to a cocktail containing both P. fluorescens and S. lentus phages effectively killed and removed the hosts from the substratum. Additionally, this cocktail approach also controlled the hosts released from the biofilms to the planktonic phase. The ability of phages to control a host population present in minority in the mixed species biofilm was also assessed. For this objective, the biofilms were challenged only with phage φIBB-PF7A, specific for P. fluorescens and the results obtained were to some extent unpredicted. First, φIBB-PF7A readily reached the target host and caused a significant population decrease. Secondly, and surprisingly, this phage was also capable of causing partial damage to the biofilms leading to the release of the non-susceptible host (S. lentus) from the dual species biofilms to the planktonic phase. The efficiency of phage treatment of biofilms was to some extent dependent on the number of cells present and also conditioned by the infection strategy (dynamic or static) utilized in the infection of the biofilms. Nevertheless, in most circumstances phages were well capable of controlling their target hosts.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Changes in the electrochemical interface as a result of the growth of Pseudomonas fluorescens biofilms on gold

In this study, variations in corrosion potential and polarization resistance of thin-film gold electrodes as a result of the growth of Pseudomonas fluorescens biofilms on them are presented. The growth of the volumetric cell fraction of biofilms, as determined by optical sectioning and digital image analysis of phase-contrast images, was found to be exponential during at least 10 hours of incubation. As a consequence of biofilm growth, an exponential decay of the corrosion potential of gold was observed. Most importantly, an increase in polarization resistance of the interface was observed following a strong linear dependence on the mean thickness of biofilms (r = 0.997), as a consequence of oxygen consumption and diffusion limitations. The results presented indicate that the measurement of polarization resistance may be a suitable technique that could be applied easily in industrial or biotechnological systems for monitoring the formation of biofilms.     

The effects of glutaraldehyde on the control of single and dual biofilms of Bacillus cereus and Pseudomonas fluorescens

Glutaraldehyde (GLUT) was evaluated for control of single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens on stainless steel surfaces using a chemostat system. The biofilms were characterized in terms of mass, cell density, total and matrix proteins and polysaccharides. The control action of GLUT was assessed in terms of inactivation and removal of biofilm. Post-biocide action was characterized 3, 7, 12, 24, 48 and 72 h after treatment. Tests with planktonic cells were also performed for comparison. The results demonstrated that in dual species biofilms the metabolic activity, cell density and the content of matrix proteins were higher than those of either single species. Planktonic B. cereus was more susceptible to GLUT than P. fluorescens. The biocide susceptibility of dual species planktonic cultures was an average of each single species. Planktonic cells were more susceptible to GLUT than their biofilm counterparts. Biofilm inactivation was similar for both of the single biofilms while dual biofilms were more resistant than single species biofilms. GLUT at 200 mg l^?1 caused low biofilm removal (<10%). Analysis of the post-biocide treatment data revealed the ability of biofilms to recover their activity over time. However, 12 h after biocide application, sloughing events were detected for both single and dual species biofilms, but were more marked for those formed by P. fluorescens (removal >40% of the total biofilm). The overall results suggest that GLUT exerts significant antimicrobial activity against planktonic bacteria and a partial and reversible activity against B. cereus and P. fluorescens single and dual species biofilms. The biocide had low antifouling effects when analysed immediately after treatment. However, GLUT had significant long-term effects on biofilm removal, inducing significant sloughing events (recovery in terms of mass 72 h after treatment for single biofilms and 42 h later for dual biofilms). In general, dual species biofilms demonstrated higher resistance and resilience to GLUT exposure than either of the single species biofilms. P. fluorescens biofilms were more susceptible to the biocide than B. cereus biofilms.     

