vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.     

A semi-quantitative approach to assess biofilm formation using wrinkled colony development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis, and Gram-negative bacteria, such as Vibrio cholerae, Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Vibrio fischeri. The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect, while strains exhibiting increased biofilm phenotypes are enhanced for colonization. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization. In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p>0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p<0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p<0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1-0.5%) and exposure period were noted (p<0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p>0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

Effect of Biocides on Biofilm Bacteria from Dental Unit Water Lines

Microbial biofilm formation in dental unit water lines (DUWL) is a phenomenon that has been recognized for nearly four decades. Water delivered by DUWL can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWL may serve as a reservoir for these microorganisms and should therefore be controlled. In this study, the effects of eight biocides were monitored on DUWL biofilms individually and in combination by epifluorescence microscopy and total viable counts (TVC). The effects of sodium dodecyl sulphate (SDS), hydrogen peroxide (H₂O₂), sodium hypochlorite (NaOCl), phenol (Phe), Tween 20 (Tw 20), ethylenediaminetetraacetic acid (EDTA), chlorohexidine gluconate (CHX), and povidine iodine (PI) were tested on DUWL biofilms alone and in combination. PI was found to have negligible effects on biofilm removal either applied alone or in combined form with CHX. Applying all biocides simultaneously did not completely eliminate viable bacteria nor did they remove biofilm. Overall, when combined, the biocides performed better than singly applied products. The most effective biocides were NaOCl and Phe (both alone and in combination).     

Effect of temperature on biofilm formation by Antarctic marine bacteria in a microfluidic device

Polar biofilms have become an increasingly popular biological issue because new materials and phenotypes have been discovered in microorganisms in the polar region. Various environmental factors affect the functionality and adaptation of microorganisms. Because the polar region represents an extremely cold environment, polar microorganisms have a functionality different from that of normal microorganisms. Thus, determining the effective temperature for the development of polar biofilms is crucial. Here, we present a simple, novel one-pot assay for analysis of the effect of temperature on formation of Antarctic bacterial biofilm using a microfluidic system where continuous temperature gradients are generated. We find that a specific range of temperature is required for the growth of biofilms. Thus, this microfluidic approach provides precise information regarding the effective temperature for polar biofilm development with a new high-throughput screening format.     

细菌生物膜研究技术

细菌生物膜是细菌生长过程中为适应生存环境而在固体表面上生长的一种与游走态细胞相对应的存在形式。只要条件允许,绝大多数细菌都可以形成生物膜。一旦形成了生物膜细菌就具有极强的耐药性,在医疗、食品、工业、军事等诸多领域给人类社会带来了严重的危害,造成巨大的经济损失。因此,细菌生物膜已成为全球关注的重大难题,也是目前科学界研究的前沿和热点。本文结合细菌生物膜研究技术的最新进展,重点介绍了几种常用生物膜发生装置及检测量化技术,并对其原理及优缺点进行了讨论。     

Initial phases of microbial biofilm formation on opaque,innovative anti‐adhesive surfaces using a modular microfluidic system

Biofilms can cause numerous problems, hence it is important to understand their formation on surfaces in order to develop resistant materials and avoidance strategies. Therefore, information is required regarding adhesion processes on surfaces generally and innovative anti‐adhesive coatings in particular. Our flow cell system allows biofilms to be monitored in continuous flow conditions, without removing material for postflow imaging. The shown laminar flow ensures the maintenance of highly controlled conditions for biofilm growth. However, carried simulations of the oxygen demands of Escherichia coli cultivated as biofilms under the chosen regime indicate that conditions may become anaerobic, at least at the outlet of the flow cell, after a certain period of time. We report data on the biofouling tendencies on coatings generated with the help of direct laser interference patterning on stainless steel surfaces. Data were estimated from images acquired by fluorescence microscopy. Differences between patterned and unpatterned surfaces were not found, which is in accordance with the attachment point theory. Nevertheless, it is particularly important to elucidate in future studies the behavior of microorganisms during their attachment and the effects of variables of potentially sensitive surfaces (such as hydrophobicity, nanotopography, and charge) on their adhesion.     

