Bacterial Biofilms on Livestock, Environment and Pharmaceutical Applications

#### **Chapter 16**

## Effect of Biofilm on Production of Poultry

*Dayamoy Mondal*

#### **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.

**Keywords:** biofilm, poultry, diagnosis, therapy, biofilmgene

#### **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* [1–3].

*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

#### *Effect of Biofilm on Production of Poultry DOI: http://dx.doi.org/10.5772/intechopen.102951*

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. Subinhibitory 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 [26–29]. 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].

#### **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,* 

*Effect of Biofilm on Production of Poultry DOI: http://dx.doi.org/10.5772/intechopen.102951*


#### **Table 1.**

*Water standards and option for correction for poultry.*

*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 *omp*A, *bap*, *bla*PER-1, *csu*E, *csg*A, and *fim*H*. 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.*

*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

#### **Table 2.**

*Bacteria cause poultry diseases and have biofilm forming capacity.*

#### *Effect of Biofilm on Production of Poultry DOI: http://dx.doi.org/10.5772/intechopen.102951*

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 *lux*S and *fla*A [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 *hly*A 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.

ODc ODof negative 3 SDof negative control = + ( × )

OD averageOD of isolate ODc isolate = −

#### **Interpretation of Result**:

OD ≤ ODc no production of biofilm. ODc < OD ≤ 2× ODC production of weak biofilm. 2× ODc < OD ≤ 4OD is moderate production of biofilm. 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**).

#### *Effect of Biofilm on Production of Poultry DOI: http://dx.doi.org/10.5772/intechopen.102951*

**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*, *Streptococcu*s 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].


#### **Table 3.**

*Therapeutic intervention against biofilm in contaminants.*

#### **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 phosphotydylcholine, 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.

#### **Acknowledgements**

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

#### **Author details**

Dayamoy Mondal Eastern Regional Station, Indian Veterinary Research Institute, Kolkata, India

\*Address all correspondence to: dayamoy21@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 17**

## Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous Cavity Leads to Recurrent Uveitis in Horses

*Bettina Wollanke and Hartmut Gerhards*

#### **Abstract**

Equine recurrent uveitis (ERU) is a disease known and feared for centuries, as it almost always leads to blindness even with careful and meticulous conservative treatment of the individual episodes of uveitis. In about one-third of horses, both eyes are affected, often necessitating euthanasia. A link between ERU and leptospiral infection has been suspected for nearly 80 years. Vitreous lavage (vitrectomy) can preserve vision in affected eyes. After surgery, no further episodes of uveitis occur in up to more than 95% of operated eyes. With routine performance of vitrectomies, numerous vitreous samples could be used for further investigations. Intraocular anti-Leptospira antibody production was proven, leptospires could be cultured from the vitreous samples, and the LipL32 gene could be detected in the vitreous samples by PCR. Thus, there was convincing evidence of a chronic intraocular leptospiral infection, which can be eliminated most reliably by vitrectomy. Recently, it has been shown that the intraocular leptospires produce biofilm in the equine vitreous. Biofilm formation explains not only the success of vitrectomy, but also the survival of leptospires in the vitreous cavity for many years despite the presence of high intraocular antibody titers and immunocompetent cells, as well as the high tolerance to antibiotics.

**Keywords:** equine recurrent uveitis (ERU), pathogenic *Leptospira* spp., biofilm formation, vitreous cavity, intraocular specimens, intraocular antibody production, leptospiral culture, real-time PCR targeting LipL32

#### **1. Introduction**

Equine recurrent uveitis (ERU) occurs in mules and horses and is a disease that has been known for a long time. From about the beginning of time-counting, ancient writings have described symptoms that are consistent with today's definition of ERU. Since the end of the nineteenth century and the beginning of the twentieth century, more and more detailed descriptions of this disease have been published [1]. In earlier times, the working power of the horse was quite crucial for the survival of men [2]. Not only during war, but also for the cultivation of the fields, for the transport of people and freight as well as for serving as living motors in preindustrial times, people were dependent on horses and mules.

Without horses, the development of mankind would not have been possible to the extent that has been achieved in the past centuries. All the more the health maintenance of the horses was of paramount importance [3]. The recurrent and painful episodes of uveitis led to reduced performance and not infrequently to blindness and thus often to unserviceability of the affected horses. For this reason, equine recurrent uveitis has preoccupied many generations of owners and veterinarians [3, 4]. There are the most diverse historical treatment approaches and theories about the causes of this disease [3, 5].

Among many causes that had not been confirmed, wet pastures and flooding as well as heritability were discussed [5–7]. An infectious etiology has been suspected for over 100 years, although *Leptospira* spp. were not known at that time [3]. Since the first description of Weil's disease in humans, "eye complications" were known to be associated with this disease [8]. A first description of leptospires was given in 1915 [9]. At that time, the identification of *Leptospira* spp. was made in Japan and Germany at approximately the same time [10].

After a link between leptospiral infections and uveitis had been established in human medicine, the Swiss ophthalmologist Gsell and coworkers studied aqueous humor from equine ERU eyes and described for the first time a link between ERU (then called "moon blindness" or "periodic ophthalmia") and leptospiral infection [11]. Since then, there have been numerous investigations addressing the leptospiral etiology of ERU.

Because antibody detection in intraocular fluids was relatively common [11–17], but uveitis bouts typically do not become apparent until months or even years after the acute systemic infection [18–22], it was assumed that the infection was a trigger of ERU, but the bacteria were no longer present when the uveitis attacks started [18, 23]. In addition, a culture of *Leptospira* spp. from equine intraocular samples failed many times [12, 16, 22, 24–28]. For this reason, ERU has also been considered by some authors to be an "autoimmune" disease [29–31].

Different causes of uveitis can occur in horses just like in other species [30, 32, 33]. However, in equine uveitis associated with painful recurrent episodes causing the typical ocular changes, chronic intraocular leptospiral infection has been found to be the cause [34, 35]. Therefore, the term "ERU" will be used hereafter to refer to leptospiralinduced recurrent uveitis.

It was not until the routine use of vitrectomy (irrigation of the vitreous chamber) in horses [36–38] and the resulting ability to obtain intraocular specimens from eyes affected with ERU [39], that the importance of leptospiral etiology in ERU was confirmed [34, 35, 40–44].

Only recently it was recognized that recurrent uveitis in horses is a biofilmmediated disease [45]. The ERU has many aspects that had raised questions and been incomprehensible before the discovery of biofilm formation of pathogenic *Leptospira* spp. in the vitreous chamber. However, knowing the characteristics of chronic and biofilm-associated infections, the pathogenesis of ERU can now be better understood [33].

*Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

#### **2. Incidence and clinical course of ERU**

Leptospiral-induced uveitis is not only in horses a late consequence of systemic infection [18, 34, 46], but also human leptospiral uveitis often occurs a long time after the acute infection [11, 46–51]. A causal relationship between uveitis and a previous leptospiral infection is often difficult to recognize when uveitis occurs, because systemic leptospirosis is predominantly inapparent in horses [19, 52] and can also be inapparent in humans [53].

ERU affects quite a lot of horses. In the United States, where there are many leopard coat pattern horses (Appaloosas), it has been reported that up to 25% of horses are affected and lose vision in one or both eyes during the course of the disease [30]. However, in that study, leopard coat pattern uveitis (Section 4.), which accounts for a large proportion of affected horses in the United States, was also classified as ERU. In other studies, the percentage of horses affected with ERU ranges from 7 to 10% [54–56], with up to one-third of the horses suffering from the disease on both sides [3, 34, 57]. The attacks of uveitis in both eyes often do not start at the same time, but with a time delay of several months up to about 2 years [34].

The first episodes of uveitis are usually noticed in younger horses between 4 and 6 years of age [34]. More rarely, however, horses can still develop ERU up to over 20 years of age. Foals up to 6 months of age typically do not develop ERU. When uveitis occurs in foals younger than 6 months, it is typically septicemia-associated and bilateral, e.g., in the course of rhodococcosis [58–61].

In ERU, recurrent episodes of uveitis occur in unpredictable intervals and oftentimes not, as the former term "periodic ophthalmia" suggests, periodically. The interval between episodes of uveitis can be less than 2 weeks and up to more than a year. In most cases, ERU episodes are associated with blepharospasm, epiphora, and photophobia, so the owner notices the eye disease and seeks veterinary advice. The severity of uveitis also varies greatly from horse to horse. Sometimes very mild episodes occur, which subside after 1–2 days. Other ERU attacks are so severe that after one or two attacks, the eye may already show significant and irreversible changes and in the worst case may even lose vision. In most horses, the clinically quiescent intervals between episodes of uveitis become shorter over time, and at the same time the uveitis bouts become more severe.

#### **3. Clinical signs of ERU**

Descriptions of the ophthalmologic findings in ERU have been given repeatedly and in broad agreement [3, 30, 32, 34, 62–64]. Acute attacks are usually painful or even very painful. Affected horses are depressed, show decreased appetite, can have a moderate rise in body temperature, severe blepharospasm, serous and later sero-mucous lacrimation, and more or less swollen eyelids. These symptoms, although typical, are not pathognomonic and can also occur with other eye lesions.

Ocular examination in horses is the easiest and most informative when a simple handheld (direct) ophthalmoscope with bright light source is used. The handheld ophthalmoscope can be used as a focal light source, magnifying glass, and slit lamp, and is most crucial for examining the posterior segment of the eye (posterior lens surface, vitreous cavity, and fundus). Since serum in horses is yellowish in color, aqueous humor and vitreous humor in acute uveitis ("leakage") are also jaundiced.

Ophthalmic examination typically reveals the following findings during an acute ERU episode:


In the inflammation-free interval, after mild ERU episodes and meticulous conservative treatment, sometimes no definite changes can be detected in early stages of the disease. However, when multiple ERU attacks have occurred, pathologic changes become increasingly apparent that are also evident during the clinically quiescent phase of the disease:


In 3% of ERU cases, the inflammation occurs primarily in the posterior segment of the eye [65]. Hardly any pain is evident in these horses, and this form of ERU is

*Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

sometimes detected only as an incidental finding during routine examinations or purchase examinations of horses. Only rarely do very observant owners notice a change in the fundus reflex of the diseased eye and call a veterinarian. In most cases, however, iritis occurs in the course of the disease, which then leads to the typical and easily recognizable pain symptoms. Depending on the changes that have already occurred in the posterior segment of the eye, the prognosis for preservation of vision is often guarded at this point. Sometimes these horses are not presented to the veterinarian until "sudden" blindness due to cataract formation or retinal detachment has occurred.

#### **4. Differential diagnosis**

A significant and strinkly common type of uveitis not caused by leptospires occurs in leopard coat pattern horses [65, 66]. This type of uveitis is strikingly common in leopard coat pattern horses. In contrast to ERU, leopard coat pattern uveitis progresses insidiously and does not present as recurrent painful episodes of uveitis. In the literature, it is therefore often referred to as "insidious uveitis," but not distinguished from ERU. Other forms of uveitis may be phacogenic, traumatic, tumor-associated, septicemia-associated, or triggered by other infectious causes such as parasites (*Micronema* (syn. *Halicephalobus*) *deletrix* or *Sertaria* spp.) or, e.g., staphylococci [33]. In addition, a chronic iritis similar to Fuchs' heterochromia iritis in humans occurs in horses [33, 67]. In most cases, all these forms of uveitis can be relatively clearly differentiated from ERU based on the clinical picture and/or the course of the disease (**Table 1**) [33].

