in vitro biofilm modules

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WELCOME

In vitro biofilm moduls:

In vitro biofilm moduls Bandeppa G S Roll. No. 4891

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A Biofilm is aggregate of microorganisms in which cells are stuck to each other and/or a surface. It is embedded within a self produced matrix of extra cellular polymeric substances(EPS). Biofilm EPS also referred as slime . A biofilm is a structured community of microorganisms encapsulated within a self-developed polymeric matrix and adherent to a living or inert surface. if it is growing in lab condition it is called as invitro biofilm. Introduction

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It may form on living or non living surfaces The cells of microorganism growing on a biofilm are physiologically distinct from planktonic cells of the same organism.

What is a biofilm?:

What is a biofilm? Biofilms are referred as structured communities of sessile microbial aggregates, enclosed in a self-polymeric matrix , attached to an inert or living surface Some of the most familiar biofilms are: the plaque on our teeth, the slippery slime on river stone, and the gel-like film on the inside of vase which held flowers for a week Biofilms have tremendous practical importance in the area of industry, medical and agriculture, exhibiting both beneficial and detrimental activities Costerton , et al ., 1995 5

Formation of Biofilm:

Formation of Biofilm Formation of a biofilm begins with the attachment of free-floating microorganisms to a surface. These first colonists adhere to the surface initially through weak, reversible Vander Waals forces . If the colonists are not immediately separated from the surface, they can anchor themselves more permanently using cell adhesion structures like pili. The first colonists facilitate the arrival of other cell by providing more adhesion sites and beginning to build the matrix that holds the biofilm together.

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During this colonization that the cells are able to communicate via quorum sensing Once colonization has begun, the biofilm grows through a combination of cell division and recruitment . In the final stage the biofilm is established and may only change in shape and size. This development of biofilm allows for the cells to become more antibiotic resistant.

MECHANISMS OF BIOFILM FORMATION:

MECHANISMS OF BIOFILM FORMATION A. Regulatory Mechanisms: 1. Quorum sensing: Plant associated bacteria frequently employ this signaling mechanism to modulate and coordinate their interactions with plants, including control of tissue maceration, antibiotic production, toxin release, and HGT Several different signals function in plant associated bacteria, including ( a) acylated homoserine lactones (AHLs) among proteobacteria; ( b) gamma- butyrolactones in Streptomyces species; (c) cis-11-methyl-2- dodecanoic acid (also called DSF) in species of Xanthomonas , Xylella , and their relatives; and (d ) oligopeptides among gram-positive microbes Von Bodman , et al ., 2003

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2. Regulation in response to phosphorus starvation: Pseudomonas aureofaciens PA147-2 shows a phoB - dependent inhibition of biofilm formation under phosphate starvation B. Phase variation: Pseudomonas brassicacearum NFM421, appears to use a flagellin -overproducing, hypermotile phase variant to colonize A. thaliana root tips Monds R, et al. , 2001 Achauak , et al. , 2004

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C. Motility and Chemotaxis : Taxis toward oxygen and other attractants promotes A. brasilense root colonization. Motility is also involved in biofilm formation of many different microbes, and although its importance is often conditional, mutants tend to be much less competitive when compared with motile strains over time Motility is involved not only in the initiation and development of biofilms, but also in their dispersal, an essential mechanism for spreading and colonizing new habitats Turnbull , et al ., 2001 & van de Mortel , et al ., 2004 Sauer , et al ., 2002

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D . Surface Adhesins : LapA (large adhesion protein) is an important adhesin in certain Pseudomonas species. In P. putida , it is involved in attachment to abiotic surfaces as well as plant seeds E . Biofilm Matrix Components : In S. meliloti and A. tumefaciens the major exopolysaccharide is SCG . It is required for nodule colonization by S. meliloti and an SCG-deficient exo Y mutant formed immature biofilms, whereas overproduction of SCG led to a thicker but less stable biofilm Espinosa- Urgel , et al ., 2000 & Hinsa , et al ., 2003 Fujishige , et al ., 2006

