BIOFILM……

4. BIOFILM

4.1 Introduction

Biofilm is defined as a structured community of microorganisms

(bacteria, fungi, algae, and protozoa) wherein the cells are enclosed in a self-

produced polymeric matrix and adherent to an inert or living surface (Davey and

O'toole, 2000; Götz, 2002). This constitutes a protected mode of growth that

allows survival of bacteria in a hostile environment, particularly in high shear

environments (i.e., rapidly flowing milieus). Many microorganisms grow on

surfaces in well protected manner, in a closed structure hence, unaffected even

when high doses of antibiotics are used. Staphylococcus biofilm has been

extensively studied in human medicine and this pathogen is considered

significant in both device associated infections and tissue infections such as

pneumonia and osteomyelitis. In dairy cattle, the prevalence of bovine

staphylococcal mastitis associated with biofilm ranges from 7% to 40%. When

biofilm is formed in a low shear environment, they are generally more sensitive

to mechanical breakage. Biofilm-associated bacteria show an innate resistance to

antibiotics, disinfectants and clearance by host defence mechanisms.

4.2 Review of literature

4.2.1 Biofilm Structure and Composition

In harsh environment, biofilm tends to be the most important necessity in

protecting microbes as it proves to enable them to survive and disperse

(Melchior et al., 2009). The way in which biofilm spreads in an environment

relies on the movement of biofilm on a surface. The below diagram, Fig 4.1.,

depicts the different movement of biofilm on a surface like rippling, streaming,

rolling and seeding dispersal.

Fig 4.1: Mobility of biofilm

Biofilm is composed primarily of microbial cells and external

polysaccharide (EPS). This primary matrix material is accounting for 50% to

90% of the total organic carbon of biofilm (Rachid et al., 2000a). EPS may vary

in chemical and physical properties, but it is primarily composed of

polysaccharides. Some of them are neutral or polyanionic, as is the case of

gram-negative bacterial EPS. The presence of uronic acids (such as D-

glucuronic, D-galacturonic, and mannuronic acids) or ketal-linked pryruvates

confers the anionic property. This property is important because it allows

association of divalent cations such as calcium and magnesium, which have

been shown to cross-link with the polymer strands and provide greater binding

force in a developed biofilm. In the case of some gram-positive bacteria, such as

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the staphylococci, the chemical composition of EPS may be quite different and

may be primarily cationic. Scientists found that the slime of coagulase-negative

bacteria consists of a teichoic acid mixed with small quantities of proteins.

EPS may associate with metal ions, divalent cations, other

macromolecules (such as proteins, DNA and lipids). EPS production is known

to be regulated by nutrient status of the growth medium; excess available carbon

and limitation of nitrogen, potassium, or phosphate promote EPS synthesis.

Slow bacterial growth will also enhance EPS production. Because EPS is highly

hydrated, it prevents desiccation in some natural biofilm. EPS may also

contribute to the antimicrobial resistance properties of biofilm by impeding the

mass transport of antibiotics through the biofilm, probably by binding directly to

these agents (Gotz, 2002). Structure may also be influenced by the interaction of

particles of nonmicrobial components from the host or environment. For

example, erythrocytes and fibrin may accumulate as the biofilm forms. S. aureus

biofilm is a complex composition consisting of PIA (Polysaccharide

intercellular adhesion), proteinaceous factors and eDNA (extracellular DNA).

4.2.2 Biofilm Formation

Biofilm formation is not restricted to certain group of microorganisms.

The various microbes involved in biofilm formation are Arcanobacterium

(Actinomyces) pyogenes, S. aureus, S. hyicus, Str. agalactiae, Corynebacterium

renale, or Corynebacterium pseudotuberculosis (Melchior et al., 2009; Quoc et

al., 2006; Yazdani et al., 2006).

Biofilm grows in a three stage process as shown in Fig 4.2. The initial

stage includes the attachment of bacteria to the substratum facilitated by cell

wall associated adhesions followed by surface motility and binary division result

in an aggregation of attached cells (Stoodley et al., 2010).