Characterization and oxidation of chromium(III) by sodium hypochlorite in alkaline solutions

Chromium exists in nuclear waste sludges and is a problematic element in the vitrification process of high-level nuclear wastes. It is therefore necessary to treat the waste sludges to remove chromium prior to vitrification, by caustic leaching or oxidation of Cr(III) to Cr(VI). The objective of this study is to investigate the effect of oligomerization of Cr(III) on its oxidation by hypochlorite in alkaline solutions.Monomeric, dimeric and trimeric Cr(III) species in solution were separated by ion exchange. The kinetics of the oxidation of the separated species by hypochlorite in alkaline solutions was studied by UV/Vis absorption spectroscopy, and compared with the oxidation by hydrogen peroxide previously studied. Results indicate that hypochlorite can oxidize Cr(III) to Cr(VI) in alkaline solutions, but the rate of oxidation by hypochlorite is slower than that by hydrogen peroxide at the same alkalinity and concentrations of oxidants. The rate of oxidation of Cr(III) by both oxidants decreases as the concentration of sodium hydroxide is increased, but the oxidation by hypochlorite seems less affected by the degree of oligomerization of Cr(III) than that by peroxide. Compared with the oxidation by hydrogen peroxide where the major reaction pathway has an inverse order with respect to C_NaOH, the oxidation by hypochlorite has a significant reaction pathway independent of [OH⁻].     

多重耐药铜绿假单胞菌群体感应系统与主动外排基因表达的研究

目的研究临床多重耐药铜绿假单胞菌群体感应(QS)系统与主动外排泵MexAB-OprM系统基因表达水平与抗生素耐药关系。方法收集苏州市立医院和上海市江湾医院2011年2月至6月间临床标本中分离的铜绿假单胞菌,定量分析细菌生物被膜形成能力;MIC法检测细菌抗生素耐药性,用多重聚合酶链反应(PCR)扩增群体感应系统lasI、lasR及主动外排泵系统mexA基因,实时定量逆转录RT-PCR检测lasI、lasR和mexA基因的相对表达量。结果临床样本分离出84株铜绿假单胞菌,其中产生物被膜菌58株,占比69%;多重耐药菌共24株,占比28.6%;多重耐药菌株中产生物被膜有11株,占45.8%;多重耐药菌中mexA基因表达上调有18株,占75%;lasI基因表达上调有8株,占33.3%。结论多重耐药菌株的生物被膜形成率显著低于非多重耐药组,多重耐药铜绿假单胞菌的主动外排泵MexAB-OprM系统基因表达出现显著上调,生物被膜菌的lasI基因表达显著上调而lasR基因的表达无明显变化。     

Cytotoxicity mechanisms of sodium hypochlorite in cultured human dermal fibroblasts and its bactericidal effectiveness

We investigated the therapeutic efficacy of the topical antiseptic sodium hypochlorite (NaOCl) for antibacterial activity and in parallel the cytotoxicity mechanisms by which hypochlorite and the chloramines generated therefrom induce oxidative tissue damage, which further influences the wound-healing process. Human dermal fibroblasts were exposed to increasing concentrations of reagent NaOCl (0.00005-0.1%) at exposure times varying between 2 and 24 h and the protective effects of fetal calf serum (FCS) determined. Antibacterial power was studied by testing a wide range of hypochlorite concentrations (0.00025-0.5%) against four isolated bacterial species. Total bactericidal effects were observed only for 0.5%; concentration range 0.25-0.025% produced partial antimicrobial activity. The early NaOCl-produced cytotoxic action on cultured fibroblasts was cell ATP depletion which occurred at 0.00005% (with FCS 2%) followed by dose- and time-dependent decreases, reaching levels below 5% of control values. Using the 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid metabolic assay to evaluate cell death, we observed that NaOCl concentrations greater than 0.05% provoked null fibroblast survival at all exposure times assayed. Hypochlorous acid proved to exert a rapid inhibitory effect on DNA synthesis, consistent with its primary role in bacterial killing by phagocytes. Cytotoxicity produced by increasing NaOCl concentrations and assessed by measuring both mitochondrial function and cell DNA synthesis was reduced with the greatest presence of FCS (10%) in culture media.     