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Plant defense compounds: The systemic plant defense signal salicylic acid acts to directly suppress virulence genes in P. aeruginosa , inhibiting attachment and biofilm formation on A. thaliana roots Phenazine : Phenazine apparently contributes significantly to biofilm maturation in vitro and on plants, in addition to its antibiotic activity in Psuudomonas chlororaphis Surfactants: P. putida strain PCL1445 forms two CLP surfactants, both of which inhibit biofilm formation of several Pseudomonas species and can even dissolve existing biofilms Kuiper , et al ., 2004 Maddula , et al ., 2004 Prithwiraj , et al ., 2005 Metabolites Affecting Bio-film Formation

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When a cell switches to the biofilm mode of growth ,it may undergoes a phenotypic shift in behavior in which large suites of genes are differentially Biofilms are also often characterized by Surface attachment, Structural heterogeneity, Genetic diversity, Complex community interactions

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The biofilm held together and protected by matrix ,the matrix protect the cells within its and facilitates communication among them trough boichemical signals ,some biofilm have been found to contain water channels that help distribute nutrient and signalling molecules Bacteria living in a biofilm usually have significantly different properties from free –floating bacteria of the same species ,as the dense and protected environment of the film allows them to cooperate and interact in many way ,one benefit of this environment is to increased resistance to detergent and antibiotic, some case antibiotic resistant increased to 1000 fold

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On your shower curtain On medical devices implanted in patients On rocks, in rivers and streams, and In your nose etc Biofilms everywhere

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Five stages of biofilm development Initial attachment, Irreversible attachment, Maturation I Maturation II, Dispersion

Fungal-bacterial biofilms and their development for novel biotechnological applications:

Fungal-bacterial biofilms and their development for novel biotechnological applications The microbes undergo profound changes during their transition from planktonic organisms to cells that are part of a complex, surface-attached biofilm . Biofilms have a unique pattern of gene expression which is different from their non- biofilm forming stages Expression of genes for polysaccharide production is up-regulated in biofilm cells compared with planktonic cells

Biotechnological applications of bacterial or fungal biofilms:

Biotechnological applications of bacterial or fungal biofilms Biofilms encompass a spectrum of applications in the medical setting, Apart from medical applications, biofilms have tremendous practical importance in environmental settings. Biofilms can also be used to produce a wide variety of biochemicals Production of anti-microbial compounds. Biological corrosion has been decreased by using beneficial biofilms

Fungal-bacterial biofilms:

Case Study Biofilmed biofertilisers : Novel inoculants for efficient nutrient use in plants Fungal-bacterial biofilms A phase-contrast microscopic view of a fungal filament attached by bacterial cells forming a Fungal-Bacterial Biofilm (FBB) ·

Biofilmed biofertilisers: Novel inoculants for efficient nutrient use in plants :

Biofilmed biofertilisers : Novel inoculants for efficient nutrient use in plants Relationships between pH and indoleacetic -acid-like substances (IAAS) production in liquid culture media by fungal–bacterial biofilms or mixed cultures with no biofilm formation of a large collection of microbes.

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Microbial preparation ARA( nmol C2H4/24h/g fresh weight) SEMIA 5019 alone Nd Penecillium spp. Alone Nd SEMIA 5019 + Penecillium spp 221± 16 Nitrogenase activity of Bradyrhizobium elkanii SEMIA 5019 alone, Penicillium spp. alone, and their combination in the biofilm , evaluated by acetylene reduction assay (ARA). Mean ± SE n=6 Nd = not detected A bradyrhizobial-Penicillium spp . biofilm with nitrogenase activity improves N2 fixing symbiosis of soybean