The effects of disinfectant foam on microbial biofilms

This investigation examined the effects of common aqueous biocides and disinfectant foams derived from them on Pseudomonas aeruginosa biofilms. Biofilms were grown on stainless steel coupons under standardised conditions in a reactor supplemented with low concentrations of organic matter to simulate conditions prevalent in industrial systems. Five-day-old biofilms formed under ambient conditions with continuous agitation demonstrated a low coefficient of variation (5.809%) amongst viable biofilm bacteria from independent trials. Scanning electron microscopy revealed biofilms on coupons with viable biofilm bacteria observed by confocal microscopy. An aqueous solution of a common foaming agent amine oxide (AO) produced negligible effects on bacterial viability in biofilms (p?>?0.05). However, significant biofilm inactivation was noted with aqueous solutions of common biocides (peracetic acid, sodium hypochlorite, sodium ethylenediaminetetraacetic acid) with or without AO (p?<?0.05). Aereation of a mixture of AO with each of these common biocides resulted in significant reductions in the viability of biofilm bacteria (p?<?0.05). In contrast, limited effects were noted by foam devoid of biocides. A relationship between microbial inactivation and the concentration of biocide in foam (ranging from 0.1?–?0.5%) and exposure period were noted (p?<?0.05). Although, lower numbers of viable biofilm bacteria were recovered after treatment with the disinfectant foam than by the cognate aqueous biocide, significant differences between these treatments were not evident (p?>?0.05). In summary, the studies revealed significant biofilm inactivation by biocidal foam prepared with common biocides. Validation of foam disinfectants in controlled trials at manufacturing sites may facilitate developments for clean in place applications. Advantages of foam disinfectants include reductions in the volumes of biocides for industrial disinfection and in their disposal after use.     

A new non-aerated illuminated packed-column reactor for the development of sulfide-oxidizing biofilms

This paper describes an illuminated reactor that allows the spontaneous development of biofilms aimed at the treatment of sulfide-containing streams. The reactor operates as a sulfidostat and is composed of an illuminated packed-column, in which microorganisms are exposed to constant low substrate concentrations, thereby avoiding inhibition due to high sulfide concentrations. The control system allows highly polluted streams to be oxidized by the microbial biofilm while ensuring the quality of the effluent produced. Both monospecies and multispecies biofilms have been developed. Biofilms undergo changes in light irradiance and sulfide load while providing a consistent reduction of the sulfide levels, down to micromolar concentrations. Both types of biofilm developed differ from stirred reactors in that their specific activities are lower, constituting systems with a slow dynamic behavior and, therefore, they are less sensitive to sudden disturbances.     

The BvgAS signal transduction system regulates biofilm development in Bordetella

The majority of Bordetella sp. virulence determinants are regulated by the BvgAS signal transduction system. BvgAS mediates the control of multiple phenotypic phases and a spectrum of gene expression profiles specific to each phase in response to incremental changes in the concentrations of environmental signals. Studies highlighting the critical role of this signaling circuitry in the Bordetella infectious cycle have focused on planktonically growing bacterial cells. It is becoming increasingly clear that the major mode of bacterial existence in the environment and within the body is a surface-attached state known as a biofilm. Biofilms are defined as consortia of sessile microorganisms that are embedded in a matrix. During routine growth of Bordetella under agitating conditions, we noticed the formation of a bacterial ring at the air-liquid interface of the culture tubes. We show here that this surface adherence property reflects the ability of these organisms to form biofilms. Our data demonstrate that the BvgAS locus regulates biofilm development in Bordetella. The results reported in this study suggest that the Bvg-mediated control in biofilm development is exerted at later time points after the initial attachment of bacteria to the different surfaces. Additionally, we show that these biofilms are highly tolerant of a number of antimicrobials, including the ones that are currently recommended for treatment of veterinary and human infections caused by Bordetella spp. Finally, we discuss the significance of the biofilm lifestyle mode as a potential contributor to persistent infections.     