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The second stage is characterized by cell multiplication and formation of

a mature structure consisting of many layers of cells, connected to each by

extracellular polysaccharides. Bacterial growth and division then leads to the

colonization of the surrounding area and the formation of the biofilm.

Fig 4.2: Stages of biofilm formation

The third stage involves maturation of biofilm, where many staphylococci

generate a glycocalyx, a slime layer that further protects the biofilm bacteria.

Mature biofilm, as shown in Figure 1, is a complex heterogeneous structure of

dormant and actively growing bacteria colonies along with further enzymes,

excretory products and small channels forming part of the overall structure.

When the biofilm reaches a critical mass, a dynamic equilibrium is reached at

which the outermost cell layer begins to generate planktonic organisms. These

bacteria are free to escape the biofilm and to colonize other surfaces (Saldarriaga

Fernandez et al., 2010).

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4.2.3 Molecular level / QS system

To form an integral part of biofilm, a bacterial cell needs to communicate

with the population and generate a proper cell density for attachment. This is

achieved by means of extra cellular signal molecules which are already present

in the environment and act as auto inducers to start genetic programs (Camilli

and Bassler, 2006). This intercellular communication principle is used by many

bacterial species to monitor cell density, hence known as Quorum-sensing (QS),

and allows individual cells to behave as a community (Schauder and Bassler,

2001). Thus, quorum-sensing is important both to start biofilm formation and to

maintain the biofilm.

Quorum-sensing is regarded as key mechanism in biofilm development

and auto inducers involved offered an attractive new target for non-antibiotic

control of biofilm infections (Kjelleberg and Molin, 2002). Autoinducers action

in different bacterial species and the complex interaction between autoinducers

of different species demonstrated that the goal of controlling biofilm infections

is not easily achievable.

4.2.4 Genetics of biofilm formation

The formation and development of biofilm occurs in a sequential manner

certain genes coding for EPS needs to be turned on and also certain enzymes and

proteins are involved in each stage of biofilm development. The composition of

exopolysaccharide or extra cellular polymeric substance (EPS) is complex

containing mainly of cellulose and β-1, 6-linked N- acetylglucosamine. Their

synthesis is highly regulated by secondary signal of c-di-GMP whose

concentration in turn depends on the activity of proteins containing a

GGDEF/EAL domain. This protein is observed to be a common factor in

biofilm development (Lasa, 2006).

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Fig 4.3: Scheme of typical biofilm formation steps (A). General

mechanism of the biofilm control (B). Biofilm control molecules action

(Milner et al., 1997)

The first stage in biofilm development is attachment of planktonic cells on

to a surface involving certain surface proteins; the first member to be identified

was Bap (biofilm Associated Protein), from S. aureus in bovine mastitis. The

Bap gene encoding the surface protein was carried in a putative composite

transposon inserted in a mobile Staphylococcal pathogenecity island and was

found only in bovine mastitis isolates. It was apparently present in only 5% of

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350 bovine mastitis and absent in all human clinical S. aureus isolates tested so

far. It was also noticed that Bap positive isolates showed high resistance to

antibiotic treatment.

Studies on S. aureus biofilm development suggested that clinical S.

aureus isolates are not dependent only on PIA but also on non-polysaccharide

component of intracellular matrix. Certain protein mediated biofilm formation

has emerged as an alternative to PIA and many surface adhesions, such as Bap

(Cucarella et al., 2004a), Spa, FnBPA, FnBPB, and SasG. It was also seen that

polysaccharide degrading enzymes, such as dispersin B and mutation in the ica

gene locus that generates PIA are least effective on PIA-independent strains

(Ziebuhr et al., 2000).

