Biofilms in Aquaculture- Benefit and Harm

Sujit kumar FB-MA2-01

Presentation outline

• What is biofilm?• Composition and str of biofilm• Dynamics of biofilm formation • Significance of biofilm formation• Biofilm and aquaculture• Impact of biofilm on health

management in aquaculture• Application of biofilm in aquaculture.

INTRODUCTION

• A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA (Hoiby N et al. 2009)

• Society of bacteria (The social life of microorganisms )

• A biofilm is a well organized, cooperating community of microorganisms.

• Biofilms are matrix-enclosed microbial accretions that adhere to biological or non-biological surfaces (NATURE REVIEWS | MICROBIOLOGY

VOLUME 2 | FEBRUARY 2004 | 95)

Biofilms

Formation of biofilms in nature

Biofilms are ubiquitous communities since it invariably develops on all solid surfaces exposed to aquatic environments(Allison and Gilbert, 1992; Rao et al., 1997)

• BIOFILMS may form:

– On solid substratums in contact with moisture– On soft tissue surfaces in living organisms 

- At liquid-air interfaces. 

Structure of biofilm• Confocal scanning laser microscope

(CSLM) - to monitor biofilm development in flow cells (allows direct observation of the biofilm without disrupting the community)- visualization of fully hydrated sample

• Biofilm is composed primarily of micro colonies of different species of microbial cells (+15% by volume) and of matrix material (+85%).

• Heterogeneous structure which includes:– cell clusters– void spaces– water channels– end slime streamers

MATRIX MATERIAL

• EPS (extracellular polymeric substance )• EPS may vary in chemical and physical

properties, but it is primarily composed of polysaccharides

• Some of these polysaccharides are neutral or polyanionic, as is the case for the EPS of gram-negative bacteria.

• The presence of uronic acids (such as D-glucuronic, D-galacturonic, and mannuronic acids) or ketal-linked pryruvates provides the anionic property

• This property is important because it allows association of divalent cations such as calcium and magnesium, which have been shown to cross-link with the polymer strands and provide greater binding force in a developed biofilm.

• In the case of some gram-positive bacteria, such as the staphylococci, the chemical composition of EPS may be quite different and may be primarily cationic.

• Hussain et al found that the slime of coagulase-negative bacteria consists of a teichoic acid mixed with small quantities of proteins.

• EPS is also highly hydrated because it can incorporate large amounts of water into its structure by hydrogen bonding.

• EPS may be hydrophobic, although most types of EPS are both hydrophilic and hydrophobic.

• EPS may also vary in its solubility. • There are two important properties of EPS

that may have a marked effect on the biofilm. – composition – structure of the polysaccharides determine their

primary conformation.

MICROBES• The basic structural unit of the biofilm is the

microcolony.• Proximity of cells within the microcolony (or between

microcolonies) provides an ideal environment for creation of nutrient gradients, exchange of genes, and quorum sensing.

• Since microcolonies may be composed of multiple species, the cycling of various nutrients (e.g., nitrogen, sulfur, and carbon) through redox reactions can readily occur in aquatic and soil biofilms.

• Biofilm formation is a response by microorganisms to alterations in growth rate, exposure to subinhibitory concentrations of certain antibiotics, and growth on solid surfaces (Brown and Gilbert 1993; Sasahara and Zottola 1993; Yu and McFeters 1994; Smoot and Pierson 1998a, 1998b; Kerr et al. 1999).

• Many bacterial EPS possess backbone structures that contain 1,3- or 1,4-β-linked hexose residues and tend to be more rigid, less deformable, and in certain cases poorly soluble or insoluble.

• Other EPS molecules may be readily soluble in water.• Second, the EPS of biofilms is not generally uniform

but may vary spatially and temporally.

• Different organisms produce differing amounts of EPS and that the amount of EPS increases with age of the biofilm.

• EPS may associate with metal ions, divalent cations, other macromolecules (such as proteins, DNA, lipids, and even humic substances).

• Slow bacterial growth will also enhance EPS production.

http://www.umb.no/statisk/konferanser/lsh_nov25.pdf

BIOFILM FORMATION

Steps in biofilm formation

• Surface conditioning• Adhesion of “pioneer” bacteria• Slime formation• Secondary colonizer• Fully functioning biofilm

• SURFACE CONDITIONING

– adherence to the surface

– neutralization of surface charge

• ADHESION OF PIONEER BACTERIA– Attachment of Planktonic

(free-floating) by electrostatic attraction and physical forces

– Some of these cells will permanently adhere to the surface with their extracellular organic matrix.