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Inoculum Plant dry weight (g/plant) Nodule dryweight (mg/plant) N accumulation (mg/plant) Shoot Root Shoot Root SEMIA 5019 alone 0.702b 0.189b 58.80b 10.13b 2.12b SEMIA 5019 penecillium spp. 0.895a 0.231a 84.33a 11. 97a 3.93a Shoot, root and nodule parameters of soybean when inoculated with a biofilmed inoculum of Bradyrhizobium elkani SEMIA 5019 and Penicillium spp., compared to the conventiona SEMIA 5019 inoculum . Mean ± SE (n=6). Cont…

In vitro formation of aggregates and biofilms by Xylella fastidiosa strains isolated from different host plants:

In vitro formation of aggregates and biofilms by Xylella fastidiosa strains isolated from different host plants STRAIN A Source of host Clumps b Strands of cells Attachment to glass b Biofilm on wood CCT citrus +++ + 0 +++ CCT Citrus +++ 0 0 + CCT Citrus +++ 0 0 ++ CCT Citrus +++ + 0 ++ CCT Coffe +++ 0 0 ++ CCT Coffe +++ 0 0 ++ CCT Almond + ++ +++ +++ CCT Plum + ++ ++ + CCT Elm + ++ ++ +++ CCT Grapevine ++ ++ +++ +++ CCT Grapevine ++ +++ +++ +++ CCT Grapevine + ++ 0 + Characterization of Biofilm Formation by Xylella fastidiosa In Vitro

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A, Biofilm formed at the liquid-air interface (arrow) by a grape strain of Xylella fastidiosa , CCT 6752T, representing biofilms attached to glass as observed for almond, elm, grape, and plum strains. B, Scanning electron micrograph of biofilms formed by X. fastidiosa grape strain CCT 6752T on the glass surface of 18-mm culture tubes.

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Biofilm formation by Xylella fastidiosa strains on wood. A, CCT 5598 (citrus); B, CCT 6752T (grape); C, CCT 6068 (grape), D, CCT 6746 (almond), E, CCT 6747 (plum) biofilm,

In vitro phase variation studies of Salmonella Gallinarum in biofilm formation :

In vitro phase variation studies of Salmonella Gallinarum in biofilm formation Sequential steps in biofilm formation by Salmonella Gallinarum. a, Free cells b, One hour (PI); c, One day PI d, Two days PI e, Two days PI f, Seven days PI (maturation and microcolony formation); g, Seven days PI (mature biofilms) and h, Seven days PI (detachment).

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Beneficial Biofilm Formation by Industrial Bacteria Bacillus subtilis and Related Species FIG. Biofilm formed by B. subtilis wild type. (a) Side view; (b) optical microscopic observation; (c) scanning electron microscopic observation.

BIOFILMS AS BIOCONTROL AGENT:

BIOFILMS AS BIOCONTROL AGENT Found in the plant rhizosphere Promotes plant growth Acts as a bio control agent Biofilm as biocontrol agent

BIOFILM AS BIOREACTOR:

BIOFILM AS BIOREACTOR Adsorbed cells form cell layers on the support, and the cell mass grows inside the reactor over time. These layers of cells form typical biofilms ,these biofilm reactors have been successful in wastewater treatment and the production of fermented products such as vinegar and acetic acid.

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Biofilm protect the plant from desiccation and other physical stress It is the biofertilizer of next generation It helps in the cycling of nutrient It helps as a biocontrol of pest and diseases Conclusions

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Biofilm provide a protective environment for microorganisms Synthesis of exopolysaccharides such as cellulose and alginate can affect the formation of biofilms on plants, but their role varies depending on the microbe It acts as pseudonodules in N 2 fixatation biologycally

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Thank you

Biofilm Resistance Mechanisms:

35 Biofilm Resistance Mechanisms There are four mechanisms a biofilm uses to resist stressors: Stewart, P. S. 2002. Int. J. Med. Microbiol. 292: 107-113. Four mechanisms of biofilm resistance: Lack of penetration, slowed metabolism, stress response, and resistor cells.