The ica ADBC operon which encodes PIA was first identified in S.

epidermidis and is also present in S. aureus and other Staphylococcal species

(Fitzpatrick et al., 2005; Lasa, 2006; Yazdani et al., 2006). A linear homoglycan

of β-1, 6-linked N-acetylglucosamine named PIA has been purified and

characterized as the main exopolysaccharide compound of S. epidermidis

biofilm matrix. PIA related exopolysaccharide PNAG, which resembles in

structure with that of PIA was described in S. aureus. Moreover, it has been

shown that PIA and PNAG are structurally and immunologically identical and

that both are synthesized by the action of four homologous proteins (IcaA, IcaD,

IcaB, IcaC) encoded by the genes organized in a single operon (icaADBC)

(Cucarella et al., 2004b; O'Gara, 2007; O'Neill et al., 2007; Tormo et al., 2005).

An overview of PIA biosynthesis by the ica operon is given in Fig: 4.4 a & b.

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Fig 4.4a: Representation of the ica Operon

Fig 4.4b: Overview of PIA biosynthesis by ica operon

4.2.5 Identification tests

4.2.5.1 Tissue culture method

Tissue culture method was discovered by Christensen et.al in 1985. Since

this method has been most widely used and considered as standard test for

biofilm formation. The organisms grown in trypticase soy broth with 1%

glucose were tested for biofilm expression. The biofilm attached to the bottom

of the plate were fixed 2% sodium acetate and stained with 1% crystal violet.

Optical densities of the stained biofilm were then obtained at 570nm using

ELISA autoreader.

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4.2.5.2 Tube method

Tube method was discovered in the year 1982 (Goldstein and Roberts,

1982). In this method, ability of S. aureus adherence to the walls of borosilicate

glass tube is noted. This method is considered to be the most primitive and first

method to check for biofilm forming strains of S. aureus and practiced till now

in diagnostic laboratory. The tubes are dried and stained with 0.1% crystal

violet. Visible stained film was seen on the tube wall and also on the bottom of

the tube. Ring formation observed at the broth interface is not considered as

positive result for biofilm formation.

4.2.5.3 Congo Red Agar test

Phenotyping the pathogenic and biofilm producing S. aureus can be

performed by Congo Red Agar test. The slime producing phenotype appears as

black colonies and can be differentiated from other non slime producers

visually. This is an easy and cost effective method and can be performed

routinely in microbiological laboratory to identify biofilm producing pathogenic S. aureus.

4.2.6 Molecular Screening

Gene level identification of biofilm forming strains of Staphylococcus

aureus is the most confirmatory test and relevant, unlike qualitative tests like

Congo red agar method which at times shows false positive results. Candidate

genes like icaD and icaA representing ica operon encoding the biofilm pathway

enzymes can be targeted in molecular screening. By quantitative PCR, the

expression level could be measured and the effect of the drug could be

evaluated. This is important since, the biofilm level is increased when the

organism is under stress like exposure to antibiotic.

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4.2.7 Eradication / elimination

Elimination of bovine mastitis is very problematic, firstly, as it is an

endemic disease i.e. a disease that is constantly present to a greater or lesser

degree in a population of a certain class or in population living in a particular

location. And secondly, apart from this there are no tests available which can

detect the mastitis disease at the early stage in single step. Visualization of the

udder and milk is not the confirmatory test for Mastitis. Depending on timing of

infection, eradication of S. aureus infection can be achieved. But during

lactation the antibiotic treatment is avoided and many of the S. aureus affected

cows become chronic and have to be culled. Preventive measures can be

adopted for the contagious and environmental mastitis.

4.2.7.1 Resistance to antimicrobials and predator living cells

Biofilms are more resistant to antibiotics compared to planktonic bacterial

cells. The cause of the resistance is not yet completely understood but several

hypotheses were proposed. One of the earliest hypotheses was about the

inability of the antibiotics to penetrate the biofilm matrix, which may contribute

to antibiotic resistance. Other hypotheses suggested that antimicrobials are

degraded in the biofilm before showing its effect, like, penicillin being degraded

by penicillinase enzyme.

Apart from biofilm being resistant to synthetic chemical or antimicrobial

compounds, they are also found to be resistant to protozoa or phagocytic blood

cells (Paape et al., 2000). The Fig 4.5. shows neutrophil attacking the biofilm

mass by grazing through the surface, where the cells located deep inside the

biofilm remain unaffected and replace the lost cells thus, maintaining the

integrity of the biofilm.