Effects of biofilms on zebra mussel postveliger attachment to artificial surfaces

Abstract. The zebra mussel is an introduced fouling organism in North American inland waters. This field study tested whether natural biofilms, formed by covering substrata with a 100-μm mesh that allows microorganisms to attach and films to develop in the absence of postveligers, influenced the attachment of zebra mussel postveligers to artificial surfaces. Low-wettable polycarbonate and wettable glass surfaces were used in the experiments over four field seasons to study biofilm formation (1997–1998) and mussel attachment (1998–2000). The presence of the mesh did not quantitatively affect biofilm development on either substratum as determined by microscopic direct counts and colony-forming units on R2A agar. Natural biofilms on polycarbonate surfaces positively influenced postveliger attachment compared to substrata that initially had no film (ANOVA, p-values ranged from ≤.05 to ≤.001). Biofilms did not influence postveliger attachment to glass surfaces (ANOVA, p>.05). Attachment to both substrata was similar on surfaces with and without previously settled postveligers. Based on these results, we conclude that biofilms can enhance postveliger attachment to some but not all artificial surfaces.     

Booster biocides and microfouling

Antifouling (AF) paints are used to prevent the attachment of living organisms to the submerged surfaces of ships, boats and aquatic structures, usually by the release of biocides. Apart from copper, organic booster biocides are the main active components in AF paints, but their use can have a negative impact on the marine environment. The direct effects of biocides on marine bacteria are poorly known. This work investigates the impact of two biocides, viz. diuron and tolylfluanid, on the growth and the viability of marine microorganisms and on their ability to form biofilms. The biocides in solution were found to inhibit growth of two strains of marine bacteria, viz. Pseudoalteromonas and Vibrio vulnificus, at a high concentration (1000 μg ml^?1), but only a small effect on viability was observed. Confocal laser scanning microscopy (CLSM) showed that the booster biocides decreased biofilm formation by both bacteria. At a concentration of 10 μg ml^?1, the biocides inhibited cell attachment and reduced biofilm thickness on glass surfaces. The percentage of live cells in the biofilms was also reduced. The effect of the biocides on two diatoms, Fragilaria pinnata and Cylindrotheca closterium, was also evaluated in terms of growth rate, biomass, chlorophyll a content and attachment to glass. The results demonstrate that diuron and tolylfluanid are more active against diatoms than bacteria.     

Studies on formation,control and application of biofilm formed by food related microorganisms

Biofilms are sessile microbial aggregates on the interfaces, and they were usually considered as microbial contamination sources in medical care and various industries. We studied the control and application of biofilms formed by food-related microorganisms, and mechanism of the biofilm formation was also investigated. We studied the biofilm formation in mixed cultures using various combinations of two strains of food-related microorganisms. There were various microorganisms that showed decreased or increased biofilm formation in the mixed culture in comparison with that in a single culture. Biofilm formed by lactic acid bacteria and yeast isolated from traditional fermented food, Fukuyama pot vinegar, exhibited unique feature in that structure and formation mechanism, and expected to be used as an immobilized microorganism in fermentation production. Here our studies on the control and application of biofilms and the mechanisms of its formation were described.     

Biofouling in water systems – cases,causes and countermeasures

Biofouling is referred to as the unwanted deposition and growth of biofilms. This phenomenon can occur in an extremely wide range of situations, from the colonisation of medical devices to the production of ultra-pure, drinking and process water and the fouling of ship hulls, pipelines and reservoirs. Although biofouling occurs in such different areas, it has a common cause, which is the biofilm. Biofilms are the most successful form of life on Earth and tolerate high amounts of biocides. For a sustainable anti-fouling strategy, an integrated approach is suggested which includes the analysis of the fouling situation, a selection of suitable components from the anti-fouling menu and an effective and representative monitoring of biofilm development.     

