Effect of Biofilm on Production of Poultry

Abstract

Attachment of bacterial biofilm to the surfaces of farm, fomites and equipments remains chance transmission of infection poultry and human through food chain. Formation of biofilm causes spoilage of poultry products during processing of eggs, meat and distribution. Biofilm may cause many bacterial species in biofilm society. The formation of biofilm deteriorates food quality, water supply system, drugs resistance, and reduces the efficacy of equipments, spread disease and lingering of disease course. Common bacteria cause biofilm in poultry farm and food industries are Salmonella sp., Staphylococcus spp., Listeria monocytogenes, Escherichia coli, Klebsiella pneumonae, Campylobacter jejuni, Streptococcus agalactiae. Formation of biofilm is under stress and regulated by several genes of bacterial. There are several methods of diagnosis of biofilm such as Roll plate method, tube method, microtitre assay, PCR assay, mass spectrometry method and Biological assay of Biofilm. Therapeutic elimination of biofilms for smooth production of poultry is chemical and environmental modifications. Water may be treated with several means, both chemical and physical ways. Food-contaminated biofilm-related treatment is done applying quaternary ammonium compounds, aldehydes, phenolics, alkyl amines, chlorine dioxide, etc. Veterinary medical therapy against biofilms is use of antibiotics with ultrasound, low electric current, phage therapy, nanodrug delivery system, antimicrobial peptides, antiadhesin, antimatrix and chelating substances.

1. Introduction

Biofilm is a complex structure of microbial populations having different bacterial colonies or monospecies cell type; adhere to the surface of growth. These cells are embedded in extracellular polymeric substances, the matrix substance which is generally composed of extracellular DNA (eDNA), proteins and polysaccharides, showed high resistance to antibiotics and physicochemical tolerance. The formation of biofilm have several impact in the poultry production, dessimination of infection and farm management system. In tropical countries different seasons such as horse summer, dry winter may acts as stress for formation of biofilm. These biofilm may affect the production performance, disease transmission and human health concern. Poultry farm and duckery where there is every chance of formation of biofilm needs special care and intervention against formation of biofilm and proper intervention for effective production and restriction of disease outbreaks.

2. Bacterial biofilming/biofouling conditions

Growth of bacterial population in colony or in a specific area or even in culture containers, the cells are stick to each other as well as with surface of growth container. The adherence materials are extracellular matters that may be composed of wide ranged components of extracellular polymers, these polymers may be with polysaccharides, proteins, lipid, pilli, flagella or even with eDNA). Not all microorganism can produce biofilm, some bacteria (both Gram negative and Gram positive), fungi and protists can produce biofilm. Most common bacteria those can produce biofilm are Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Bacillus subtilis, Pseudomonus fluorescens, Streptococcus mutans, Streptococcus salivaris, Acitenobacter baumanni [1, 2, 3].

Ornithobacterium rhinotracheale is a Gram-negative bacillus that causes respiratory disease in birds, and directly affects the poultry industry producing biofilm uncertain conditions [4]. Some common example of a biofilm are dental plaque, heart muscle, pond scum. Biofilms grow in rain forests, in desert as “desert varnish”, ocean bottom as deep sea vent, glaciers in Antartic They have been found at the bottom of the ocean as early colonizers of new deep-sea vents and living on glaciers in the Antarctic. The biofilm may grow in normal conditions in industrial infrastructure, hospital, different living tissues and organs of animal and human. Biofilm formation at the air-liquid and solid-liquid interfaces are very common [5]. The origin of biofilm is not just in recent thought; it was present in the primitive earth condition for prokaryotes as defense mechanism. Inside the host, the extra cellular matrix protects biofilm making bacteria from expose to innate immune defenses (phagocytosis, opsonization and antibiotics [6]. Biofilm also helps against desiccation, antibiotics and host body defense immunity.

2.1 Abiotic condition for biofilm formation

Several conditions that may alter the formation of biofilms are temperature (37–40°C), presence of CO₂ (5%), low nutrient supplements in the media, water deprivation/hydrodynamics, osmolality of the medium, concentration metals such as iron and ambient acidity [7]. Several other factors also determine the biofilm formation, presence of toxicants, oxygen concentration, antibiotics and salinity of the environment affects for motion biofilm. Nature of substratum environment of the surface of attachment, glass and stainless steel surface are more hydrophilic for growth of biofilm than hydrophobic rubber, Teflon surface.

2.2 Biotic condition for biofilm formation

The biofilm formation may contain several communities of microbes with different species and class of organisms. The community composition where there may be several microorganisms like bacteria, fungi, algae in a biofilm population. Host stress is another factors growth of biofilm. Microbial genetic factors also a determinant. Several genes are responsible for attachment at the surface and subsequent maturation and dispersion of microbes particularly in E. coli. Population of microbes is another determinant that also affects the formation biofilm. Quorum sensing (QS) has big role on production and release of signal molecules called autoinducers. Production of several extracellular proteases that helps in dispersal of biofilm is regulated by QS system in Staphylococcus aureas and B. subtilis. Production of microbial byproducts such as metabolites like antibiotics, pigments, and siderophores also check the formation of biofilms. Antimicrobial peptides can restrict the development of biofilms. The antimicrobial peptides (bacteriocenes) such as dermicidin, tachyplesins are this kind of antibacterial peptides that may prevent for formation of biofilms and has potential clinical application against drug resistance and against biofilm formation [8].