• SLIME FORMATION– Extracellular polymers consisting of

charged and neutral polysaccharide groups cement the cell and act as an ion exchange system for trapping and concentrating trace nutrients

– Accumulation of nutrients promotes reproduction of pioneer cells. The daughter cells then produce their own exopolymers, greatly increasing the volume of ion exchange surface.

• SECONDARY COLONIZERS

– The exopolymer web snares other types of microbial cells through physical restraint and electrostatic interaction. These secondary colonizers metabolize wastes from the primary colonizers as well as produce their own waste which other cells then use

• FULLY FUNCTIONING BIOFILM

• A complex, metabolically cooperative community made up of different species each living in a customized microniche

• The mixed species work cooperatively to carry out complex tasks which otherwise cannot be performed by a single species

Role of Biofilm in Microbial Communities

• Protection from Environment• Nutrient Availability• Acquisition of New Genetic Trait

BIOFILMS

FACTORS AFFECTING GROWTH AND ACTIVITY OF

MICROORGANISMS

ENVIRONMENTAL FACTORS

• Temperature• Aerobic/anaerobic conditions• pH• Dynamic conditions

WATER AVAILABILITY

Flow velocity

Nutrient availability

Mechanisms of biofilm resistance to

antimicrobial agents

Drug resistance in biofilms. A schematic of mechanisms that can contribute to the resistance of biofilm grown bacteria to antimicrobial agents. The extracellular polysaccharide is represented in yellow and the bacteria as blue ovals.

• Biofilms are marked by their heterogeneity and this heterogeneity can include gradients of nutrients, waste products and oxygen (illustrated by colored starbursts).

• Mechanisms of resistance in the biofilm include increased cell density and physical exclusion of the antibiotic.

• The individual bacteria in a biofilm can also undergo physiological changes that improve resistance to biocides.

• Various authors have speculated that the following changes can occur in biofilm-grown bacteria: – (1) induction of the general stress response (an rpoS

dependent process in Gram-negative bacteria)– (2) increasing expression of multiple drug

resistance (MDR) pumps; – (3) activating quorumsensing systems; and – (4) changing profiles of outer membrane proteins

(OMP)

• There are multiple mechanisms, which vary with the bacteria present in the biofilm and the drug or biocide being applied.

• These mechanisms include physical or chemical diffusion barriers to antimicrobial penetration into the biofilm, slow growth of the biofilm owing to nutrient limitation, activation of the general stress response and the emergence of a biofilm-specific phenotype.

Failure of the antimicrobial to penetrate the biofilm

• It has been suggested by several studies that matrix prevents the access of antibiotics to the bacterial cells embedded in the community.

• Either reaction of the compound with, or sorption to, the components of the biofilm can limit the transport of antimicrobial agents to the cells within the biofilm.

• Chlorine, a commonly used disinfectant, did not reach >20% of the bulk media’s concentration within a mixed Klebsiella pneumoniae and P. aeruginosa biofilm, as measured by a chlorine-detecting microelectrode.

Slow growth and the stress response

• When a bacterial cell culture becomes starved for a particular nutrient, it slows its growth.

• Transition from exponential to slow or no growth is generally accompanied by an increase in resistance to antibiotics.

• Slow growth of the bacteria has been observed in mature biofilm.

• Because cells growing in biofilms are expected to experience some form of nutrient limitation, it has been suggested that this physiological change can account for the resistance of biofilms to antimicrobial agents.

• Gilbert and colleagues examined growth-rate-related effects under controlled growth conditions for planktonic cultures and biofilms of P. aeruginosa, Escherichia coli and S. epidermidis.

• They made the general observation that the sensitivities of both the planktonic and biofilm cells to either tobramycin or ciprofloxacin increased with increasing growth rate, thus supporting the suggestion that the slow growth rate of biofilm cells protects the cells from antimicrobial action.

General stress response

• Recently, it has been suggested that the slow growth rate of some cells within the biofilm is not owing to nutrient limitation per se, but to a general stress response initiated by growth within a biofilm.

• The stress response results in physiological changes that act to protect the cell from various environmental stresses.

• Thus, the cells are protected from the detrimental effects of heat shock, cold shock, changes in pH and many chemical agents.