Sometimes recurrent keratitis is misinterpreted as ERU, as some types of keratitis can also cause painful with miosis and responds to the same conservative therapy as ERU. However, in keratitis cases, medical dilation of the miotic pupil results usually more rapidly and completely than in ERU. In recurrent keratitis, however, the changes that almost always are evident in ERU after several episodes of uveitis, even in the inflammation-free interval, are absent.

If an ocular disease is clinically not clearly assignable to an etiology (e.g., "recurrent keratitis" or "uveitis of unknown cause"), it is possible to take aqueous humor during the inflammation-free interval [33]. In horses, approximately 1 ml of aqueous humor can be safely collected and then used for laboratory tests [33, 68–70]. To investigate for the presence of ERU, testing for both anti-*Leptospira* antibodies and by PCR for, e.g., LipL32 is advisable [35, 71–73]. For scientific questions, a leptospiral culture can additionally be performed [34, 35]. Depending on the laboratory findings, a decision can then be made on the further course of action. In case of positive leptospiral findings, vitrectomy is indicated. With negative leptospiral laboratory findings, a leptospiral infection of the vitreous cavity can be excluded with a high probability. These horses would not benefit from vitrectomy—except to remove vitreous opacities that impair vision. In this case, however, a preoperative aqueous humor examination would be superfluous—just as in the case of unequivocal findings in terms of ERU.

For the detection of intraocular anti-*Leptospira* antibodies, the microscopic agglutination test (MAT) is used in most cases. The MAT is highly sensitive and specific when examining aqueous humor or vitreous samples [34, 35, 40]. In addition, other antibody tests can be used, which are either commercially available or available as in-house ELISA tests. Specific anti-*Leptospira* immunoglobulin class A (IgA) antibodies are particularly reliable for detecting intraocular leptospiral infection [72, 74]. Another well-suited test is the SNAP Lepto, which detects anti-LipL32



*Different types of uveitis in horses, symptoms, therapy, and prognosis.*

*Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

antibodies and is neither immunoglobulin-specific nor serovar-specific. It can be used for samples from different species. With its easy handling and the result visible within 10 minutes, this is a very useful test with a sensitivity and specificity comparable to MAT for intraocular specimens [73, 75]. In contrast to MAT, which is too unspecific for serum testing, SNAP Lepto is well qualified as a screening method even when serum is tested [71].

Antibody detections are equally reliable in vitreous and aqueous humor samples [70, 76, 77]. Both PCR and leptospiral culture are somewhat more reliable when testing vitreous humor samples compared with testing aqueous humor samples [34, 35, 78]. However, the collection of a vitreous sample is disproportionately risky and should be rejected for a preoperative diagnosis, because the aqueous humor analysis is overall very informative [33]. In rare cases, e.g., no anti-*Leptospira* antibodies are detectable in the aqueous humor, but at the same time the PCR yields a positive result. In routine diagnostics, culture has been largely replaced by the much faster and less expensive PCR.

If time is not an issue, but economic reasons have to be taken into account, a reasonable approach for the examination of aqueous humor samples is to first perform an on-site rapid test for the detection of anti-*Leptospira* antibodies. If this test is negative, the MAT can be commissioned externally if necessary. If the MAT is also negative, further antibody tests (e.g., specific in-house ELISA tests) and a PCR can be performed. The more laboratory tests are performed, the fewer "false negatives" can be expected, but the higher the costs for laboratory diagnostics will be.

#### **5. Interpretation of intraocular antibodies**

In eyes with a history of recurrent inflammations, but without clear evidence of ERU, and thus without aqueous or vitreous humor opacities, protein levels are typically not elevated. If protein levels in intraocular fluids are not elevated, leakage from the blood can be excluded. In these cases, even very low MAT titers are indicative of intraocular antibody production. The authors consider a MAT result of 1:50 as sufficient indication for vitrectomy in these cases. In eyes with obvious aqueous humor and vitreous opacities, however, the diagnosis of ERU is usually unambiguous even without aqueous humor examination. In cases of doubt, the Goldmann-Witmer coefficient can be used to differentiate leakage from intraocular antibody production [79]. It is crucial that not only the intraocular and the serum titer are evaluated, as it often could be read lately [80–86], but that—as described by Goldmann and Witmer—a reference value is determined both in the aqueous humor and in the serum. Any other antibody titer (e.g., tetanus) can be used as a reference value, provided that antibodies are present in the serum. Alternatively, the total IgG content or, if necessary, even the total protein content can be used as a reference value [33, 34].

#### **6. Therapy of acute uveitis**

Acute ERU is treated in the same way as any other equine uveitis [30, 32, 57, 58]. First of all, it is important to achieve mydriasis to avoid posterior synechiae and resulting cataract formation. Atropine is the drug of choice for this purpose and can be used as of 1–2% eye drops or eye ointment. Since the ophthalmic ointment adheres slightly better and acts more protracted, ointment is preferable, if available.

#### *Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

Atropine should initially be given several times daily or even hourly until the pupil dilates. Thereafter, the intervals can be adjusted to the pupil width and often considerably prolonged. Systemic side effects associated with the topical use of 1–2% atropine in horses do not play a significant role in the authors' experience and after having treated thousands of horses over a 30-year period. Colic, e.g., due to an impaction or a meteorism, can occur in any hospitalized horse, not just ophthalmic patients. By feeding mash and monitoring the fecal consistency, an impaction can be detected early and countermeasures (e.g., administration of laxatives) can be taken to avoid more serious colic.

Apart from mydriasis, anti-inflammatory treatment is important. Topical application of ophthalmic ointments containing dexamethasone is particularly effective, provided the corneal epithelium is intact. If corneal defects are present, topical corticosteroids must not be given.

In addition, the administration of a nonsteroidal anti-inflammatory drug (NSAID) orally is indicated. Only in exceptional situations and in case of very significant diffuse vitreous opacification, systemic administration of prednisolone (1 mg/kg per os) for several days may be considered additionally. In these particularly severe cases with significant diffuse vitreous opacification, adjunctive therapy with a systemically given antibiotic, e.g., enrofloxacin [87], can also be performed, to eliminate at least part of the intraocular bacteria—even if this does not completely eliminate the infection [88].

Other measures accompanying the therapy are keeping the horse in a dark place and resting in the stall or just light exercise until the acute inflammation has subsided. If it is not possible to keep the horse in the dark, wearing a light absorbing mask can be considered.

#### **6.1 Brief historical overview of the development of conservative treatment of uveitis in horses valid today (without treatment proposals that did not prove successful or were even questionable from an animal welfare point of view)**


#### **7. Vitrectomy during the quiet intervals**

The most effective treatment for ERU is vitrectomy (removal of diseased vitreous and irrigation of the vitreous cavity). This surgery is performed exclusively in intervals without acute inflammation. Mechanical removal of the vitreous opacities caused by inflammation and accessible vitreous parts very reliably and permanently eliminates the leptospires in the biofilm. Postoperatively, up to 98% of eyes remain free of recurrences when surgery is performed properly [98]. If, exceptionally, further episodes of inflammation occur after surgery, a second vitrectomy can, if necessary, permanently eliminate the infection and prevent further episodes.

Vitrectomy as a vision-preserving procedure is a demanding surgery, having a relatively long learning curve. Prerequisites for successful performing vitrectomies are solid training, availability of for equine ophthalmo-surgery optimized, custommade instrumentation and equipment as well as careful and intensive perioperative examination and conservative treatment. Any complication may have devastating consequences and can lead to blindness or even enucleation. Only rarely eyes that are already blind undergo surgery in order to prevent both future painful uveitis attacks and removal of the globe, which is cosmetically unsightly.

In order to perform vitrectomy with minimal complications, an experienced team (surgeon, sterile and nonsterile assistant, skilled anesthesiologist) is required, as well as expensive equipment and instruments specially adapted to the dimensions of the horse's eye. For this reason, only a few specialized equine clinics perform vitrectomies to date. In clinics in which vitrectomy is performed as a routine procedure, it is a quick (total anesthesia time is about 40 minutes, the surgical instrument is in the eye <10 minutes) and relatively safe procedure with a very good prognosis [38, 39, 89, 99].

#### **8. Other treatment options for ERU**

Apart from vitrectomy, other treatment options have been described, of which two in particular are favored in recent publications. One consists of an intravitreal gentamicin injection. However, the recommended dosage for this purpose (4–6 mg) [80, 100, 101] is 3–4 times the drug concentration that was found to be "safe" with regard to retinal toxicity in experimental studies [102]. So far, there are no long-term results after these injections and the number of horses treated in this way is still limited. Surprisingly, gentamicin injection is not recommended exclusively for equine eyes with intraocular leptospiral infection; other forms of uveitis are also treated with this injection. Improvement after the intravitreal injection is also thought to result from the antibiotic gentamicin having immunomodulatory effects [103].

The second therapeutic option described since the turn of the millennium is the deep intra- or subscleral implantation of a cyclosporine device [104–107]. These implants lead to less frequent and milder episodes of uveitis over a period of up to about 2 years. However, the uveitis does not stop completely, and if the effect wears off, a new implant may have to be inserted. Like gentamicin injection, implantation of cyclosporine devices is performed independently of leptospiral infection in the vitreous cavity. Only individual authors differentiate and use the implants exclusively when no leptospiral infection is detectable [86]. Attention should also be paid to the drug law in its current version, which currently prohibits the import of cyclosporine devices, at least in the EU [108].

*Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

However, neither gentamicin injection nor implantation of cyclosporine devices can remove the dense vitreous floaters that often lead to impaired vision. Over time, these deposits also often adhere to the posterior capsule of the lens and, just like extensive posterior synechiae, can lead to a cataract formation.

#### **9. Results of the examination of intraocular specimens**

In the literature, before the introduction of vitrectomy to the therapeutic measures against ERU, there were only very sporadic reports of cultural detection of leptospires in intraocular specimens from eyes affected with ERU [109, 110]. Numerous investigators failed to obtain cultural evidence of leptospires, casting doubt on chronic intraocular leptospiral infection. It was rather assumed that although leptospires somehow trigger ERU, the inflammations are not subsequently maintained by the presence of the pathogen [22, 24, 63, 68, 111, 112].

Vitrectomy was initially performed to remove vitreous opacities. The aim was to improve vision in the eyes affected by ERU [36, 37]. However, it soon became apparent that vitrectomy was surprisingly effective in preventing further episodes of uveitis. Therefore, more and more horses were sent to the clinic for vitrectomy.

It was only with the routine performance of vitrectomy that it had become possible to examine numerous vitreous samples from horses suffering from ERU. The peculiarity was that the samples were predominantly from eyes still able to see at an early stage of the disease. By collecting the first milliliters aspirated from the vitreous cavity before opening the intraocular infusion line, it was possible to use undiluted vitreous material for investigations. The results of these examinations, in turn, provided insights into which ocular findings were associated with leptospiral infection and which were not. It was also shown that the prognosis in terms of postoperative absence of recurrences was best when eyes with an intraocular leptospiral infection were treated by vitrectomy [98]. In this way, on the other hand, the indication for vitrectomy was optimized.