A novel micellar formulation based on natural plant extracts enhances the efficacy of hydrogen peroxide against biofilms of Staphylococcus spp. and Pseudomonas aeruginosa

Abstract

The antibacterial efficacy of hydrogen peroxide encapsulated in micelles (mH₂O₂) against biofilms was compared with that of hydrogen peroxide alone and of three commercially available aqueous biocides. The activity of mH₂O₂ on 24-h biofilms of reference strains of Staphylococcus spp. and Pseudomonas aeruginosa was tested in a static microtiter plate model. The biofilms were incubated with mH₂O₂ (17% v/v H₂O₂, 2% lactic acid, 0.3% phytoextract, H₂O) and its individual ingredients and compared with three aqueous biocides at different concentrations and times of exposure. After 5-min exposure, 10% mH₂O₂ (corresponding to 1.7% v/v H₂O₂) achieved > 8 log₁₀ reductions against all the test strains, while 1.7% H₂O₂ achieved a maximum of 1.5 log₁₀ reduction. After 5-min exposure, none of the commercially available biocides tested showed themselves to be capable of completely eliminating the test strains embedded in biofilms. Hydrogen peroxide encapsulated in micelles demonstrated enhanced activity against planktonic cells and biofilms of Staphylococcus spp. and P. aeruginosa.     

On the production of glycogen by Pseudomonas fluorescens during biofilm development: an in situ study by attenuated total reflection-infrared with chemometrics

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor Pseudomonas fluorescens biofilms in situ, non-destructively, in real time, and under fully hydrated conditions. Changes accompanying the metabolic evolution of the sessile bacterial cells from the nascent biofilm monolayer to the beginning of the multi-layered structure in the presence of nutrients were identified via the ATR-FTIR fingerprints of the young biofilm on the ATR crystal. The ATR-FTIR spectra were analysed by classical methods (time evolution of integrated intensities and profile evolution of specific bands), and also by a multivariate curve resolution, Bayesian positive source separation, to extract the pure component spectra and their change of concentration over time occurring during biofilm settlement. This work showed clearly the overproduction of glycogen by sessile P. fluorescens, which had not previously been described by other research groups.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.     

Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to natural biofilms

Settlement and metamorphosis of pediveliger larvae of Mytilus coruscus in response to natural biofilms was investigated in the laboratory. Pediveliger larvae settled and metamorphosed in response to biofilms and post-larval settlement and metamorphosis increased with biofilm age. The activity of the biofilm was positively correlated with biofilm age, dry weight, bacterial density and diatom density, but had no apparent relationship with chlorophyll a concentration. The change in bacterial community composition corresponding to biofilm age may explain differences in the age-dependent inducing activities of biofilms, which in turn may play an important role in larval settlement in this species.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

Morphological and chemical changes in the attached cells of Pseudomonas aeruginosa as primary biofilms develop on aluminium and CaF2 plates

AIMS: To investigate the morphological and chemical changes in attached cells of Pseudomonas aeruginosa (ATCC 14886) at different stages of biofilm development on two different types of substrata. METHODS AND RESULTS: The development of primary biofilm on aluminium plates representing metals and on CaF(2) discs representing dielectric materials was monitored by FTIR microscopy, ESEM, EDAX and protein analysis by SDS-PAGE. A unique cellular feature similar in morphology to pili was observed on the surface of P. aeruginosa adhering on aluminium but not on CaF(2). Results derived from FTIR analysis confirm on both substrata the successive importance of polysaccharides and proteins during the biofilm development. These results also revealed that the increase of the ratio of carboxylates to amide I was higher with the aluminium plates than with the CaF(2) discs. The number of cells adhered and the amount of oxygen incorporated in adhered cells on the latter materials were, respectively, less and almost nil in comparison with the former. Protein analysis of the lysates of cells by SDS-PAGE revealed that expression of one protein with a molecular weight of 45 kDa, was greatly enhanced in attached cells on both substrata. However, expression of another protein with molecular weight of 35 kDa was up-regulated only in cells adhering on CaF(2) but not in those on aluminium. CONCLUSION: Depending on the nature of the surface, new proteinaceous complexes and cellular features were formed in the attachment process of P. aeruginosa. SIGNIFICANCE AND IMPACT OF THE STUDY: The pattern of P. aeruginosa cells adhering onto CaF(2) discs and aluminium plates is different. Formation of biofilm is more difficult on CaF(2) than on aluminium.