3. Formation of biofilm

For the formation of biofilm an attachment with a surface is necessary, surface may be biotic or abiotic. Attachment at the surface may be with weak Van der Wales force and hydrophobic effect. In case of initial mild attachment is not disturbed, colonies are attached permanently with the cell adhesive structures like pilli, hami (archeal pilli like structure), flagellum [9]. Both motile and nonmotile as well as Gram positive and Gram negative bacteria aggregate together to form biofilm easily. During surface colonization (adhesion) bacterial cells can communicate by quorum sensing (cell to cell communication) traits like virulence factor [10] with the help of products such as N-acetyl homoserin lactone. Once the colonization begins on the settlement surface, the biofilm grows by a combination of cell division and cell recruitment. Besides quorum sensing molecules, several other signals trigger biofilm formation are secondary metabolites of bacteria such as antibiotics, pigments, siderophores. Sub-inhibitory concentration of antibiotic imipenem and tobramycin induce production of biofilm [11].

The composition of the biofilm is mostly with polysaccharides matrix (d-glucose; d-mannose; l-rhamnose [12] which is encloses bacteria forming cocoon like condition. In addition to polysaccharide in biofilm matrix there may be other materials such as protein, eDNA, extracellular enzymes like aminoglycoside modifying enzymes (AMEs), β-lactamase. Gram +ve and Gram –ve bacteria can produce biofilm. Few bacteria are more prone to form biofilm while some are less.

3.1 Stages of biofilm

There are several stages of biofilm formation starting from initial attachment. Five stages are there for complete formation of biofilm. They are stage of initial reversal attachment, irreversible attachment, maturation phase-I, maturation phase-II and dispersion. Other than bacteria protozoa, fungi, algae and archaea can produce biofilm. The common niche where the biofilm produced are slow sand filler, for water purification plant, percholating filler, mammalian intestine, animal and human organs such as urinary tract, endocardium, joint and articulations, heart valve, medical and veterinary tools and devices used may be affected with surface attachment in urinary catheter, prosthetic joints, pacemakers, stomach tube, teat syphone, milking machine etc.

In animal and veterinary medicine biofilm has tremendous impact in livestock industry and animal health that leads to tremendous economic loss. The most challenges posed with biofilm production causes antibiotic resistance which also a big threat to human health through food chain. A wide ranges of bacterial infections in veterinary importance are resistant to antibiotic therapy. Secondly, diseases are not responding to antibiotics when applied on certain disease conditions. Such pathological conditions are mastitis due Streptococcus and Staphylococcus species infection. Other diseases those also cause less healing response in pasturella pneumonia, enteritis on E. coli and Salmonella spp., urinogenital tract infection with E. coli, periodontal disease (Staphylococcus spp.), caseous lymphdenitis (Corynaebacterium pseudotuberculosis), wound infection (Pseudomonus, Staphylococcus spp. etc), pyomettra (E. coli) and others [2].

In poultry industries, several bacterial infections such as Salmonellas sp., produces biofilm in poultry meat industry that also cross contaminate public health impact [13].

4. Formation of biofilm in poultry industry

In poultry processing-during poultry processing the carcasses may come contact with many solid surfaces and forms biofilms. Bacteria may attach from carcass to the wet equipments. This may acts as continuous cross infection. Poultry plants and equipment’s solid surface have different affinity for bacterial attachment and formation of biofilm. An increased extracellular matrix of fibrils and debris are connected with individual bacterial cell. Many bacteria of same species or different species may aligned in side to side pattern. Increase attachment of bacterial population and formation continuous biofilm may act as concern in poultry plant sanitization and pathogen control [14]. Eleven different species of bacteria have been isolated and identified from meat processing unit [15]. This may acts as constant source of infection to other carcasses that lead to public health concern too. Biofilm with slime layers with matrix enclosed bacterial population like population of a metro city. On the same bacterial surfaces similar and different species can adhere each other’s side or interfaces. These bacterial population show community homeostatis, primitive circulatory cooperation and exchange of genetic materials as well as metabolic cooperation [16, 17]. Formation of biofilm on equipments and poultry plants cause damage of equipment, product contamination, loss of food energy and dissemination of infection. The microbes those affect the poultry industries are E. coli., Listeria monocytogenes, Pseudomonus fluorecens, Acenetobactor harbinensis, Arthrobactor sp., Brochothrix thermosphacta, Carnobacterium maltaromaticum, Lactococcus piscium, Mycobacterium spp., Campylobacter jejuni, Pseudomonus fragi, Psycgrobacter spp., Rhodococcus erythropolis, Stenotrophomonas sp.[15, 18]. In food processing environment, bacteria in biofilm as well as suspended forms undergo stresses such as dehydration, temperature variations, antimicrobial agents, therefore, their morphology is changed than their planktonic relatives. As a result they become more resistant (up to 500 times) to antimicrobials [16]. These bacteria also show slow growth not due to nutrient deficiency but due to stress. In the biofilm city/society all the species remain but some of the species contribute to enlarge the size of the biofilm. The formation of biofilm depends on different surface material made up of and nutrients content in the media. It has been reported that glass surface, stainless steel and plastic surface varies. Biofilm can be grown in any surface of stainless steel, glass, rubber, polycarbonate, polyurethane, polystyrene, polypropylene, Teflon, nitrile rubber, titanium, aluminum, ceramic, and wood for developing countries in poultry farms and industries [19]. The formation of biofilm of the above article surfaces remained 96,144 and 240 h with 10⁶ cfu/cms for salmonella isolates [20]. Due to contamination of biofilm in poultry and poultry industries several diseases may occur.