With careful assessment of the indication for vitrectomy and examination of undiluted vitreous specimens, MAT titers of 1:100 or higher were detected in 382 of 426 vitreous samples (90%) examined [34, 35]. In some MAT-negative specimens, specific anti-*Leptospira* antibodies (especially immunoglobulin class A) could be detected by an in-house ELISA [74]. Leptospires were culturally detected in 189 of the undiluted vitreous samples from 358 eyes (53%) affected with ERU [34, 35]. The positive cultures had been obtained only after optimization of the sampling technique and immediate sterile inoculation into a transport medium for mailing to a laboratory. The sensitivity of PCR is in between culture and antibody detection. In 70–77% of vitreous samples from eyes affected by ERU, the PCR result was positive [35, 73, 75, 113].

In Germany and neighboring countries, infections with leptospires of the serogroup Grippotyphosa are dominating, accounting for about 80% of intraocular infections in horses suffering from ERU. Infections with leptospires of the Australis serogroup account for about 13–14% of intraocular infections. Less frequently, leptospires of the serogroups Pomona, Sejroe, and Javanica were also detected in the vitreous samples from ERU eyes [34, 35, 114].

Vitreous samples obtained during vitrectomies from eyes affected by ERU were also used for histological and ultrastructural studies. It has been shown that the leptospires in the vitreous of eyes affected with ERU are surrounded by a homogeneous layer, which is lacking the leptospires from culture [115]. This homogeneous layer surrounding the

leptospires could be extracellular matrix. In another study, in addition to phagocytosed leptospires, dense roundish structures were detected in vitreous material from eyes affected with ERU [116]. Some of these roundish structures had been phagocytosed, but others of these structures were so large that phagocytosis was impossible. These dense round structures could represent mature leptospiral biofilm constructs.

In 1971, Williams reported on immunologically mediated tissue damage in cases of equine uveitis [22]. However, autoimmune reactions that can be detected at the same time as the leptospiral infection [117–121] must be autoimmune phenomena accompanying the infection, since they cease as soon as the infection has been eliminated [33, 35]. Thus, there is no evidence of autoimmune disease following ERU.

#### **10. Pathogenic** *Leptospira* **spp. and biofilm**

Since many chronic infections are associated with biofilm formation, it has long been suspected that leptospires also form biofilm in vivo. In in vitro studies, biofilm formation of pathogenic *Leptospira* spp. was observed [122], and a detailed description of the three-dimensional structure of these biofilms was given [123]. The main focus with regard to in vivo biofilm formation was on small rodents, which are considered the main vectors of pathogenic leptospires and are chronic shedders. Following experimental infections, evidence of biofilm formation in the proximal renal tubules had been observed [124, 125]. Recently, there was also a description of in vivo biofilm formation in naturally infected rats [126]. At about the same time, biofilm formation of leptospires in vitreous samples from eyes affected with ERU could be demonstrated by immunohistochemistry (IHC) [45].

#### **11. Characteristics of biofilm infections in ERU**

Recurrent episodes of uveitis and the concomitant intraocular persistence of leptospiral infection over a long period of time meet the criteria of a biofilm infection [127, 128] very well [129].

The infection primarily affects the vitreous cavity. Possibly following the vitreous clearance, leptospires (more rarely) can also enter the anterior chamber of the eye and be detected there [33–35, 130, 131]. However, the infection obviously remains limited to the eye, there is no evidence of further spreading. As with other local biofilm infections, IgA antibodies are of particular importance in diagnostics [72, 132–135].

One criterion of biofilm infections is the difficult cultural detection of the causative pathogen and ERU meets this criterion. Despite urgent suspicion of leptospiral infection in ERU (high intraocular antibody titers, intraocular antibody production), however, cultural detection of leptospires is demanding and often failed [24–26, 136].

In the vitreous of horses suffering from ERU, there are not only high antibody titers, but also immunocompetent cells (besides lymphocytes, especially plasma cells, macrophages, and granulocytes) [116, 137, 138]. The epithelium of the ciliary body shows many plasma cells in eyes affected by ERU [139]. In the area of the ciliary body and the iris root, even lymph follicles develop during the course of ERU, which contain B lymphocytes in the center [30, 140, 141]. Nevertheless, the immune system fails to eliminate the infection from the large vitreous chamber of the horse.

#### *Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

*Leptospira* spp. could be visualized in vitreous samples from eyes affected with ERU by immunohistochemistry (IHC). The infecting bacteria are bound to each other, and extracellular matrix could be demonstrated around and in between the bacteria [33, 45, 115, 142]. *Leptospira* spp. could be demonstrated in planktonic forms as well as in smaller and larger cell aggregates and in larger biofilm structures [33, 45].

Leptospires localized in the vitreous chamber show high tolerance to antibiotics. The first cultures were performed with samples from the entire lavage fluid collected during vitrectomy [41, 42, 44]. In the lavage fluid, the vitreous material was diluted about 10-fold and the lavage fluid contained 0.08 mg gentamicin/ml. This concentration had been shown to be 100 times the minimum inhibitory concentration (MIC) for WHO strains of pathogenic leptospires in vitro [143]. Cultures with these vitreous samples were less frequently positive than in later studies performed with undiluted vitreous samples [34, 35, 88], but nevertheless several culture sets eventually became positive after further inoculations and thus dilution of the antibiotic concentration [41, 42, 44].

Similar results were found in a study in which horses had been treated preoperatively intravenously with enrofloxacin. In the undiluted vitreous samples obtained at vitrectomy, the enrofloxacin content was above the MIC. Compared with the control group, in which more than 50% of the cultures were positive for pathogenic *Leptospira* spp., only 30% of the cultures in the group treated with enrofloxacin were positive. Thus, although the probability of a positive culture had been reduced with antibiotic treatment, reliable elimination of the infection was not achieved.

#### **12. Discussion**

ERU with persistent intraocular leptospiral infection over a long period of time meets all criteria of an infection associated with biofilm formation. The most likely route by which leptospires enter the vitreous cavity during acute systemic infection is by the fenestrated capillaries in the Pars plicata region of the ciliary body [30, 33]. In the healthy vitreous with its collagen fibers and viscosity, there are ideal conditions for the formation of leptospiral biofilm (**Figure 1**) [129].

The vitreous body is 98% water and contains a collagen fiber scaffold. It has been shown that plant fibers in rice fields are important sites for biofilms [144]. The vitreous fibers [138] might also serve as "surfaces" to which *Leptospira* may adhere and start biofilm production. Furthermore, viscous media promote biofilm production of *Leptospira* spp. [145], and healthy vitreous humor is such a viscous substance. With the collagen fiber scaffold and viscous consistency, the vitreous thus represents an ideal medium for biofilm formation of *Leptospira* spp. [33, 45, 129, 145].

Another factor to consider is that the vitreous cavity of the horse has a volume of approximately 28 ml, making it a large immunologic niche [34]. In addition, there is the immune privilege of the eye [146, 147], which effectively suppresses the immune defense. In this way, pathogenic *Leptospira* spp. can remain clinically unnoticed in the eye for a long time. The latency period can be many months or several years. It probably varies with individual factors of the host, the amount of *Leptospira* spp. in the vitreous, and possibly the leptospiral serovar involved.

Only after months or years, when a threshold is exceeded due to gradual multiplication of the leptospires and increase of immune reactions despite the ocular immune privilege, a uveitis attack with disturbance of the blood-aqueous barrier or blood-ocular barrier becomes apparent [33, 34]. The immune response that occurs in conjunction with the

#### **Figure 1.**

*Schematic illustration of the discussed pathogenesis of equine recurrent uveitis (ERU) caused by a leptospiral biofilm infection in the vitreous chamber. Each uveitis bout leads to increasing damage to the intraocular structures. 1. Infection of horses with Leptospira spp. may occur on humid and muddy pastures or by drinking from standing waters. The bacteria can enter the blood stream via intact mucous membranes (e.g., oral cavity) or small skin lesions (e.g., on the legs). 2. Leptospira spp. most probably enter the vitreous chamber (VC) via the fenestrated capillaries of the pars plicata of the ciliary body (CB). 3. Leptospira spp. within the vitreous chamber attach to each other and to vitreous fibers, starting biofilm production. 4. Transmission electron microscopy using a vitreous sample from an ERU eye: Leptospira spp. are surrounded by extracellular matrix (reprint of [115] courtesy of Schluetersche specialized media GmbH, Hanover, Germany). 5. Most Leptospira spp. are protected within the biofilm, single planktonic bacteria are in the vitreous chamber. 6. Vitreous samples from ERU eyes, containing visible inflammatory products ("vitreous floaters"); the yellow color indicates increased permeability of the blood-ocular barrier. 7. Threshold exceeded, immune privilege of the eye temporarily suspended, clinically apparent uveitis bout (left: Epiphora and blepharospasm; right: Much fibrin in the anterior chamber).*

inflammation likely results in the elimination of some planktonic bacteria. Other bacteria in the biofilm outlast the inflammatory bout. After the inflammation subsides under antiphlogistic treatment and with the help of intraocular immunosuppressive mechanisms, a clinically apparently inflammation-free interval occurs, which, however, does not represent a totally quiescent phase immunologically [137].

There are reports, and some own experiences seem to support this, that episodes of uveitis can be triggered by exposure to stressful situations (e.g., competitions, long-distance transport, change of stables, general anesthesia and major surgery). It is conceivable that endogenous cortisol release in stressful situations further reduces the immune defense in the eye (in addition to the ocular immune privilege). This in turn might increase the number of planktonic *Leptospira* spp. in the vitreous cavity after a stress situation and lead to contact with the uvea—which then causes an exaggerated immune reaction resulting in a uveitis attack.

A gradual spread of biofilm structures in the vitreous cavity could explain that ERU episodes occur at shorter intervals and become more severe over time. In addition, there are immune reactions that fail to eliminate the leptospires but may result in damage to the ocular structures adjacent to the vitreous chamber. One example is neutrophil extracellular traps (NETs), which have been detected in vitreous samples from eyes affected by ERU [148]. These NETs are formed by granulocytes to remove

#### *Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

pathogens too large for phagocytosis [149]. A disadvantage of the formation of NETs is that tissue-damaging substances are also secreted, which in turn promote an inflammatory reaction of the surrounding tissue [150, 151], which in ERU cases is the uvea.

The high MAT titers in eyes affected by ERU certainly also play a crucial role in the course of the disease, as they promote agglutination of planktonic leptospires. However, since complete elimination of the bacteria is usually not possible, this agglutination can also be the starting point for new biofilm formation. During agglutination, leptospiral aggregates are formed, extracellular matrix is produced after surface contact of bacteria with each other, and thus new biofilm structures can be built. In this way, the agglutinating antibodies could accelerate the biofilm formation of pathogenic *Leptospira* spp. [33].