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Fig 4.5: Neutrophils on surface of biofilm.

4.2.7.2 Antibiotics and biofilm: A strategy to overcome difficulties

For the eradication of biofilm formed by various microbes, a wide range

of antibiotics are used. But it is observed that plank tonic cells showed

sensitivity than biofilm, thus it necessitated to develop drugs that targeted that

quorum sensing system and disrupted the formation and activation of auto

inducers. Olsen et.al used 12 different antibiotics to check their effect on biofilm

composed of Arcanobacterium (Actinomyces) pyogenes, S. aureus, S. hyicus,

Str. agalactiae, Corynebacterium renale, or Corynebacterium

pseudotuberculosis microbes.

4.2.7.3 Combo drug

Combo drug denotes different combination of antibiotics used to eradicate

biofilm formed during Mastitis. This mechanism is possible due to synergy

between the antibacterial agents. Scientists observed that combination dosage of

farnesol and gentamicin treated on static biofilm depleted bacterial population

by more than 2 log units

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4.3 Materials and Methods

4.3.1 Materials

The Staphylococcus culture (Staphylococcus aureus-MTCC3160)

obtained from Microbial Type Culture Collection and Gene Bank, Institute of

Microbial Technology, Chandigarh was used as positive control for the analysis

of biofilm expression.

4.3.1.1 Congo Red Agar

Composition

Congo Red

Muller

Agar

Sucrose

Agar

gm/l

0.8

Hinton 21

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15

The final volume was made up to 1l and the pH was adjusted to 7.4 ± 0.2 at

250C. Sterilization was carried out by autoclaving at 1210C for 15 min.

4.3.1.2 Reverse Transcriptase - Polymerase Chain reaction

Random primer 1.25mM/µl,

dNTPs 2.5mM

MMLV Reverse transcriptase 40units/µl

10 picomoles of forward and reverse primers,

1.25 units of Taq

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4.3.2 Methods

The biofilm phenotypic expression of S. aureus isolates were evaluated by

Congo red agar and quantified. Total RNA was extracted from the bacterial

cultures and subjected to reverse transcriptase PCR using icaD specific primer

representing the biofilm synthesis by the ica operon.

4.3.2.1 Congo Red Agar

The mannitol salt agar positive colonies screened for biofilm producing

capacity by Congo Red Agar. Congo red agar was prepared by adding the dye to

Muller Hinton agar. The test organisms were streaked onto Congo red agar

plates and incubated at 370C for 24 h. The biofilm producing colonies exhibited black colonies while those failed were of yellow color colonies.

4.3.2.2 Quantification of Biofilm

S. aureus culture was initiated with 50µl of 104CFU cells in a 96-well

polystyrene plate containing 150µl of Congo red broth and incubated for 24h at

370C without shaking. After 24h, the plates were washed vigorously three times with PBS and dried for 1h at 56˚C prior to staining with a 0.4% crystal Violet

solution. The absorbance of the adhered, stained cells producing biofilm was

measured at 492nm using Multiskan plate reader (Thermo systems, USA).

4.3.2.3 RNA isolation

Total RNA isolation was performed by TRIZOL method with slight

modifications. The frozen bacterial cells were thawed to room temperature and

centrifuged at 10000X for 10min at 4˚C. The supernatant was discarded and the

cell pellet was resuspended in 150µl of trizol. The cell was homogenized using

Tissue Tearor (Biospec Products INC, USA) at 30,000rpm for 30 sec and

incubated at room temperature for 5 min. To this, 100µl of Chloroform was

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added and centrifuged at 13,000X for 20 min. The upper aqueous phase was

transferred to a fresh 1.5ml micro centrifuge tube and added equal amount of

isopropanal. Incubated at -200C for one hour, then centrifuged at 13,000X for 15

min at 40C. The supernatant was discarded and the pellet was washed with 500µl

of 75% ethanol by centrifugation at 10,000X for 5min at 40C. The supernatant

was discarded and the pellet was dissolved in 20µl of double autoclaved water.