• The central regulator of this response is the alternate σ factor, RpoS, originally thought to be expressed only in stationary phase.

• However, recent studies suggest that RpoS is induced by high cell density and that cells growing at these high densities seem to have undergone the general stress response, as judged by the production of trehalose (an osmoprotectant) and catalase.

• As cells in a biofilm experience high cell density, it is logical to propose that these cells would express RpoS.

• It has been shown by RT-PCR that rpoS mRNA is present in sputum from CF patients with chronic P. aeruginosa biofilm infections

• Another link between RpoS and biofilms was recently identified: E. coli cells that lack rpoS are unable to form normal biofilms whereas planktonic cells are apparently unaffected by the absence of this σ factor

Quorum sensing

• The role of quorum sensing in biocide resistance is not yet clear.

-Previous work by Davies and colleagues showed that a mutant in the lasR–lasI quorum-sensing system in P. aeruginosa was unable to form a biofilm with normal architecture.

Moreover, these authors presented data showing that lasI mutant biofilms were abnormally sensitive to treatment with SDS, although the question of whether these mutant biofilms had

altered antibiotic resistance was not addressed.

Induction of a biofilm phenotype

• A biofilm-specific phenotype is induced in a subpopulation of the community that results in the expression of active mechanisms to combat the detrimental effects of antimicrobial agents.

• This phenotype might be induced by nutrient limitation, certain types of stress, high cell density or a combination of these phenomena.

• Multidrug efflux pumps can extrude chemically unrelated antimicrobial agents from the cell.

• In E. coli, upregulation of the mar operon results in a multidrug-resistant phenotype.

• The efflux pump thought to be responsible for this resistance is AcrAB.

• Another resistance mechanism that can be induced in biofilm cells is the alteration of the membrane-protein composition in response to antimicrobial agents.

• This change could result in decreased permeability of the cell to these compounds.

BIOFILM IN AQUACULTURE

PROBLEMS AND BENEFIT

Problems

• Biofilms form at the water/solid interface of all components of aquaculture systems.

• Some bacteria found in aquaculture system biofilms are essential in removal of metabolic toxins harmful to fish, such as ammonia and nitrites.

• Studies have shown that bacteria in biofilms can alter cellular functions as an adaptive response to adverse environmental conditions, such as sublethal antimicrobial treatments or poor nutritional levels (Brown and Gilbert 1993; Sasahara and Zottola 1993; Yu and McFeters 1994; Smoot and Pierson 1998a, 1998b; Kerr et al. 1999; Stoodley et al. 1999)

• The differences between sessile biofilm cells and their planktonic counterparts include different cellular enzymatic activity, cell wall composition, and surface structures (Smoot and Pierson 1998a, 1998b; Wong 1998)

• Biofilm bacterial cells have been found to be resistant to antimicrobials including antibiotics, surfactants (or detergents), heavy metals, phagocytic predators and drying (Brown and Gilbert 1993; Ronner and Wong 1993; Yu and McFeters 1994; Liltved and Landfald 1995).

• The presence of pathogenic bacteria in aquaculture system can make the system a potential unacceptable public health risk.

• The periodic sloughing of pathogens may also cause recurring disease in stressed fish or can lead to the presence of infected asymptomatic fish being consumed.

• Another, perhaps more hazardous occurrence, would be the possibility of more pathogens with increasing antibiotic resistance if chemotherapeutics were employed to treat symptomatic fish.

Pathogenic bacteria associated with biofilms in commercial aquaculture

facilities

BENEFIT

• Help in establishment of periphyton

Treatment of water through bioremediation.

Biofilm vaccine

• Certain bacteria form biofilms on substrates (Costerton, 1984), and these have been found to be resistant to antibiotics (Anwar & Costerton, 1990), phagocytosis and the killing effect of whole blood and serum (Anwar et al., 1992) due to a protective glycocalyx layer.

• The glycocalyx matrix of the biofilm vaccine is believed to protect the antigens against gastric destruction.

REFERENCES

• http://classes.biology.ucsd.edu/old.web.classes/bimm124.SP05/mechanisms_biofilm_resistance.pdf

• Robin K. King,Bacterial Pathogens in Biofilms Pose Health Risks in Recirculating Systems, GLOBAL AQUACULTURE ADVOCATE OCTOBER 2002

DISCUSSION

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