High levels of serum amyloid A (SAA) [152] and the formation of AA amyloid [153, 154] were detected in intraocular samples from eyes affected with ERU. The formation of amyloid is a good explanation for the fact that the dense vitreous floaters in ERU fail to resolve, but instead increase as the disease progresses. Besides the collagen fibers of the vitreous scaffold, the NETs and the amyloid fibers provide additional fiber structures that could be used for biofilm formation. The formation of NETs and biofilm promote each other [155, 156]. Similar to what has been described for otitis media [157], these numerous fibers could be incorporated into the biofilm and help to reinforce the biofilm scaffold, so that therapeutically only mechanical removal is promising.

With knowledge of the successful cultivation of leptospires from vitreous specimens that contained an active level of gentamicin or enrofloxacin above the MIC, it is questionable whether intraocular gentamicin injections, which are performed therapeutically by some veterinarians, provide lasting success. Biofilms can increase tolerance to antibiotics up to 1000-fold compared with planktonic bacteria [158, 159]. The described improvement of eyes suffering from ERU after gentamicin injection could be due to the fact that planktonic bacteria are eliminated. However, it is questionable whether the bacteria in the biofilm can really be eliminated by the injection. It could also be that the structure and composition of the biofilm change accordingly, so that the bacteria survive protected in the biofilm and then lead to ERU relapses again after some time. With the therapeutically used cyclosporin-devices, spread of the leptospiral biofilm in the vitreous cavity could even be favored, since immune reactions of the host, including those directed against the bacterial pathogen, are suppressed.

In vivo biofilm formation has also been described for other spirochetes. In human medicine, for example, chronic Lyme disease with its various organ manifestations plays an important role [160, 161]. In patients with Lyme disease, in vivo biofilm formation was shown to be associated with the long-term persistence of *Borrelia* spp. [162], and biofilms were found to contain both *Borrelia* spp. and Chlamydiae [163]. For example, alginates have been found in biofilms of *Borrelia* [164]. Alginates induce a distinct immune response [165] and result in the biofilm being more pathogenic than the planktonic bacteria. For lymphocytoma [166] and Alzheimer's disease [167, 168], there are detailed descriptions of biofilm formation and indications for improved treatment options. In Alzheimer's disease, *Borrelia* bacteria in planktonic form do not appear to cause noticeable harm. Here, too, it is the biofilms that create the pathology [167]. Biofilm formation and approaches to improve therapy have also been demonstrated following experimental *Borrelia* infections of mice as a model for Lyme disease [169].

The composition of leptospiral biofilms in the vitreous cavity in ERU is still largely unknown. Neither alginates nor curli fibers (bacterial amyloid) could be detected in

the in vitro *Leptospira* biofilms [123]. The in vitro biofilms of *Leptospira* spp. consisted predominantly of extracellular DNA. However, the composition of in vivo biofilms of leptospires could be quite different [170]. It is possible that further analysis of the leptospiral biofilms in the vitreous cavity of horses suffering from ERU may provide further information on how to disperse these biofilms in a manner that is as tissue (retina, lens capsule) compatible as possible. This could provide new insights for the treatment of other biofilm-associated infections that are also relevant to human medicine.

### **13. Conclusions**

ERU is a spontaneously occurring intraocular leptospiral biofilm infection. For centuries, only symptomatic conservative treatment was possible, which has become increasingly effective with the availability of modern anti-inflammatory drugs. However, even the most potent anti-inflammatory treatment could not prevent recurrences of uveitis, which led to gradual damage and even destruction of the affected globe. It was not until the introduction of vitrectomy in equine ophthalmology that causative therapy had become possible. Samples containing leptospiral biofilm can easily be collected in the course of therapeutic vitrectomy. Not only can these samples be used for laboratory diagnostics regarding intraocular leptospiral infection, but further studies can be performed on the composition of the biofilm. There could be significant differences between the composition of the biofilm formed in vitro and that formed in vivo, as host tissues (here: vitreous material and collagen fibrils) and interactions with the host immune system (e.g., agglutinating antibodies, macrophages, granulocytes, NETs, fibrin, and amyloid) influence the composition of the biofilm. ERU provides possibilities for investigation of an in vivo biofilm infection without the need for animal experiments and, thus, could serve as a naturally occurring entity for further research.

### **Author details**

Bettina Wollanke\* and Hartmut Gerhards Ludwig-Maximilians-University, Munich, Germany

\*Address all correspondence to: b.wollanke@lmu.de

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Chronic Intraocular Leptospiral Infection Relying on Biofilm Formation inside the Vitreous… DOI: http://dx.doi.org/10.5772/intechopen.104527*

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#### **Chapter 18**

## Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties for Pharmaceutical Approaches

*Lakshmi Singh*

#### **Abstract**

Cyanobacteria also known as Blue Green Algae (BGA) are widely distributed in environments. Cyanobacteria or BGA commonly being aquatic are also reported from terrestrial ecosystems like sub-aerial surface of temples, monuments and building facades etc., represent their versatile habitats and extremophilic nature. These organisms are the excellent material for primary and secondary metabolites has been investigated by ecologists, physiologists, biochemists and molecular biologists. Scientists and young researchers require knowledge of the potential cyanobacteria and their exploitation in order to formulate effective natural compound or drug remedies. A large number of reports in literature stress have acknowledged the use of Cyanobacteria in pharmaceutical and industries, due to the production of different secondary metabolites with diverse bioactivities. However, very less study is being carried out with respect to exploitation of these sub-aerial Cyanobacteria group for production of different secondary metabolites with biological activities. Since many cyanobacteria are also able to survive most type of stress/and or extreme, they may become even more important as antimicrobial agents of pharmaceuticals in the future. Hence, special attention is paid to these groups of organisms.

**Keywords:** sub-aerial cyanobacteria, extreme environment, antimicrobial agents, pharmaceutical sector

#### **1. Introduction**

The appearances of multi drug resistance among pathogens growing day by day. This could be attributable to prolonged and indiscriminate use of antibiotics and chemotherapeutic agents, over and/or under use of drugs, use of antibiotics without prior knowledge of antibiotic sensitivity pattern of the pathogens, non-completion of dose. In addition, prolonged use of antibiotics and chemotherapeutics results in many side effects too. So there has been a growing demand in search of some new source group of alternative antibiotics. Most of the academicians and researchers all over the world, starting from the ancient age, exploited medicinal and aromatic plants,

to a great extent for treatment of diseases and discovery of new antimicrobials or compounds with bioactivities. Based on the complexity in composition, extractions of compounds from microorganisms now are studies again for new antimicrobial compounds. A greater interest has been raised in the field of research towards bioactive compounds from algae. Secondary or primary metabolites of algae consist of diverse groups of chemical compounds. The antibiotic activity of algae has been reported since 1944 [1]. More than 164,784 algae species and infraspecific taxa are reported from all over the world in AlgaeBase whereas, regarding Cyanobacteria, 5152 species have been reported [2]. Among them few have been identified or tested for their efficiency. Algae are sources of amino acid, terpenoids, phlorotannins, steroids, phenolic compounds, halogenated ketones and alkanes and cyclic polysulphides [3]. The natural products from a wide variety of taxa have been isolated and tested for their potential biological activities [4]. Sub-aerial cyanobacteria are one of the important taxa of prokaryotic algae; distributed in extreme habitats need to be explored for their efficiency with respect to bioactivities, as prior research in this area has been inconclusive.

#### **1.1 Cyanobacteria distribution in diverse habitats**

Cyanobacteria (BGA) are gram negative photoautotrophic bacteria found in almost all ecological habitats, of aquatic and terrestrial origin. Aquatic forms are abundantly found in both marine and fresh water ecosystems including stagnant water bodies, under running water bodies, lagoons etc. Brackish water bodies also harbor a large number of Cyanobacterial species. Terrestrial habitats, including extreme environments also reflects the tolerance of Cyanobacteria have been reported as biofilms/ or crusts on the exposed surfaces of solid substrata in almost all climatic zones [5]. These organisms grow as epiphytes on tree bark, as epiliths on rocks and stones, and also on anthropogenic surfaces such as facades, concrete floors of roofs and other artificial surfaces of buildings where they cause esthetically unacceptable discolouration of the structures [6]. Such growths are common in humid places on uneven surfaces such as holes, crevices and also on damp building walls due to leaking, roof guttering, inadequate drainage of flat areas or from adjacent water courses. Their adaptation on surfaces of both modern and ancient buildings as well as old monuments represents them as sub-aerial Cyanobacteria/extremophiles since enduring extreme environments. They are particularly abundant in tropics as compared to temperate regions due to their capacity to resist very harsh conditions such as very high temperature, prolonged dry periods, extreme light intensity and UV radiation as they are the prolific producers of secondary metabolites, extracellular glycans, heat shock proteins and, UV pigments such as Mycosporine like amino acids (MAAs) and Scytonemins [7–9]. This population has been reported to have a characteristics appearance and develop a large number of photosynthetic pigments (chlorophyll, carotenoids and phycobiliproteins) including UV - absorbing compounds and pigments which play a key role in their protection and adaptability [10, 11]. These are certain attributes for their colonization and also have of great importance implications in scientific research and for human welfare.

According to literature stresses, the organisms those occur on such substrata mainly consists of coccoid forms of the order Chroococcales (*Chroococcidiopsis*, *Chroococcus*, *Gloeocapsa*, *Myxosarcina*), filamentous forms of the order Oscillatoriales (*Plectonema*, *Leptolyngbya*, *Lyngbya*, *Microcoleus*, *Oscillatoria*, *Phormidium*, *Pseudophormidium*, *Schizothrix*) and Nostocales (*Calothrix*, *Nostoc*, *Scytonema*,

*Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*

*Tolypothrix*) etc. Several researchers have studied and reported this type of forms from almost all climatic zones. Examples are building facades in Greece [12], buildings in South Eastern, Spain [13–18], building in American countries [19], building facades in France [20, 21], stone monument and building facades in Italy [22–25], monuments in Portugal [26, 27], monuments in Slovakia [28, 29] and modern/old monuments, India [30–39]. In general, the knowledge on sub-aerial Cyanobacteria diversity colonizing building facades and their exploitation in different applications is still limited. To our knowledge, few reports have been published that deal specifically with the presence of secondary metabolites and pigments of sub-aerial Cyanobacteria and algae isolated from facades of buildings, on structural of cultural heritage and on rock surfaces of different monuments. No systematic scientific approach has been taken yet in India including other countries on this subject. A few research workers have worked on the microorganisms from facades of buildings, cultural heritages and monuments and other material in different parts of the globe [40]. However, no effective chemical or compound which can be employed as an antimicrobial agent from sub-aerial species in Pharmaceutical and Nutraceuticals industries has not yet been reported for which search is on.