5. Advantage and difficulties of biofilm formation in poultry industry

The bacteria show biofilm formation for their survival and to overcome hardship and stress. The extracellular polymeric substances (EPS) of biofilm is negatively charged and hydrophobic in nature helps to keep concentrated ions and dissolved carbon compound from the bulk fluid medium. The advantageous points for bacteria are (i) protection from antibiotics, and antimicrobials (ii) increased availability of nutrition’s for their growth (iii) increased capacity of binding water molecules and avoiding dehydration (iv) keep close contacts with progeny, relatives and other bacteria for strategic ecology and transfer of plasmid (v) avoid adverse environments such as temperature, changed pH, antiseptics, disinfectants etc. Biofilm bacteria are more resistant than the planktonic ones, this due to acquisition of resistant genes in plasmid which also transmitted to other species in the biofilm colony.

6. Poultry farm, fomites and water supply system

Biofilm produced by bacterial species and population firmly adhere to the surfaces of attachment with its matrix EPS. These bacterial communities survive for long time and create resistance to various antibiotics; antimicrobials and disinfectants. These being potential contaminants in farm and fomites extend dessimination of infection to other population of birds, animals and human. The contaminants may be at any stage of farm and poultry industry particularly with very common organisms of Salmonella and Campylobactors [21]. The accumulation of biomass of biofilm affect major constrains in water supply in poultry industry. The bacterial biofilm may disturb in area of walls, floors, pipes, watered, drain, feed trough and utensils made up of steel, aluminum, nylon, rubber, plastic, glass and polystyrine [22]. In poultry industry particularly broiler farm, slaughter house, meat processing units, produces large amount of residues mainly proteins and lipids those are accumulated on the surface of containers, drains and waste chambers generate biofilm that eventually target the public health concern. Whatever the top most farm management may be for the poultry farming, there is every chance to be contaminated and formation biofilm with endemic pathogens with E. coli, Pseudomonus sp., Salmonella sp., Coliform bacteria and Enterobacter.

There is also chance of formation of biofilm and transmission of infection with L. monocytogenes, Campylobacer jejuni associated with poultry industry and diagnostic kit wares.

7. Biofilm potential source of economic loss

Production of biofilm in poultry industries cause huge economic loss, through food spoilage with constant source of potential infection sources and damage of water supply installations, equipments and water supply lines. A wide range of disease conditions causing by food contaminations such as gastroenteritis, abdominal colic, fever, indigestion and several other systemic diseases like respiratory disease, flaccid paralysis in human and veterinary importance. During high summer, the poultry units use cool ventillary system like cooler and wet straw cooling system which may have preexisting biofilm or it may generate biofilm that also spread infection to poultry population and poultry products. The chemicals and supplements used in poultry unit through feed and water may help in the propagation of biofilm.

The biofilm infection also causes certain condition in animals and birds. They are chronic inflammation, impaired wound healing, chronic skin diseases, formation of infectious emboli and antibiotic resistance. Poultry hatchery particularly duck hatchery is also a big sources of biofilm formation epicenter. Several instruments such as incubator, brooder, hover, brooder guards, and humidity chamber may be contaminated with biofilms [23].

Egg cold storage where eggs are stored, packaged and transported, may also be a potential source of biofilm producing concern. Eggs may be kept in trays and basket may be having preformed biofilm that also acts as potential constant source of infection for human through food chain and for next generation chicks/ducklings. Poultry pathogens like Salmonella enterica, can cause biofilm formation through feces of chicken and turkey and acts as very possible antimicrobial resistance [24].

8. Poultry drinking water standard

All the water supplied for poultry should be maximum cleaned and hygienic, there should be minimum level of microbe content and mineral composition in water. If the water content for microbial population and minerals are high, there should be option for big correction. More microbes and minerals content induce health hazard [25]. Several microbial loads that affect water quality for poultry farms due to different bacteria such as E. coli, Salmonellas spp., Pseudomonas spp., Campylobacer jejuni, coliform bacteria, Enterobacter spp., L. monocytogenes, Staphylococcus spp., are more common contaminants [26, 27, 28, 29]. The microbial contents in the water vary with species and their numbers. The minimum and maximum level of bacteria usually occurs and permissible are 0–100 CFU/ml of water (Table 1). More bacterial nuclei in the water deviates the standards of health of birds and also the taste of water as well as amount of water used by the birds. There may be restriction in common salts content in water such as sodium (50–150 mg/L), sulphate (15–200 mg/L), nitrate (1–25 mg/L), zinc (0–1.5 mg/L), calcium (60 mg/L), ferrus salt (0.2–2.5 mg/L). The mineral contents in drinking water for poultry needs a standard with restriction of minimum and maximum level [30].