The quality and quantity of the isolated RNA was checked by 2% agarose gel

electrophoresis.

DNA contamination if any seen with RNA preparations was removed by

the DNase treatment. The reaction volume was set up to 20µl containing 9U of

DNase. It was incubated at 370C for 30-45 min then 20mM of 2µl EGTA was

added and further incubated at 650C for 10min. Sodium acetate (1/10 V) and

absolute ethanol (2V) was added and incubated at -20˚C for 1 h. Then it was

centrifuged at 12000xg for 20 min at 40C, the supernatant was discarded and the pellet was washed with 500µl of 75% ethanol. Air-dried pellet was dissolved in

20µl of DEPC treated water and used for RT-PCR.

4.3.2.4 Expression analysis of ica Operon

The biofilm expression by S. aureus cultures was analyzed by amplifying

the ica D mRNA representing the ica Operon gyrB as housekeeping gene. The

primers for icaD and gyrB genes of S. aureus were designed based on the

available sequence (Gene Bank Accession no: NC_002592) using Genetool lite

software (Gene twist, USA). Total RNA was converted into cDNA using a

reaction mixture containing MMLV Reverse Transcriptase, RNase Inhibitor,

Random hexamer as primers, dNTPs and 1X Buffer. The cDNA synthesis was

carried out at 420C for 60 min and extension at 750C for 10 min. The prepared cDNA was used as template to amplify gyrB and ica D genes. The primers

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gyrBF and gyrBR amplified a 232 bp of gyrB gene while icaDF and icaDR

primers amplified a 238 bp product of icaD gene. The temperature profile of

icaD gene was: initial denaturation for one cycle at 940C for 2 min, followed by

30 cycles of 940C for 15 sec, the annealing at 52.5ºC for 15 sec, extension at

720C for 30 sec and a final extension for one cycle at 720C for 1 minute. The

same temperature profile was used for gyrB gene except that the annealing

temperature was at 630C. The amplicons were purified with a gel extraction kit (GF-1 GEL DNA recovery Kit, Vivantis, Singapore) and subjected to

sequencing in both directions using big dye terminator-sequencing kit (Applied

Biosystems, USA).

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4.0 Results

The S. aureus isolates biochemically confirmed were further evaluated for

their biofilm expression. The phenotypic confirmation was obtained by congo

red agar and at molecular level, the ica operon encoding the genes responsible

for the biofilm synthesis was screened.

4.4.1 Congo Red Agar

In this selective medium, congo red in muller hinton agar interacts with

biofilm and generates black colonies. The S. aureus isolates that are negative for

biofilm expression exhibit pale yellow colonies. Out of 180 confirmed S. aureus

isolates 101 (56%) were found to express biofilm (Table 4.1 and Fig. 4.6). This

constitutes 31.5% of the total mastitis samples. Among the very few S. aureus

isolates obtained from normal milk samples, the two isolates from HF breed

were found to have biofilm expression. Others were negative for biofilm

synthesis.

4.4.2 Expression analysis of ica Operon encoding biofilm

The enzymes in major synthetic pathway for biofilm are encoded by ica

iperon which is comprised of four genes namely ica A, D, B & C. The ica D was

considered as representative gene for the ica operon and analyzed by RT-PCR

among the S. aureus isolates. The expression of icaD was compared with that of

the housekeeping gene gyrB as internal control. Out of 101 isolates confirmed

by congo red agar, 88 (87%) were found to express icaD mRNA (Table 4.1 &

Fig.4.7). This is about 49% (88/180) of the S. aureus isolates and 27.5% of the

study sample size of 320 milk specimens. The S. aureus isolates obtained from

the normal HF milk samples were positive for icaD expression.