#### **2. Bioactive compounds from cyanobacteria**

Literature stresses isolation and identification of Cyanobacteria from a diverse environment with bioactivities, but only few research has focused on a variety of bioactive compounds produced by Cyanobacteria after analysis of a great number of marines [41–43], freshwater [44–46], terrestrial [47, 48], and hot spring [49, 50]. Cyanobacterial natural products still seem to prevail followed at much lesser proportions by alkaloids, aromatic compounds, cyclic depsipeptides, cyclic peptides, cyclic peptide, cyclophane, fatty acids, linear peptides, lipopeptides, nucleosides, phenols, macrolides, polyketides, polyphenyl ethers, porphinoids and terpenoids [51]. These interesting and biochemically active compounds possess biological activity covering a wide range of antibacterial [52–55], antifungal [56], antialgal [56], antiviral [57], anticancer effectiveness [58–60], and immunosuppressive [61] activities. Some bioactive lead compound are bastadin, bis-x-butyrolactone, hapalindole, didehydromirazole, kawaguchipeptin B, muscoride, noscomin, nostocine A, scytophytin, and lipids [62] exhibited with antibacterial activity and, ambiguines, calothrixin, cyanobacterin, fischerindole A, hapalindole, hassallidin, phytoalexin, scytophycin, tjipanazole and *Y*-lactone [63, 64] with antifungal activity and few compound such as 4,4′-dihydroxybiphenyl, norhamane pyrido (3,4-*b*)indole, beta-glucan, bacteriocin, ambiguines, parsiguine, scytoscalarol, hapalindole [65] which have been reported to show antimicrobial activity. However, only few of them have been investigated in details [66, 67] are described under this subpoint 2.1. Some known bioactivities as per reported are listed below (**Table 1**). Thus, screening efforts aimed to identify antimicrobial agents in sub-aerial Cyanobacteria which might reveal promising compounds.

#### **2.1 Bioactive compounds and its inhibitory activity with actions**

Earlier reports indicate that bioactive compounds contradict synthetic drugs in their composition and their arrangement of radicals and atoms. However, their inhibitory activities are much more depends on the nature of interaction between donor and target organisms. They may inhibit growth or photosynthesis, kill the competitor or exclude


*Focus on Bacterial Biofilms*


#### *Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*


#### *Focus on Bacterial Biofilms*


### **Table 1.**

*Bioactive molecules or compound produced by various cyanobacteria on database.*

#### *Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*

it from the donor vicinity, may be potent in inhibiting protein–protein interactions resulting in effective immune response, signal transduction; mitosis and ultimately apoptosis without causing much harm to living organisms [68, 69]. A large number of novel antimicrobial agents have been identified with antimicrobial, antibacterial and antifungal activities globally represented in (**Table 1**). However, few compounds like ambiguines, calothrixine A, cyanobacterin, fischerindole L, hapalindole, hassallidin, muscoride, noscomin, nostocine, phytoalexin, scytophycin, scytoscalorol and tjipanazole etc., either synthetized by ribosomal pathways or by non-ribosomal pathways [70] have attained importance for their antimicrobial activity in the field of pharmaceutical sector. Most of the cyanobacteria bioactive compound reported here are generally soluble in organic solvents and with low molecular weight. With respect to their mode of action, a relatively limited number of compounds have been studied or identified based on growth inhibition against target organisms. Kawaguchipeptin B, an antibacterial cyclic undecapeptide isolated from the cultured cyanobacterium *Microcystis aeruginosa* (NIES-88) showed antibacterial activity by growth inhibition towards gram positive bacterium *Staphylococcus aureus* at a concentration of 1 μg/mL (MIC) [71]. Ambiguines reported from *Fischerella ambigua* and *Haplosiphon hibernicus* was found to inhibit bacteria like *Mycobacterium tuberculosis* and *Bacillus anthracis*, and fungi such as *Aspergillus oryzae*, *Candida albicans*, *Penicillium notatum*, *Saccharomyces cerevisiae* and *Trichophyton mentagrophytes* [72–74]. Hassallidin reported with various types (hassallidin A, hassallidin B, hassallidin D, hassallidin 12, hassallidin 14 and hassallidin 15) from three different species, *Tolypothrix*, *Anabaena* strain (BIR JV1 and HAN7/1) and *Nostoc* strain (6sf Calc and CENA 219) showed as a potent antifungal agent against *Aspergillus fumigatus* and *C. albicans* [75] through inhibiting growth. Similarly, many other compounds such as gamma lactone from *Scytonema hofmanni* [76], didehydromirabazole from *Scytonema mirabile* [77], bastadin and Bis-x-butyrolactones from *Anabaena basta* and *A*. *variabilis* [78, 79], tjipanazole from *T*. *tjipanasensis* [80], muscoride from *Nostoc muscorum* [81], fischerellin A produced by *Fischerella muscicola* [82], nostofungicidin, noscomin and nostacine A from *Nostoc commune* and *Nostoc spongigaeforme* TISTR 8169 against *Bacillus cereus*, *Staphylococcus epidermidis* and *Escherichia coli* [83–85], fischerindole L and Parsiguine from *Fischerella muscicola* and *Fischerella ambigua* [86–88], and Scytophycins from *Scytonema pseudohofmanni*, *S*. *hofmanni* PCC7110, *Nostoc* sp. HAN11/1 and *Anabaena cf. cylindrica* (BIR JV1 and HAN7/1) [89, 90] are demonstrated with antibacterial /or antifungal activity based on growth inhibition but the type of target organisms and mode of action is unclear. However, few compounds have been shown to exhibit their mode of action through inhibition of photosystem - II, or enzyme or nucleic acid synthesis and/ or cellular paralysis. Phytoalexin from *Scytonema 0cellatum* exhibited inhibition of fungal enzymes and mycelial growth including cytoplasmic granulation, disorganization of the cellular contents and rupture of the plasma membrane of fungi like *Aspergillus oryzae*, *C. albicans*, *Penicillium notatum* and *S. cerevisiae* [89]. Cyanobacterin from *Scytonema hofmanni* and *Nostoc* sp., both found to inhibit the photosystem II-mediated photosynthetic electron transfer [91, 92]. Calothrixine A from *Calothrix* sp., as antifungal activity leads to growth inhibition because of RNA synthesis inhibition [92]. Two alkaloids, hapalindole a polycyclic isothiocyanate and 12-*epi*-hapalindole E isonitrile from *Fisherella* sp., and *Nostoc CCC537* have pointed to inhibition towards bacteria (*Bacillus subtilis*, *M. tuberculosis* H37Rv, *S. aureus* ATCC25923, *Salminella typhi* MTCC3216, *Pseudomonas aeruginosa* ATCC27853, *E. coli* ATCC25992 and *Enterobacter aerogenes* MTCC2822) and fungi (*C. albicans*) based on RNA polymerase, DNA and protein synthesis [92, 93]. β-glucans, a beta-D-glucose polysaccharides from *Chrococcus turgidis* exhibited phagocytic activity and resistance

*Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*

towards *B. subtilis*, *E. coli*, *P. aeruginosa* and *S. aureus* and have shown chronic wound healing activity either directly or indirectly by modulating the activity of diverse cells and growth factors to reparative process [94, 95]. Bacteriocin, an antimicrobial protein/ or peptide toxin isolated from *Nostoc* sp. 78–11 A-E found to be inhibits protein and its actions against bacteria and cyanobacteria [96]. Other bioactive molecules like 4–4′-dihydroxybiphenyl (*Nostoc insulare* 54, 79), Norhamane pyrido (3,4-b) indole (*Nodularia harveyana*), Pentadecane (*Anabaena oryzae*), 6-pentadecanol and octadecyl acetate (*Synechococcus* strain), m-Xylene, 2,6,10,14-Tetramethylheptadecane, 2-Ethoxy2 methylbutane, propanedioic acid dimethyl ester (*Oscillatora* sp.), hexaethylene glycol dimethyl ether, propylene glycol trimer 3 and phthalic acid mono-(2-ethylhexyl) ester, (3E)-3-Icosene, (Z)-14-Tricosenyl formate (*Stigonema ocellatum*), 6- Octen-1-ol 3,7-dimethyl- acetate and 9-Hexadecenoic acid octadecyl ester [97–100] are reported with activity, although the mode of that action is still unknown.

#### **3. Bioactivity of sub-aerial cyanobacteria**

Many sub-aerial Cyanobacteria are known to tolerate environmental extremes as they possess a great capacity for producing biologically active compounds. Researchers are in believe that more harsh and extreme conditions lead to a wider production of a diverse range of more or less, specific substances thus pointing towards these organisms as brilliant candidates for antimicrobial properties. A few numbers of sub-aerial cyanobacteria compounds are found to inhibit the target organisms, making them an attractive source of antimicrobial agents. Some known bioactivities from ten subaerial cyanobacteria as per reported are listed below (**Table 2**). The chloroform fraction of *Scytonema* br1 isolated from wall and Terrace, Konark Temple, Puri, Odisha showed significant anticyanobacterial activity against *Anabaena* BT2 and *Nostoc* pbr01 and antialgal activity against a green alga *Bracteacoccus* [55]. The lipids extract from *Toxopsis calypsus* and *Phormidium melanochroun* isolated from caves established good antibacterial activity against *Enterococcus faecium* (VRE), *Enterococcus faecalis* (ATCC) and *S. aureus* (MRSA) by disrupting cellular membranes [101]. Another study reported the chloroform extracts of *Scytonema hofman* isolated from building facades showed antibacterial activity against *E. coli*, followed by *Klebsiella pneumonia* and *P. aeruginosa*, *S. aureus* [102]. There is a report that acetone extract of sub-aerial species, *Scytonema ocellatum* isolated from sub-aerial habitats exhibits antibacterial activity towards *E. coli*, *B. subtilis* and *S. aureus* and GC analysis showed 98% and 95.6% purity antibiosis [103]. The sub-aerial Cyanobacteria *Anabaena* sp. (VBCCA 052002) as dominant species on terracotta monuments of Bishnupur showed highest antibacterial activity against *S. aureus*, *Salmonella typhimurium* and *E. coli* with a MIC value of 100 μg/ml against *S. aureus* and 150 μg/ml against *S. typhimurium* [104]. In another study reported three different type of bioactive compounds such as 2, 4-Bis (2-methyl-2-propanyl) phenol - phosphorous acid (C42H69O6P:Mw- 700 g/mol) as phenolic, and other two compound Ergost-5-en-3-ol (C28H38O4: Mw-704 g/mol) and 7, 11-dihydroxysolasodine (C27H43NO4: Mw-413 g/mol) as steroidal alkaloid from three sub-aerial cyanobacteria species, *Tolypothrix rechingeri*, *Scytonema hyalinum* and *Scytonema ocellatum* respectively which exhibiting antimicrobial activity against *E. coli*, *P. aeruginosa*, *S. aureus*, *C. albicans* and *Epidermaphyton flocossum* etc. [105]. Out of ten, one of the sub-aerial cyanobacteria, *Fischerella* sp. (NCBI Accession number MN593556) reported with most potent active compound with Rf value 0.96 of acetone fraction showed complete growth inhibition against *E. coli* and moderate


#### **Table 2.**

*Bioactive molecules or compound produced by various sub-aerial cyanobacteria on database.*

activity to *C. albicans*, was identified as, Iron (2+) amino (cyclopenta 2,4 diene-1-ylidine) methanolate 1,2,3,4,5-pentaphenycyclopenta-2.4, dien-1-ide (Pentaphenyl ferrocene carboxamide), C41H31FeNO: Mw-610 g/mol and was found to be non-toxic against cells lines of *Catla thymus macrophage* and osteoblast precursor cell line of *Mus musculus* up to 72 hours*,* with a concentration range of 0.875 - 4 mg/ml indicated their potentiality for development of new antimicrobial compounds [106].