Component of water	Level expected	Lower acceptable level	Higher acceptable level	Correction
Total microbes	0	100	300	Chlorination, sanitizing cleaner for safe water
Total aerobic plate count (cfu/ml)	0	<100	<1000	Water sanitizing
Total coliform (cfu/ml)	0	50	<50	Water sanitizing
Fecal coliform (cfu/ml)	0	0	0–1	Water sanitizing
E. coli (cfu/ml)	0	0	0–1	Water sanitizing
Pseudomonas (cfu/ml)	0	0		Water sanitizing
pH level	5–8	6.5	7.8	pH increase with soda, Na2Co3,NaOH,Ca(OH)2, pH decrease withHPO3, H2SO4,HCL,citrate,vinegar.
Total water hardness	0–17	60	150	Acidification and use of polyphosphate to softenwater
Calcium salt mg/L	60	60–80	110	As above
Iron mg/L	0.2	0.3	0.4	Filtration and chlorination
Sodium mg/L	50	100	150	Reverse osmosis, sanitization
Sulphate mg/L	15	40	150	Treatment with oxidizing sanitizers then filtration
Lead mg/L	0.1	0.1	0.1	Water softeners and activated carbon can reduce lead
Nickel mg/L	0.01	0.01	0.05	Water softeners and activated carbon can reduce lead
Cadmium mg/L	0.5	3	5	Zinc oxide (ZnO), manganese oxide (MnO2), titanium oxides (TiO2), magnesium oxide (MgO)

Table 1.

Water standards and option for correction for poultry.

9. Bacterial biofilm and gene regulation in poultry industry

In poultry farming and meat processing industries several bacterial contaminations may be a common sequlae where large numbers of microbial contamination and transmission may occurs through egg, meat, fomites, machinery, water supply system and utensils used in this sector. Very common infections those affect the birds health could be forming biofilms. These may cause various economical and health concern in poultry industries. Salmonella is common pathogens in poultry system. Salmonella gallinarum, S. typhimurium and S. enteritidis are prevalent. The genes responsible for biofilm formation are csgD and bcsA adrA, gcpA [20, 31]. Lipopolysaccharide (LPS) producing gene is rfbA that helps formation biofilms. Using transposon mutagenesis, several genes such as metE, ompR, rpoS, rfaG, rfaJ, rfaK, rfaP, rfbH, rhlE, spiA, and steB are found to be associated with biofilm formation of S. enteritidis [32]. Similarly, there are several genes in E. coli bacterial genomes where many genes controls the formation of biofilms. Several adherence genes such as luxS, iha, papC, aatA, aggR fimC have been described [33]. Many other genes also have role in the biofilm formation. They are fliC. csfA, luxS, adrA, gcpA [20, 34]. Common poultry contaminate E. coli have many genes responsible for biofilm formation. Gene like fliC, csgA, fimA, luxS, his, papC, aatA, aggR, fimC, help in the formation and adhesion of bacterial growth on surface [3, 33].

Klebsiella pneumonia causes pneumonia, septicaemia and liver abscess in poultry. They parasitize in respiratory and gastrointestinal system. Formation of biofilm in different organs of poultry and poultry industry is very high by the organism (upto 93.6%). The samples that may transfer the biofilm through surgical wound, feces and other discharges [35, 36]. The genes responsible in Klebsiella pneumonae for formation of biofilm in poultry are treC, sugE which produce more capsular saccharides (cps) that helps in biofilm formation [37]. The Enterococcus sp. procuses biofilm frequently. The quorum sensing peptide pheromones (cpd, cob, fsr, ccf) are secreted by the cell to induce conjugate apparatus of doner cell. The bacteria transfer the pherome responsive plasmid which carry virilence genes promotes biofilm formation. Enterococcus fecalis, E. faecium, E.durans, E. hirae, and E. cecorum show biofilm formation in poultry. The most genes responsible for biofilm formation are ebpB, ebpC and srt. Acenobacter baumanni have some gene that cause biofilm. Serotypes have several gene that regulate biofilm are ompA, bap, blaPER-1, csuE, csgA, and fimH. Proteus mirabilis cause several diseases in poultry such as cellulitis, digestive disorder, urinary infection and hydronephrosis [31]. Several biofilm producing genes in poultry due to Proteus sp. are mrpA, pmfA, ucaA, atfA, zapA, ptA, hpmA, and ireA, ureC, zapA, rsmA, hmpA, mrpA, atfA and pmfA (Table 2) [45, 48]. Pseudomonas aerugenosa is very common poultry pathogens causes diarrhea, septicaemia and respiratory diseases. The bacteria may transmit from animals and inanimate objects where they form biofilm. Several virulent genes have been isolated responsible for disease production. Ggenes responsible for biofilm formation are katA and kpsM.