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Group Breed Diagnosis +ve Congo Red ica D expression

Table 4.1: Gene expression analysis on S. aureus Biofilm

S. aureus

samples*

Agar

n % n %

Mastitis

HF

Clinical 82 53 65% 47 57%

Sub clinical 24 7 29% 6 25%

Jersey

Kangayam

Normal HF Jersey

Kangayam

Clinical 48

Sub clinical 5

Clinical 19

Sub clinical 2

2

1

1

28 58%

2 40%

10 53%

1 50%

2 100%

0 0%

0 0%

23 48%

2 40%

9 47%

1 50%

2 100%

0 0%

0 0%

* ‐ Number of samples positive for S. aureus as described in Table 3.3.

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4.5 Discussion

Many common bacterial pathogens exist in animals as biofilm and their

infections are generally chronic and often difficult to treat. Biofilm associated

microorganisms behave differently from planktonic organisms with respect to

growth rates and ability to resist antimicrobial treatments and therefore pose a

major health problem. S. aureus with phenotypic expression of biofilm is known

for its disease severity with increased resistance to the antibiotics and blocking

the lumen of the duct. The synthesis of biofilm is by two different pathways, ica

operon dependent and independent (Gotz, 2002).

Earlier studies have reported that 20-30% of mastitis is associated with S.

aureus infection. In this study 320 milk samples from clinical and subclinical

mastitis cows were screened for S. aureus infection. The conventional

microbiological and biochemical methods employed confirmed 56% of them

positive for S. aureus. The selection medium, congo red agar identified 31.5% of

the S. aureus isolates exhibiting biofilm. The molecular screening recognized

27.5% of the samples had biofilm expressed by functional ica operon. The

incidence recorded by this study is in accordance with the published reports.

The biofilm produced by S. aureus confers antibiotic resistance, blocking

the secretion duct and the inflammation, emphasizing the fact that preventing the

biofilm synthesis is the primary requirement in controlling S. aureus infection

associated mastitis. Since, synthesis of biofilm depends on two different

pathways, it is important to consider the genes associated with the pathways. On

evaluating the role of the candidate genes belonging to the pathway in a given

geographical location, drugs could be developed/identified and targeted.

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Bacteriological cure rate for the treatment with antimicrobial agents is

ranging between 0% and 80% as indicated by earlier epidemiological studies.

Bacterial species with biofilm synthesis exhibit an innate resistance to

antibiotics, disinfectants and clearance by host defense mechanisms. Despite its

higher incidence, expression of the ica locus and biofilm formation, seems to be

highly variable among staphylococci isolates. Sub-inhibitory concentrations of

tetracyclines and quinuprestin-dalfopristin, as well as high temperature and

osmolarity increase the ica promotor activity. In contrast, penicillin, oxacillin,

chloramphenicol, clindamycin, gentamicin, ofloxacin, vancomycin and

teicoplanin seem to have no effect (Rachid et al., 2000b; Ziebuhr et al., 2000).

Comparatively, bacteria growing in a biofilm can become 10–1000 times

more resistant to the effect of antimicrobial agents than planktonic growing

bacteria of the same strain (Amorenaa et al., 1999; O’Toole, 2003). Several

mechanisms are known to be responsible for resistance of biofilms to

antimicrobial agents, including delayed penetration of the antimicrobial agents

through the biofilm matrix, altered growth rate of biofilm organisms and

physiological changes due to the biofilm mode of growth.

The relapsing nature of biofilm infections can be extrapolated from the

dynamic features of biofilm formation and shedding of cells from one biofilm to

form a new biofilm. Added to this there is a high prevalence of the ica genes

among S. aureus mastitis isolates. This emphasizes the need for extending

antibiotic therapy to interrupt the dynamics of biofilm formation. antibiotics had

a greater effect on young biofilms and those grown in milk, than on older

biofilms and those grown in broth (Amorenaa et al., 1999). These experiments

did show also that gentamicin and erythromycin were the least effective

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antibiotics against S. aureus biofilms. Mastitis pathogens are able to form

biofilms, we must accept that current NCCLS guidelines on testing sensitivity of

pathogens to antibiotics have a poor predictive value, and that current

therapeutic regimes might not be appropriate.