#### **4. Sub-aerial Cyanobacteria: as a source of antimicrobial compounds towards pharmaceutical approaches.**

In modern research, a number of significant advancements have been made in Cyanobacterial pharmacologically active compounds from natural resources like marine, freshwater**,** and very few terrestrial etc., and has received ever increasing interest. A large number of antibiotic compounds, many with novel structures, have been isolated and characterized, but few compounds such as dolastatins, soblidotin, Tasidotin, cryptophycin, curacin D and micropeptins exhibited very interesting results and successfully reached Phase II and Phase III of clinical trials [107–111]. Isolation of these compounds

#### *Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*

from cyanobacteria species like *Symploca* sp., *Nostoc* sp., and *Lyngbya majuscule* offers great opportunity and a platform for the discovery of anticancer and antitumor agents. Furthermore, a few have focused on baseline information for promoting the use of cyanobacterial bioactive compounds as drugs using the computational approach. They can be profitable to mankind in multidirectional ways and probably they constitute a principal group of organisms for biotechnological exploitation, especially for valuable products, processes and services, with significant impact in food and pharmaceutical industries as well as in public health. However, still the active principles and their mode of action are yet unknown in most cases. Since there is a direct need for an alternate antimicrobial drug due to the emergence of multi drug resistant pathogens throughout the Globe, as one of the major concerns. Literature stresses the study of emerald compound of algae including Cyanobacteria having antimicrobial property. The search of new active substances with antimicrobial activity from Sub-aerial Cyanobacteria (BGA) of extreme environments, form a major group among algae too are the potential and promising candidates. It is of its kind to mention here that, In the past [33] a number of sub-aerial Cyanobacteria from old temples, monuments, caves, building facades were isolated to accelerate their survival strategies and control mechanisms; only few made an effort for their bioactivity [55]. Few are proved to be antiviral drug, anticancer drug, antibacterial drug and or antifungal drug too [112, 113]. In this review, ten major activities of sub-aerial cyanobacteria have been listed from the literature (anticyanobacterial, antialgal, antibacterial and antimicrobial activities) as describe in **Table 2**. However, to the best of knowledge these sub-aerial Cyanobacteria of unique environment are not explored for their biotechnological applications in terms of bioactivities and/or antimicrobial activities to find out their possible use in pharmaceuticals for development of new antimicrobial compounds which need to be further analyzed.

#### **5. Conclusion**

Nowadays, the production of secondary metabolites from extreme enduring cyanobacteria has catapulted this group of organisms into the midst of intense research. The survival strategies of cyanobacteria to various stress fixed secondary metabolites sources in term of growth, physiology and different metabolic processes are of great interest as they able to secrete different metabolites with environmental stress and ability for their adaptation to extreme environments. No systematic scientific approach has been taken yet on secondary metabolite with their antimicrobial properties from sub-aerial cyanobacteria in India or other countries on this subject. A few research workers have worked on the bioactive compound and their approaches in pharmaceutical sectors of these sub-aerial cyanobacteria to represent as a new source of biologically active compounds in the form of secondary metabolites with production of different antimicrobial compounds, further more studies are desired to find its way for use in pharmaceutical industries, for development of newer antimicrobials, against costly harmful antibiotics and chemotherapeutics, in order to enjoy the benefits and/or the fruits of this investigation for future uses. However, this knowledge may be important in developing strains of sub-aerial cyanobacteria with higher efficiency for antimicrobial properties.

#### **Funding**

No funding was received for this contribution.

### **Conflicts of interest**

The author declares no conflict of interest.

### **Author details**

Lakshmi Singh

Department of Botany, College of Basic Science and Humanities, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, India

\*Address all correspondence to: lakshmisinghouat@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Sub-Aerial Cyanobacteria: A Survey of Research with Antimicrobial Properties… DOI: http://dx.doi.org/10.5772/intechopen.102696*

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[96] Flores E, Wolk CP. Production, by filamentous, nitrogen- fixing cyanobacteria, of a bacteriocin and of other antibiotics that kill related strains. Archives of Microbiology. 1986;**145**(3):215-219

[97] Volk RB. Screening of microalgal culture media for the presence of algicidal compounds and isolation and identification of two bioactive metabolites excreted by the cyanobacteria *Nostoc insulare* and *Nodularia harveyana*. Journal of Applied Phycology. 2005;**17**(4):339-347

[98] Vestola J, Shishido TK, Jokela J, Fewer DP, Aitio O, Permi P, et al. Hassallidins, antifungal glycolipopeptides, are wide spread among cyanobacteria and are the endproduct of a non-ribosomal pathway. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**:E1909-E1917

[99] Seddek NH, Fawzy MA, El-Said WA, Ragaey MM. Evaluation of antimicrobial, antioxidant and cytotoxic activities and characterization of bioactive substances from freshwater blue-green algae. Global NEST Journal. 2019;**21**(3):329-337

[100] Amaral R, Fawley KP, Němcová Y, Ševčíková T, Lukešová A, Fawley MW, et al. Toward modern classification of Eustigmatophytes, including the description of Neomonodaceae fam. Nov. and three new genera. Journal of Phycology. 2020;**56**(3):630-648

[101] Lamprinou V, Tryfinopoulou K, Velonakis EN, Vatopoulos A, Antonopoulou S, Fragopoulou E, et al. Cave cyanobacteria showing antibacterial activity. International Journal of Speleology. 2015;**44**(3):1-9

[102] Panigrahi S, Sethi AK, Samad LK. Antibacterial activities of *Scytonema hofman* extracts against human pathogenic bacteria. International Journal of Pharmacy and Pharmaceutical Science. 2015;**7**(5):123-126

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Bishnupur, West Bengal. International Journal of Pharmaceutical Science and Drug Research. 2020;**12**(2):1333

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[106] Pattnaik S, Mishra BB, Singh L. Isolation and characterization of a novel antimicrobial compound from sub-aerial cyanobacterium *Fischerella* sp., isolated from building facades. Applied Biological Research. 2021;**23**(1):1-12

[107] Luesch H, Moore RE, Paul VJ, Mooberry SL, Corbett TH. Isolationof dolastatin 10 from the marine cyanobacterium Symploca species VP642 and total stereochemistry and biological evaluation of its analogue symplostatin 1. Journal of Natural Product. 2001a;**64**:907-910

[108] Edelman MJ, Gandara DR, Hausner P, Israel V, Thornton D, DeSanto J, et al. Phase 2 study of cryptophycin 52 (LY355703) in patients previously treated with platinum-based chemotherapy for advanced nonsmall cell lung cancer. Lung Cancer. 2003;**39**:197-199

[109] D' Agostino G, Del Campo J, Mellado B, Izquierdo MA, Minarik T, Cirri L, et al. A multicancer phase in study of the cryptophycin analog LY355703 in patients with platinum-resistant ovarian cancer. International Journal of Gynecology Cancer. 2006; **16**:71-76

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[113] Abdo SM, Hetta MH, El-Senousy WM, El Din RAS, Ali GH. Antiviral activity of freshwater algae. Journal of Applied Pharmaceutical Science. 2012;**2**(2):21-25

#### **Chapter 19**

## Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology Application

*Galina Satchanska*

#### **Abstract**

This Chapter discusses the entrapment, growing and biofilm formation by an environmental bacterium immobilized in polyethyleneoxide cryogel to be applied in environmental biotechnology. The KCM-R5 bacterium was isolated from the heavy metal-polluted environment near a large Pb-Zn smelter, also producing precious metals in Bulgaria. Molecular-genetic analysis revealed affiliation with *Pseudomonas rhodesiae.* The strain is capable of growing in high concentrations of phenol and different phenol derivatives. Polyethylene oxide was found to be friendly and nontoxic to bacteria polymer enabling bacteria easy to penetrate in it and fast to grow. KCM-R5 biofilms were grown for 30 days in batch culture with phenol (300-1000 mg L−1) dissolved in the mineral medium. The bacterium was able to involve phenol in its metabolism and use it as a single carbon supplier. The results obtained in the study showed 98% phenol biodegradation using the biotech installation described. The proposed PEO cryogel-*P. rhodesiae* KCM-R5 bacterium biotech biofilter can be used for environmental biotechnology application in industrial wastewater detoxification.

**Keywords:** PEO cryogels, environmental bacterium, biodegradation, phenol derivatives, biofilms

#### **1. Introduction**

In recent years, the growing amount of polymer-encapsulated bacteria and engineered bacterial biofilms have enhanced both wastewater management and biodegradation of industrial pollutants. Amidst the aromatic substances, monocyclic phenol and its nitro- and chlorophenol derivatives represent one of the most harmful environmental pollutants. Phenol (**Figure 1**) is a by-product of benzene production and widely exploited in the chemical industry.

The continuous application of phenol and its derivatives such as *ortho*-nitrophenol (*о*-NP), 2,4- dinitrophenol (2,4-dNP), 2,5-dinitrophenol (2,5-dNP), penthachlorophenol (PCP) and 2,4- dichlorophenoxyacetic acid (2,4-D) (**Figure 2**) in the

**Figure 1.** *Structural formula of phenol.*

#### **Figure 2.**

*Structural formulas of nitro- and chlorophenol derivatives.*

chemical, agricultural, woodworking and oil processing industries has resulted in their persistent presence in the environment.

Worldwide, high concentrations of phenol and phenol derivatives were detected in industrial wastewaters, which further flow into rivers, seas and oceans. Bisphenol A (BPA) as phenol derivative is amid he most prominent plasticizers and is omnipresent in surface and ground water. This toxic substance is detected in many aquatic organisms. Mathieu-Denoncourt *et al*. [1] reported that BPA was the most toxic (96 h LC50s) to aquatic invertebrates (0.96-2.70 mg/L) and less toxic to fish (6.8- 17.9 mg/L). It plays toxic effect on amphibians being more noxious to embryos than to juveniles. It plays neuro-toxic and reproductive effect reported by Santoro *et al.* [2].

Phenol is harmful to human causing blood pressure increase, leukemia, skin necrosis and pores creation, damages of the phospholipid bilayer, heart arrhythmia, tight junctions disruption, liver and kidney injury, earlier child birth and gastro-intestinal perforations [3, 4]. Phenol did not demonstrate a carcinogenic effect (**Figure 3**) [4].

It is estimated that the median *lethal dose* of phenol in humans is 14-214 mg kg−1or 1-15 g [4].

*Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

**Figure 3.** *Harmful effect of phenol on human organs.*

Among bacteria, bacterial species like *Pseudomonas* [5–7], *Bacillus* [8] and *Geobacter* [9] are capable of effective phenol biodegradation. Besides bacteria, fungi of the genera *Aspergillus* [10], *Trichosporon* can also successfully degrade phenol. Most authors describe mainly the degradation by free planktonic cells, but data about degradation by encapsulated bacteria are scarce. Both natural or synthetic polymers can be used as bacteria carriers. Biodegradation of phenol is accomplished *via ortho*or *metha* cleavage of the aromatic ring. First step is conversion of phenol to catechol by attachment of additional hydroxyl group (**Figure 4**).

Further the catechol is degraded either *via* the *metha*-mechanism, a process catalyzed by the enzyme catechol 2,3-dioxigenase or *via* the *ortho*-mechanism using catechol 1,2-dioxigenase [11].