Name of organism	Causes disease in poultry	References
Escherichia coli	Avian colibacillosis	Grakh et al. [38]
Riemerella anatipestifer	Epizootic infectious disease	Sun et al. [39]
Salmonella enteritides; S.typhimurium	Salmonellosis	Afshari et al. [40]
Klebsiella pneumonae	Respiratory disease	Ammar et al. [41]
Listeria monocytogenes	Septicemia, encephalitis	Ossaili et al. [42]
Campylobacter jejuni	Transient diarrhea in chicks	Shanes [43]
Pseudomonas auruginosa; P. fragi	Gastroenteritis and zoonotic infection	Wafaa and Ghany [44]
Proteus mirabilis	Cellulitis, GI disorder	Sanches et al. [45]
Staphylococcus aureus, S. intermedius, S. schleiferi, S. pseudointermedius, S. lutrae	Arthritis, synovitis, and osteomyelitis	Marek et al. [46]
Staphylococcus aureus, Staphylococcus hyicus, Streptococcus agalactiae	Septicaemia, peritonitis, salpingitis, tropical infection and endocarditis	Olson et al. [47]

Table 2.

Bacteria cause poultry diseases and have biofilm forming capacity.

Campylobacter is also a pathogenic bacterium in poultry flock and several genes responsible for production of biofilm in surfaces of stainless steel and polystyrine articles at different temperatures and oxygen concentration. The genes responsible for production of biofilm are bhpC, cadF, clpP, dnaJ, docA., flaA, flaB, katA, kspM, luxS, racR and sodB [49].

Ornithobacterium rhinotracheale is a Gram positive bacterium, causes respiratory disease in poultry and other birds that affect the productivity. All serovar A-E can produce biofilm at optimal condition of 40°C after 72 hours of incubation in elevated CO2 concentration [4].

Listeria monocytogenes is an important poultry bacterium that causes septicemic condition in poultry. The bacteria has significant role in the public health concern through egg and meat food chain. Samples collected from different poultry outlets revealed biofilm forming capacity [50]. The high capability for biofilm formation in this organism derived out of several genes such as luxS and flaA [51]. The ability of L. monocytogenes have adaptability in refrigerated environment in poultry slaughter houses and industry, food processing unit, fish processing unit as well as in vegetable processing industries [52]. It has been found hlyA gene may have role in the formation of biofilm in stainless steel and polypropyline surface [53]. Different Mycobacterium sp. are also have role in the biofilm formation process in poultry farm and meat food industries. Many species other than Mycobacterium tuberculosis are involved in the formation of biofilm. Mycobacterium avium, M. fortuitum, M.smegmatis produce biofilm and transmission of diseases to new hosts. The Mycobacterium spp. may produce biofilm in the variable temperature and conditions (Table 2).

10. Diagnosis of biofilm formation

Formation of biofilm in veterinary and medical related instrument, tools and in different tissues and in vitro structures may be due to various methods. Several methods of direct and indirect methods are there for detecting the biofilm formation. In direct methods, observing the microbial colonization with several techniques such as contact plates, enzymatic reaction, electron transmission (transmission electron microscopy, TEM), scanning electron microscopy, (SEM), laser scanning confocal, epifluorescence microscopy. Indirect methods of detection of biofilm where it may be done based on detaching the microorganism from the surface before counting them.

For detection of biofilm formation several instruments and devices have been developed for clinical microbiological investigation. Some of the instruments are modified Robins device, Calgary biofilm device, flow well disc reactor, profusion biofilm fermenter, model blade etc. The substratums of the tools cited above are mainly made up of sialic (silicon), plastic, teflon stuff and cellulose derivatives. Biofilm in urinary catheter can be detected directly by Scanning electron microscopy or transmission electron microscopy (SEM/TEM). The rate of biofilm formation on model system i.e. in different tools may be altered with the composition of medium used such as amount of glucose, iron, antimicrobial agents, cation of Ca⁺⁺, Mg⁺⁺ present [54].

Several methods of studies have been used to detect and determination of biofilms. The methods are tube method, Congo red agar method, microtitre plate assay, plate counting of biofilm covered bacteria (Sessile bacteria), PCR study, mass spectrometry etc. Some of the methods used for detection of biofilm are as follow.

10.1 Microscopic observation

Both light and electron microscopic studies can be made for direct observation of biofilm. The confocal laser scanning microscopy (CLSM), scanning (SEM) and transmission electron microscopy (TEM) are done for observation of microorganism adhere on surface, fluorescent dye can be used for clarity of organism and biofilm materials and their thickness. Indirect observation of biofilm of bacterial origin can be observed by various methods, they are roll plate method, Congo red agar method, tube method, microscopic assay etc.

10.2 Roll plate method

In Roll plate method, where the development of biofilm on the surface of cylindrical device and tools such as urinary catheter and vascular graft. It is not considered the growth of microorganisms inside the tubular device. The Congo red agar method is a qualitative test for detection of biofilm producing bacteria, the colony color is changed in the medium. Blackish crystalline colonies are produced by the biofilm forming sessile organism while the planktonic bacterial cells produced red in the medium [55].