Biofilm Formation by Psychrobacter arcticus and the Role of a Large Adhesin in Attachment to Surfaces

Psychrobacter arcticus strain 273-4, an isolate from a Siberian permafrost core, is capable of forming biofilms when grown in minimal medium under laboratory conditions. Biofilms form at 4 to 22°C when acetate is supplied as the lone carbon source and with 1 to 7% sea salt. P. arcticus is also capable of colonizing quartz sand. Transposon mutagenesis identified a gene important for biofilm formation by P. arcticus. Four transposon mutants were mapped to a 20.1-kbp gene, which is predicted to encode a protein of 6,715 amino acids (Psyc_1601). We refer to this open reading frame as cat1, for cold attachment gene 1. The cat1 mutants are unable to form biofilms at levels equivalent to that of the wild type, and there is no impact on the planktonic growth characteristics of the strains, indicating a specific role in biofilm formation. Through time course studies of the static microtiter plate assay, we determined that cat1 mutants are unable to form biofilms equivalent to that of the wild type under all conditions tested. In flow cell experiments, cat1 mutants initially are unable to attach to the surface. Over time, however, they form microcolonies, an architecture very different from that produced by wild-type biofilms. Our results demonstrate that Cat1 is involved in the initial stages of bacterial attachment to surfaces.     

Removal of Burkholderia cepacia biofilms with oxidants

Iodine is used to disinfect the water system aboard US space shuttles and is the anticipated biocide for the international space station. Water quality on spacecraft must be maintained at the highest possible levels for the safety of the crew. Furthermore, the treatment process used to maintain the quality of water on research must be robust and operate for long periods with minimal crew intervention. Biofilms are recalcitrant and pose a major threat with regard to chronic contamination of spacecraft water systems. We measured the effectiveness of oxidizing biocides on the removal and regrowth of Burkholderia (Pseudomonas) cepacia biofilms. B. cepacia, isolated from the water distribution system of the space shuttle Discovery, was grown in continuous culture to produce a bacterial contamination source for biofilm formation and removal studies. A 10(7) CFU ml-1 B. cepacia suspension, in distilled water, was used to form biofilms on 3000 micrometers2 glass surfaces. Rates of attachment were measured directly with image analysis and were found to be 7.8, 15.2, and 22.8 attachment events h-1 for flow rates of 20.7, 15.2, and 9.8 ml min-1, respectively. After 18 h of formation, the B. cepacia biofilms were challenged with oxidants (ozone, chlorine, and iodine) and the rates of biofilm removal determined by image analysis. Fifty percent of the biofilm material was removed in the first hour of continous treatment with 24 mg l-1 chlorine or 2 mg l-1 ozone. Iodine (48 mg l-1) did not remove any measurable cellular material after 6 h continuous contact. After this first removal of biofilms by the oxidants, the surface was allowed to refoul and was again treated with the biocide. Iodine was the only compound that was unable to remove cellular debris from either primary or secondary biofilms. Moreover, treating primary biofilms with iodine increased the rate of formation of secondary biofilms, from 4.4 to 5.8 attachment events h-1. All the oxidants tested inactivated the B. cepacia associated with both primary and secondary biofilms. The amount of biocide needed to inactivate 50% of planktonic B. cepacia in 10 min at 25 degrees C was 8.4, 0.5, and 0.2 mg l-1 for iodine, chlorine, and ozone, respectively. The data suggest that iodine maynot be the best chemical for treating of biofilms when removal of cellular material is required.     

Combating biofilms

Biofilms are complex microbial communities consisting of microcolonies embedded in a matrix of self-produced polymer substances. Biofilm cells show much greater resistance to environmental challenges including antimicrobial agents than their free-living counterparts. The biofilm mode of life is believed to significantly contribute to successful microbial survival in hostile environments. Conventional treatment, disinfection and cleaning strategies do not proficiently deal with biofilm-related problems, such as persistent infections and contamination of food production facilities. In this review, strategies to control biofilms are discussed, including those of inhibition of microbial attachment, interference of biofilm structure development and differentiation, killing of biofilm cells and induction of biofilm dispersion.     

Signals,Regulatory Networks,and Materials That Build and Break Bacterial Biofilms

Summary: Biofilms are communities of microorganisms that live attached to surfaces. Biofilm formation has received much attention in the last decade, as it has become clear that virtually all types of bacteria can form biofilms and that this may be the preferred mode of bacterial existence in nature. Our current understanding of biofilm formation is based on numerous studies of myriad bacterial species. Here, we review a portion of this large body of work including the environmental signals and signaling pathways that regulate biofilm formation, the components of the biofilm matrix, and the mechanisms and regulation of biofilm dispersal.