The current chapter discusses the variety of natural and synthetic polymers used for bacterial entrapment; the content, development and structure of bacterial biofilms, and encapsulation of the xenobiotic degrading bacterium *Pseudomonas rhodesiae* KCM-R5 in PEO cryogels, creating a biofilter, bacterial biofilm formation and phenol degradation by said polymer-bacterium biofilter.

**Figure 4.** *Mechanism of phenol degradation [11].*

#### **2. Natural and synthetic polymers used for entrapment of bacteria**

Natural polymers most commonly used are: (i) Alginate: alginate, alginate/ soy protein isolate (SPI), algnate/cashew gum, (ii) Cellulose derivatives: cellulose acetate, ethyl cellulose, cellulose fibers, (iii) Chitosan: chitosan, the binary system beta-cyclodextrin modified chitosan, chitosan/synthetic poly(ethylene oxide), (iv) Starch and maltodextrin: gum acacia/maltodextrin, Arabic gum/maltodextrin/starch, (v) Whey protein, (vi) Fibroin: fibroin/poly-caprolactone, (vii) Gelatine. The main advantages of natural polymers are their biocompatibility and nontoxicity to living cells and biological structures, e.g. essential oils [12].

Synthetic polymers used for bacterial encapsulation are polyvinylchloride, polylactic acid, polycaprolactone, polycaprolactone/hydroxiapatite composites, poly(methil methacrilate), poly(vinyliden fluoride), poly(ethileneoxide), poly(ethylene brassilate-co-squaric acid) [12–14]. A limited number of studies have been reported for phenol degradation by bacterial biofilms formed by immobilized bacteria. Immoblilization of bacteria was conducted in polyacrylamide [15], polyurethane [16], polyamide [17], polyacrylonitrile [18, 19] or polyvinyl alcohol [20].

In the last 20 years, different organic carriers for bacterial immobilization were investigated [15, 16]. Among synthetic polymers, poly(ethylene oxide) hydrogels are excellent candidates because they are nontoxic and biocompatible materials which meet all of the requirements for strength, absorbency, flexibility and adhesiveness [17]. Hydrogels of poly(ethylene oxide) have been synthesized *in situ* by applying a facile optimized protocol, which will be further described.

#### **3. Structure and development of bacterial biofilms**

Biofilms are an excellent strategy for bacterial survival in a sessile way and 40-80% of bacteria on earth can form biofilms [21–26]. The first to observe under a microscope microbes living on the surfaces of teeth was the Dutch merchant Antony van Leuwehoek. He can also be considered the first discoverer of bacterial biofilms. The invention of the electronic microscope in the 1930-ies provided an insight into the structure and organization of biofilms. Biofilms colonize different surfaces like plant and animal tissues, medical devices, potable water pipes, and natural lakes and rivers. In the early 1970-ies, the ambiguous role of disinfectants in the disruption of bacterial biofilms was proved, a finding published by [24]. The authors discussed bacterial resistance to chlorine, one of the most widely used disinfectants, due to bacterial biofilms.

Bacterial biofilms are complex living communities composed of a wide range of components and molecules such as bacterial cells, their polysaccharides, proteins, lipids, DNA and RNA. The external DNA (eDNA) in particular plays an important role in the early phase of biofilm arrangement [27].

Several factors can influence biofilm generation [28, 29]. The main factors are related to the bacterial surface and its charge. Hydrophobicity is a main factor influencing the adsorption and change in the surface tension of bacteria. Biofilm formation involves all flagellar and non-flagellar bacterial structures - fimbriae, pilli and flagella [30]. Investigations on the structure of fimbriae show that they contain predominantly residues of hydrophobic amino acids, such as valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and cystein [23]. Fimbirae also contain *Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

adhesion molecules [29] which attach to substrates and thus bacteria can deliver nutrients for their metabolism. Temperature and substrate availability also impact biofilm formation.

It is important to note that bacterial adhesion [29] and biofilm formation increase on rough surfaces compared to smooth surfaces. The larger surface area and the weaker shear forces facilitate biofilm formation. As Donlan [29] described, the physicochemical properties of the surface is of great importance in biofilm build-up. Bacteria attach more easily to hydrophobic, nonpolar, rough surfaces like Teflon or plastics than to hydrophilic surfaces like glass or steel [31–33]. Bacterial biofilms develop on tooth enamel in the oral cavity. The pellicle contains albumin, lipids, glicoproteins, gingvinal fissure liquid, lysozime and bacteria dwelling in the oral cavity. Mittelman [34] discussed in his publication that the host produces complex bacterial biofilms as saliva, respiratory secretion, tears, urine and blood, which strongly influence bacterial attachment. The development of bacterial biofilm is shown in **Figure 5**.

As shown in **Figure 5**, the stages of bacterial biofilm development include the crucial initial steps of finding, interacting with, and adhering of planktonic bacteria to a surface [35, 36]. Once irreversibly attached to a surface, bacteria form microcolonies. Biofilm matures and when it has completely matured it is affected by shear forces and undergoes rupture resulting in free planktonic cells. The liberated planktonic cells fall on new surfaces and colonize them, forming new biofilms [22, 37].

Both pH and the high amount of nutrients increase the concentration of ferric, sodium and calcium cations. These cations affect the adhesion of *Pseudomonas fluorescens* reducing the chemical forces between the negatively charged bacterial cells and the glass surface [29]. Several studies reported that mycolic acid-containing bacteria like *Mycobacterium* [38], *Corynebacterium* [39] and *Nocardia* [40] attach more intensively than non-mycolic ones. The longer chain length of mycolic acid correlates with high and rapid bacterial adhesion. Silva and de Ataujo [41] discussed the inhibitory role of lectins on biofilm formation. Lectins are proteins which bind to carbohydrates

**Figure 5.** *Bacterial biofilm development.*

and polysaccharides of the outer membrane of bacteria. Lectins are ubiquitous in nature and can be found in large amounts in cereals and legumes.

Depending on the affinity of motile and nonmotile bacteria to adhesion, motile bacteria are capable of more active attachment. Nonmotile bacteria are slower in forming biofilms. The flagella of motile bacteria are crucial for the early stages of biofilm formation [30, 42].

*Pseudomonas aeruginosa*, one of human opportunistic pathogens was used as model organism to study bacterial biofilm formation cells [43]. Authors show that three extracellular polysaccharides (EPS) - alginate, Psl, and Pel are mainly responsible for the biofilm formation. EPS can represent between 50% and 90% of the total organic carbon in the biofilm [44]. EPS composed of polysaccharides are neutral biopolymers [45]. When EPS contain uronic acids such as D-glucoronic or D-galacturonic acid, they contribute to their anionic nature. The anionic property is important for the association with calcium and magnesium bivalent cations, which cross-link and provide greater strength to the bacterial biofilm. EPS can be either hydrophobic or hydrophilic but are generally highly hydrated due to water accumulation *via* the hydrogen bonding. This is the reason why natural biofilms can hardly be desiccated. In addition to divalent cations, EPS can bind to metal ions, proteins, DNA or lipids. Some EPS can even bind to humic acids [46].

The main extracellular polymeric substances which bacteria produces when exposed to phenol are PN (exopolymeric protein) and PS (lower polysaccharides) as described by Gao *et al.* [47]. During the biotransformation of heavy metals synthesize as EPS both homopolysaccharides and heteropolysaccharides [48]. Among the homopolysaccharides are identified dextrane, mutane, alternant, reuteran, gurdlan, levan and inulin. Gupta *et al.* reported the most abundant amidst heteropolysaccharides - alginate, xanthan, hyaluronan and sphingans [48]. *P. aeruginosa* responds to chlorine-based disinfectants by synthesis of alginate-based EPS as described by Xue *et al.* [49].

Undoubtedly, the architecture of each bacterial biofilm is unique. They can be mono-, double or multi-layer thick. When consisting of several layers, a network of many water channels can be observed inside the biofilm. According to the bacterial diversity, biofilms can consist of one bacterial strain but most often they contain mixed bacterial cultures. Different bacteria form thicker or thinner biofilms. Sometimes, when the biofilm is formed in the human body, it can also include nonbacterial compartments like erythrocytes or fibrin. Such types of biofilms form on heart valves. Bacterial biofilms formed on urinary catheters are known to consist of bacteria capable of urease-catalyzed degradation of urea, resulting in the release of ammonia. Ammonia induces precipitation of the calcium and magnesium inside the biofilm, leading to encrustation and catheter blockage [50].

Bacterial biofilms are perfect structures for plasmid DNA horizontal transfer, which occurs more easily between cells in biofilms than between planktonic cells because of the tighter cell-to-cell contact [51]. Quorum sensing also plays an important role in attachment or detachment of the biofilm [52].

#### **4. Industrial area where the environmental bacterium KCM-R5 was isolated**

KCM-R5 is an environmental bacterial isolate collected from a Pb-Zn smelter area and successfully entrapped in a synthetic polymer – poly(ethylene oxide) hydrogels (PEO) [53]. PEO hydrogels are macroporous polymers with high molecular weight and *Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

appropriate for bacterial immobilization due to their biocompatibility, strength and adhesiveness [54]. Additionally, they demonstrate nontoxicity, flexibility and durability. Initially, PEO hydrogels were obtained *in situ* by γ-irradiation of aqueous solutions [55], and two decades later, *via* methods based on chemical crosslinking [56]. UV crosslinking at cryogenic temperatures contributes to an important feature of the PEO hydrogels, namely the formation of macroporous structure. This macroporous structure is highly compatible with bacteria and enable their easy penetration, movement, hence, biofilm generation inside the hydrogels. The second main advantage of poly(ethylene oxide) hydrogel synthesis under cryogenic conditions than at room temperature is the extraordinarily high yield of gel fraction and better crosslinking [55, 56].

The environmetal bacterial isolate KCM-R5 was isolated from a soil sample collected at the industrial area of KCM Pb-Zn smelter (plant for production of non-ferrous metals), located in Central Bulgaria, near the town of Plovdiv. This plant is the biggest smelter on the Balkan Peninsula and producer of Pb, Zn, Au, Ag and Pt and their alloys since 1962. At approximately 1 km away is the pesticide factory AGRIA Ltd., founded in 1932. Both plants have been polluting the environment with heavy metals and hydrocarbons for years. Recently, the new wastewater treatment plant operating at KCM has reduced the outflow of polluted water. The produce of both plants is sold on the local market but is mainly exported worldwide. After the isolation, the bacterium was successfully cultivated in nutrient broth and nutrient agar and on selective media containing various heavy metals and 2,4-Dichlorophenoxyacetic acid (2,4-D).