The tube method of qualitative assay of detection of biofilm formation. In this assay a visible film is developed around the glass tube of culture of bacteria with tryptic soy broth. The sessile bacteria form biofilm on the wall of the polystyrene test tube which may be stained with Safranine for 1 h dye exposure. The plankotonic cells are discharged by waiting twice with Phosphate-buffered saline (PBS). The sessile bacterial test tube showing visible stained at the bottom while the Planktonic cells contain bacterial culture tube become clear after washing with PBS.

10.3 Biofilm assay by microtitre assay

Microtitre plate assay is quantitative test to determine biofilm production by microplate reader. Bacterial broth suspension is prepared in Muller Hintone broth (MHB) with 1% glucose solution. An amount of 20 μL of bacterial isolate is in 180 μL MHB. Microplate with 96 well polystyrene stuff is incubated at 37°C for 24 h. The sessile bacterial form biofilm on the wall of the wells those can be stained with Safranine for 15 min. The planktonic cells well are rinsed with PBS (pH 7.2) and air dried at 60°C for an hour. Biofilm of well can be fixed with 150 μL methanol for 20 min. Air dry of micropipette is resolubilized by 150 μL of 95% ethanol, or 33% of glacial acetic acid. The study is repeated in triplicates. Microplates are measured photometrically at 570 nm filter in spectrophotometer by microreader. Uninoculated well with MHB medium is considered negative control as blank [56]. The cut off value (ODc) can be categorized of the isolates by biofilm producer or not.

$$\mathrm{O}\mathrm{D}\mathrm{c}=\mathrm{O}\mathrm{D}\mathrm{of}\mathrm{negative}+\left(3\times \mathrm{S}\mathrm{D}\mathrm{of}\mathrm{negative}\mathrm{control}\right)$$

$${\mathrm{O}\mathrm{D}}_{\mathrm{isolate}}=\mathrm{average}\mathrm{O}\mathrm{D}\mathrm{of}\mathrm{isolate}-\mathrm{O}\mathrm{D}\mathrm{c}$$

Interpretation of Result:

1. OD ≤ ODc no production of biofilm.

2. ODc < OD ≤ 2× ODC production of weak biofilm.

3. 2× ODc < OD ≤ 4OD is moderate production of biofilm.

4. 4× OD < OD is indication of strong production of biofilm.

10.4 PCR based biofilm detection

Amplification of target gene helps in species diagnosis for microbiological studies; similarly genes responsible for biofilm formation can be identified using gene specific primers. Biofilm related genes are amplified by PCR machine as qualitative real time PCR. Several species specific gene of different microbial species have different gene segment that express the biofilm formation. Several genes in different bacterial species have been discussed in the text.

10.5 Mass spectrometry method

The extracellular polymeric substances (EPS) composed of polysaccharides and proteins (extracellular enzymes) are produced in biofilm The proteins in biofilm matrix can be detected and characterized by mass spectrometry (MS), complex biological structures like EPS can be characterized by MS. The matrix assisted laser desorption ionization (MALDI) and Electrospray ionization (ESI) is similar to that of massspectrometry. The time of flight mass spectometer (TOF) with which mass is analyzed by ion desorped in cacuum chamber. If these two techniques (MALDI and TOF) are combined called MALDI-TOF) can help in the analysis of biofilm mass. In recent years matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potential tool for microbial identification and diagnosis [57]. Here, the matrix mass is ionized and vapourized by laser beam, depending on mass/charge ratio of the samples molecules are measured by TOF. Bacteria are identified by expressing of proteins like surface proteins, Co-enzymes (β-lactamase) response to antimicrobial can be monitored.

10.6 Biological assay of biofilm

Biofilm colony may produce numbers of bacterial species and wide range of biological products. Estimation of biofilm embedded products and characterization of the products. The planktonic and sessile bacterial producers are very similar. Standardization of curves of each microorganism tested needs to be formed. Estimation of total protein at 550 nm or 950 nm ansorbance. Estimation of tryptophan fluorescence, urease, formazan and endotoxins are also assayed.

11. Treatment of biofilm intrigue

The biofilm causes several inefficacy of equipments and infrastructures as well as spread of infection and resistant to antimicrobial therapy. The biofilm cause equipment inefficient and corrosion that reduces the efficacy any equipment. Biofilm in industry causes better insulator which is scale type, this insulator increases energy cost. It also changes the water passing capacity in the water supply in poultry industry. Biofilm hamper the water distribution system with disinfectant residual, increase bacterial level, reduction of O₂ level in water, reduce water taste and produce bad odor. Red and black water problem due to iron and sulphate reducing bacteria.

Chemical and environmental modification is the main tools to prevent biofilm formation. Several antibiotics, biocides, and ion coating are commonly used against biofilm in veterinary and human medicines. Biofilm prevention is two types; prevention of growth and prevention of surface attachment. Microbial growth can be preventing giving antimicrobial coating in indwelling, medical device etc. Several antibiotic, biocides and ion coating are used. All these coating remains effective for few days to week, later they disperse. Silver ions have antibacterial property for water purification in reverse osmosis process. Other way of purification of water is electric deionization, exposure of UV light and application of ozone.

11.1 Therapeutic intervention for poultry production

Several microorganisms affect poultry production both egg and meat through infection and diseases production. Besides getting infection, other risk factors for biofilm production in poultry farm and meat industry are scarcity of quality water, negligence of biosecurity standard, co-existence of other animals in vicinity of poultry premises, inadequate infrastructures and their condition. A scarcity of water is lethal for growth of biofilm, interrupts water supply through drinking fountain and drips are sufficient source of biofilm bacteria. Control of water distribution system reduces the microbial load and infection. Several chemicals such as chlorine, chlorine dioxide, organic acid, hydrogen peroxide may be used but they are used in some occasion. Intermittent used of such antibacterials and unhygienic used of water supply invites biofilm formation.

11.2 Water purification

The equipment and water supply system will be such that the coating of the equipment and water supply pipes will be free from corners, cracks, valve, joint and pores. A mechanical sensor system have been developed to monitor biofilm formation in the system where production of gas due bacterial fermentation will be alarmed by the device. Once biofilm is established it may be dismantled through cleansing by physical and chemical means and disinfection of tools and fomites are to be done regularly. Water can be purified applying Ozone exposure (1.0–2.0 mg/L). It disintegrate bacterial cell into fragments. Chlorine and chloramine are highly effective method of water disinfection, but in the pipes it produces small amounts of chemicals dirt if the water contains much impurities and the taste of water is also changed. The amount of chlorine used is 4 milligrams per liter (mg/L i.e. 4 ppm). Different chlorines used are chlorine gas, sodium hypochlorite, and calcium hypochlorite. The biofilm polymeric surface charged can be modified by electrostatic charged particle that will repeal other particle of same charge. The electrostatic charge and biofilm polymeric charge are negative so, they dispel each other.

11.3 Food industries

In food industries, most disinfectants used are quaternary ammonium compounds (amphoteric compounds, hyperchlorides, peroxides (H₂O₂, peracetic acid), aldehydes (formaldehyde, glutaraldehide), phenolics, alkyl amines, chlorine dioxide etc. [58].

11.4 Veterinary medical therapy and biofilm

Antimicrobials can be on the medical devices surface using long flexible polymeric chain. The chain forms a covalent bonds with device surface killing microbial organisms. Several such antibacterial materials used are N-alkylpyridimidinium bromide can act against E. coli, Streptococcus epidermidis, Pseudomonas aurugenosa. The dispersion force dispel the organism on the surface of the device to prevent adhesion and biofilm formation. For effective result with biofilm infected patients, combination of antibiotics and antibiofilm can be used in poultry and veterinary therapy. Usually, quorum sensing mechanism binds the whole biofilm population of the society through a complex cascade of events which unit the biofilm population. So antibiotic and use of ultrasound device that enhance the antibiotic activities. The ultrasound helps to pass energy weave through the cell of biofilm particularly in tropical infection. Several antibiotics along with application of different antibiofilm agent and their use are presented (Table 3).

Therapy	Action	Usefulness	Response against bacteria
Ultrasound	Destroy bacterial cell penetrating biofilm	Helps to penetrate antibiotics in deep seated bacteria	Staphylococcus aureas
Phage therapy	Destroy bacteria penetrating biofilm	Generates EPS degradating enzymes that destroy bacteria	Pseudomonas auguginosa; Streptococcus sp, Listeria sp, Proteas sp
Drug delivery system	Nano carrier drug delivery system prolongs the drug stability	Different nanoparticles like silverAg, Zn,Ti, Au with antibiotics	Fusebacterium nucleatum
Antibacterial peptides	Some peptides have antibacterial properties	Peptides can be used to check bacterial infection	Staphylococcus sp, Pseudomonas auruginosa
Antiadhesin agents	Inhibits formation of biofilm preventing surface attachment by pilli, flagella etc	Prevent adhesion on surface by bacteria	E. coli, Salmonella sp; Proteus spp etc
Antimatrix agents	Disintegration of biofilm by enzymes	Digest the matrix of biofilm and remove it	E. coli, Pseudomonas sp Klensiella pneumonae, Staphylococcus epidermides, Enterococcus fecalis
Chelating agent	Chelates out of the matrix metallic ions	Helps in disintegration of biofilm matrics	Staphylococcus aureus, Staphylococcus epidemidis; Pseudomonas aeruginosa

Table 3.

Therapeutic intervention against biofilm in contaminants.

Use of ultrasound can destruct the bacterial cell penetrating the biofilm and the antibiotic can pass through the biofilm to reach the bacterial cell and act upon it

Low electric current-Passing of low level of electric with antibiotic can provide effective response in biofilm Society that may be situated in tissues. Electromagnetic pulse may increase the antimicrobial response of cationic antibiotic against biofilm. Gentamicin with mild electric current cans synergistic effect against Staphylococcus aureas.