#### **5. Molecular-genetic analysis of KCM R5 bacterial isolate**

A molecular-genetic analysis of the bacterial DNA was conducted aiming the identification of the bacterium. 16S rDNA of the KCM R5 strain was amplified, restricted with the frequently cutting endonuclases *Msp*I, *Hae*III and *Rsa*I (New England BioLabs, UK) and sequenced. PCR amplification was performed using the primers 8F (forward) (5'-AGAGTTTGATCCTGGCTCAG-3′) and 1513R (reverse) (5'-GGTTACCTTGTTACGACTT-3′). This primer pair is preferable because it generates the longest amplicon ofapproximately 1400 bp. The amplification protocol consisted of one cycle of initial denaturation at 95°C for 3 min, 35 cycles of DNA denaturation at 94°C for 90 sec, primer annealing at 55°C for 40 sec, and primer extension at 72°C for 1.5 min, ending with a final extension step at 72°C for 20 min. Sequencing was accomplished with an automated sequencer 310 ABI-PRISM (Applied Biosystems, USA). The sequences obtained were analyzed using BLAST program and the bioinformatic analysis showed that the 16S rDNA sequence of KCM-R5 is affiliated with *Pseudomonas rhodesiae* with 99.9% identity. The 16S rDNA sequence of the strain was submitted to the Gene Bank-EMBL Database under the accession number AJ 830707. **Figure 6** presents the dendrogram of the strain *P. rhodesiae* KCM-R5 (Gamma- Proteobacteria) with its closely related relatives.

Members of the genus *Pseudomonas* are heterotrophs, rod-shaped, psychrotrophic and motile. According to Gram staining, pseudomonads are Gram-negative. Gram staining of the bacterial isolate KCM R5 shown in **Figure 7** demonstrated that it is a Gram-negative bacterium.

Ubiquitous in nature, the size of the bacteria of genus *Pseudomonas* varies between 1 and 5 micrometers in length and 0.5-1.0 micrometers in width. Bacterial flagella and pilli are important for the adhesion process. Pseudomonads are known to produce a vast amount of extracellular polysaccharides (EPS) [57]. They are able to produce biofilms even on smooth stainless steel surfaces, multiplying alone in the biofilm or co-existing with other bacterial species [58]. The biodegradation of phenol in wastewater by immobilized cells of *Pseudomonas putida* was described by [7, 59, 60].

When pseudomanads exist in mixed biofilms, they are more stable. In such biofilms *P. aeruginosa* or *P. fluorescens* synthesize a blue toxic substance called pyocianin (**Figure 8**) able to kill bacteria competing pseudomonads [27]. Norman et al. [61] demonstrated that

**Figure 6.** *Dendrogram of the strain Pseudomonas rhodesiae KCM R5.*

**Figure 7.** *Gram staining of Pseudomonas rhodesiae KCM R5.*

*Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

**Figure 8.** *Structural formula of pyocyanin.*

pyocyanin influenced the functional diversity of a crude oil-degrading culture containing *P. aeruginosa and* affected the overall degradation of the crude oil.

#### **6. Heavy metal tolerance and growth of Pseudomonas rhodesiae KCM R5 on phenol and phenol derivatives as planktonic cells**

The tolerance of planktonic cells of *P. rhodesiae* KCM-R5 to phenol and phenol derivatives was studied by cultivation of the strain on phenol, *o*-nitrophenol, pentachlorophenol, 2,4-dinitrophenol, 2,5-dinitrophenol and 2,4-dichlorophenoxiacetic acid (2.4-D) added to mineral media of Furukawa and Chakrabarty [62]. The medium contained per liter 5.6 g К2HPO4x3H2O, 3.4 g КH2PO4, 2 g (NH4)2SO4, 0.34 g MgCl2x6H2O, 0.001 g MnCl2x4H2O, 0.0006 g FeSO4x7H2O, 0.026 g CaCl2x2H2O and 0.002 g Na2MoO4x2H2O. Phenol was applied at a concentration of 100 mg L−1 while its five derivatives were added at a lower concentration of 20 mg L−1 due to their higher toxicity and carcinogenicity, which may cause bacterial cells death. Xenobiotics were metabolized as a sole carbon source with no glucose or other carbohydrate addition. The investigation was performed for 144 h at 28°C. **Figure 9** shows

the growth of *P. rhodesiae* KCM R5. The strain demostrated the most intensive growth on 2,5 - dinitrophenol, 2.4-D, and pentachlorophenol (**Figure 9**).

#### **7. Cryogels preparation**

PEO cryogels necessary for bacteria entrapment were kindly supplied by Prof. Petar Petrov, DSc, Institute of Polymers, Bulgarian Academy of Sciences. Polyethylene oxide was dissolved in distilled water and polymerized by adding a photo initiator (4-benzoylbenzyl) trimethylammonium chloride. The obtained solution was poured into Teflon dishes forming layers 50 mm in diameter. The layers were further placed at -20°C for 2 h and irradiated with UV–VIS light for 2 min. PEO cryogels were extracted in distilled water for 7 days and freeze dried at -55°C, adopted from Doycheva *et al*. [55]; Petrov *et al*., [56], Satchanska *et al*., [63], Berillo *et al*., [54].

#### **8. Entrapment of the bacteria into the PEO cryogels**

The dried PEO cryogels were swelled by soaking without shaking in Furukawa and Chakrabarty medium for 24 h. The strain *P. rhodesiae* KCM R5 was prepared for entrapment in the PEO cryogels by cultivation in Furukawa and Chakrabarty mineral medium with added 0.1% sterile glucose and 100 mg/L phenol until reaching OD 0.550. Then the bacterial culture was mixed with the pre-swollen PEO cryogels and shaken mildly at 100 rpm for 48 h. The resulting PEO-KCM R5 unit consisting of cryogel and immobilized inside bacteria was gently placed inside the sterile Top Filter 45 mm, 500 ml system (Nalgene, Rochester, USA) and the locking rings were softly screwed up in order to avoid cutting of the cryogels, adopted from Satchanska *et al*., [63]; Donelli *et al.,* [64] and Berillo *et al*., [65]. In the control swelled but empty (without immobilized bacteria inside) PEO cryogel was used.

#### **9. Phenol biodegradation by PEO cryogel-***P. rhodesiae* **KCM R5 biofilter and biofilm formation**

The phenol biodegradation by the PEO cryogel-*P. rhodesiae* KCM R5 biofilm occurred *via* a sequencing batch process [66, 67]. The cycle of feeding *via* the upper container (phenol inflow) was 24 h and phenol concentrations was increased from 300 to 1000 mg L−1. Volume of phenol inflow was 250 mL. The experiment was conducted 28°C, in triplicate. Every 24 hours 250 mL sterile medium that contained increasing phenol concentrations on the following scheme: 7 days with 300 mg L−1, 5 days with 400 mg L−1, 4 days with 600 mg L−1 and 12 days with 1000 mg L−1 phenol was poured into the upper funnel. The experiment lasted 28 days. No pressure was applied to the phenol-containing liquid and it run through the PEO cryogel-*P. rhodesiae* KCM R5 biofilm by only its gravity force, adopted by Satchanska *et al*., [63].

Inside the PEO cryogel-*P. rhodesiae* KCM R5 biofilm phenol degradation occurred and the solution of degraded phenol flowed out into the lower container (phenol outflow) [51, 52]. Phenol concentration in both phenol inflow and outflow was

*Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

measured in succession at every 24 hours for a period of 28 days. Assessment of the phenol concentration in both inflow and outflow was carried out by colorimetric method using pyramidone. The protocol can be briefly described as follows: 0.125 ml phenol outflow liquid, 0.250 ml ammonium chloride buffer pH 9,3, 0.125 ml 3.5% pyramidone and 0.375 ml ammonium persulfate pH 7.0 were added to 12.375 ml distilled water to obtain 13 ml total volume. The reaction was incubated at room T o C for 45 min and its absorption was measured with a UV/VIS spectrophotometer at

**Figure 10.** *Phenol degradation by PEO cryogel-P. rhodesiae KCM R5 biofilter.*

**Figure 11.** *Macrostructure of swelled PEO cryogel without bacteria.*

**Figure 12.** *Bacterial P. rhodesiae KCM R5 biofilm0 engeneered inside the biofilter.*

540 nm. In the control, instead of the phenol outflow liquid 0.125 ml distilled water was added adopted by Satchanska *et al*., [63].

The phenol amount and biodegradation was calculated according to a standard curve and phenol biodegradation was calculated according the equation:

$$\text{Efficiency in (X)}\,\text{hour}\,(\text{\(\%\)}=Ci-\text{\(\(\%\)}\,\text{Ci}\times\text{100}\,\tag{1}$$

Data about phenol biodegradation [54–57] by the PEOcryogel-*P. rhodesiae* KCM R5 biofilm is presented in **Figure 10**.

After 28 days of biodegradation, the PEO-KCM R5 biofilter was disassembled and the cryogel with bacteria degrading phenol inside was taken out and subjected to Scanning Electron Microscopy analysis (SEM). The biofilter sample was covered with an Au microlayer and observed at JSM-5510 Scanning Electron Microscope (Jeol, Japan) in vacuum at 10000 V voltage and under different magnifications ranging from x500 to x20 000 (**Figures 11** and **12**).

#### **10. Conclusions**

Our molecular-genetic analysis showed that the environmental bacterium KCM-R5 is affiliated to *Pseudomonas rhodesiae.* The strain is tolerant to xenobiotics and can grow as planktonic cells on phenol and nitro- and chlorophenol derivatives as sole carbon sources. The constructed PEO cryogel-*P. rhodesiae* KCM R5 biofilm is capable of phenol degradation at a concentration of 1000 mg L−1/24 h. Phenol biodegradation is due to the biofilm formed by *P. rhodesiae* KCM R5 inside the PEOcryogel approved by observation using Scanning Electron Microscope. The so engineered PEO cryogel-*P. rhodesiae* KCM R5 biofilm can be used for environmental biotechnology application in industrial wastewater detoxification.

*Growing Environmental Bacterium Biofilms in PEO Cryogels for Environmental Biotechnology… DOI: http://dx.doi.org/10.5772/intechopen.104813*

#### **Acknowledgements**

The author is grateful to Prof. P. Petrov, Institute of Polymers, Bulgarian Academy of Sciences, for supplying the PEO-cryogels and to Prof. Maria Angelova, Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, for the critical reading of the manuscript.

The author thanks RIDACOM Ltd., Bulgaria for the financial support in publishing the chapter.

#### **Conflict of interest**

The author declares no conflict of interest.

#### **Author details**

Galina Satchanska Department of Natural Sciences, New Bulgarian University, Sofia, Bulgaria

\*Address all correspondence to: gsatchanska@nbu.bg

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Theerthankar Das*

Bacterial biofilms are colonies of bacterial cells embedded in their self-produced matrix composed of polysaccharides, DNA, and proteins. They protect bacterial cells against antibiotics, antibacterial agents, soaps and detergents, and shear stress. Some of the most common biofilm-associated infections in humans include urinary tract infections, infection of wounds and surgical sites, diabetic foot ulcers, dental caries (tooth decay) and gingivitis (gum inflammation), ventilator-associated infections, sinusitis, microbial keratitis, secondary infection related to Covid-19 and other viral infections, and so on. Bacterial resistance to common antibiotics (e.g., penicillin, gentamycin, erythromycin, ciprofloxacin, etc.) is driving us to a catastrophic failure of our health systems. Strategies to develop novel antibacterial agents and technology must be prioritized to combat and eradicate biofilms and their associated challenges. This book provides a comprehensive overview of biofilms with chapters on bacterial virulence factors, quorum sensing in bacteria, antimicrobial resistance in bacteria, strategies to develop new antibacterial agents, and much more.

Published in London, UK © 2022 IntechOpen © AIFEATI / iStock

Focus on Bacterial Biofilms

Focus on Bacterial Biofilms

*Edited by Theerthankar Das*