11.5 Phage therapy

The phage virus may act on biofilm bacteria penetrating the biofilm through diffusion and even propagation of phage into biofilm environment. The function of phage virus also depends on nature of biofilm matrix, species of bacteria etc. Usually phage generates EPS degradating enzymes (depolymerases) that may digest the matrix. Another function of phage is that in biofilm the bacteria remains under several stress condition, this stress can enhance the phage to disintegrate the biofilm Community, particularly in Pseudomonas aurugenosa and Fusebacterium nucleatum, Streptococcus sp., Proteus mirabilis., Listeria sp., E. coli etc. The phage can be used are pyobacteriophage, phage PB-1, T4 etc. The antibiotic and phage combination acts suitably in complicated cases of infection [59].

11.6 Chelating agents

Several metalic ions such as Ca⁺⁺, Mg⁺⁺ and Fe⁺⁺ are abundant in the biofilm matrix for their integrity. Chelating agents such as Sodium citrate, trisodium citrate, Na-EDTA can be used to chelate out the cations from the biofilms matrix and this helps in disintegration of biofilm society.

11.7 Drug delivery system

Encapsulated nano carrier drug delivery system that prolongs the activity of active molecules against a Fusebacterium nucleatum bacteria. Such combination of antibiotic such as gentamicin, ciprofloxacin, ampicillin, along with nano carriers of phosphotydyl-choline, polyethylene glycerol, polyamidoamine are used. Silver nanoparticle has also antibacterial property. This is due to positive charge of Ag‑ and –ve charge of biofilm attract and a strong bacteriocidal action of nano silver (Ag) provides antibacricidal function. Other nanoparticles used are zinc (Zn), titanium (Ti), gold (au) nano particle.

11.8 Antimicrobial peptides

Antimicrobial peptides (AMPs) are small peptides that widely exist in nature and they are an important part of the innate immune system of different organisms. The AMPs have a various inhibitory effects against microorganisms. The emergence of antibiotic-resistant concern and the increasing of concerns about the use of antibiotics resulted in the development of AMPs, which have a good application prospect in veterinary medicine, food Science, agriculture, aquaculture and human medicine. It could be novel types of antibacterial in the regime of antibiotic resistance. Antibacterial peptides must be assayed before use about their spectrum and mechanism. Several peptides such as SMAP-29 (Sheep myoloid antimicrobial peptide), BAMP-28(bovine antimicrobial peptide), BAMP-27 have property to reduce significant biofilm reduction property against multidrug resistant Pseudomonas aurugenosa. These peptides kill the microorganism in the beginning of biofilm formation [60]. High efficacy of α-helical cecropin/melitin hybrid peptide CEME reported against Staphylococcus aureas. Due to increasing concern of AMR with different antibiotics, the use of antibacterial peptides in poultry have been tried and found that 2 truncated cathelicidins and 4 avian β-defensins are potent peptides against bacterial infection and immunomodulatory effect [61].

11.9 Antiadhesin agents

Several antiadhesion agents could be used against biofilm in-vivo and in-vitro. Use of mannocides, pilicides and culicides. Mannocides are small molecules of drug that contains mannose sugar group. The bacterial fimbri bound to mannose. Mannocide fits the FimH mannose binding pockets and completely inhibits FimH site to the host receptor. Similarly pillicides are those chemical that inhibits the formation of the pilli of bacteria. Pillicides are designed such that interfare the process of pilli formation through inhibition of export of pillin subunits. The curli is a protenaciuos fiber that produced by certain bacteria like E. coli, Salmonella spp. It helps in the formation biofilm. Curlicides are those chemicals which inhibits formation of curli. All these three forms of antiadhesins agents are used in upper urinary tract infection with E.coli, Proteas sp. etc.

11.10 Antimatrix agents

Bacterial matrix aggregation in the biofilm colony with extracellular matrix is a hardle for therapy and elimination of bacterial propagation. Several natural and engineered enzymes and used of bacteriophage that can disintegrate the biofilm society and matrix. The N-acetyle-D- glucosamine-1 phosphate acetyle transferase (GlmU) can be used against E.coli, Pseudomonas aurogenosa, Klensiella pneumonae, Staphylococcus epidermides, Enterococcus fecalis. Other enzymes have potential use are Dnase, dispersinB etc.

11.11 Chelating agents

Several metalic ions such as Ca⁺⁺, Mg⁺⁺ and Fe⁺⁺ are abundant in the biofilm matrix for their integrity. Chelating agents such as Sodium citrate, trisodium citrate, Na-EDTA can be used to chelate out the cations from the biofilms matrix and this helps in disintegration of biofilm society.

12. Conclusion

Biofilm formation is a real problem in the therapeutic and poultry management. In poultry a large numbers of bacteria that form biofilm have several direct and indirect effects on disease transmission and resistance to antibiotic therapy. Several infectious diseases whose course remains longer might be due to biofilm formation. Besides therapeutic difficulties poultry industries and water supply system also hampered. To avoid biofilm formation and treatment with different areas of biofilm have been discussed. Regular investigation for biofilm formation and therapeutic interventions as deem fit should be taken regularly.

Acknowledgments

The author is very thankful to the director, Indian Veterinary Research Institute for providing facilities to write this chapter.