Characterization Of Biofilm Formed By Escherichia Coli

Published Date: 02 Nov 2017

Patients that are subjected to the use of catheters in order to prevent urine disorders or other purposes become prone to urinary tract infection. Thus, the treatment or precautions to such conditions becomes necessary. The relationship between the catheters as a substrate, for inducing Escherichia coli biofilm growth has long been established. But, recent increase in the growth of the pathogenic strains of E.coli has been a concern for researchers, recently. A RRL-36 strain has been observed in recent clinical isolates. But, little is known of the biofilm forming capabilities of the Escherichia coli RRL-36 strain, the study becomes necessary to treat and cure CAUTI.

Introduction

Bacteria tend to colonize the exposed surfaces to aqueous solutions as a mode of survival by the formation of microbial consortia or biofilms. The consortia are formed in a phased manner, beginning with the attachment of single bacterial cells to the surface, followed by cell proliferation and exo-polysaccharide matrix formation. The substrate on which these sessile communities are formed determines the deleterious or beneficial role of these biofilms. When these biofilms are formed on medical devices or insertions they set the trend for the evolution of antimicrobial resistance, as biofilms have been reported to be inert to administered antimicrobials. Also, when a pathogenic strain forms biofilms on the surfaces of the host cell upon entry, it becomes difficult to eradicate them from the system are they are internalized and make the host prone to secondary infections.

Therefore the main way to access the pathogenicity and its contributing virulence factors is by identifying the genetic and the phenotypic differences between the pathogens and the closely related non-pathogenic bacteria. Most of the current insights available on the evolutionary and population genetics of E.coli have been obtained from the studies carried out on the wild strains from healthy hosts. Thus the genetic structure and the level of variation observed among the commensal populations of E.coli have served as the basis to classify the bacteria as pathogenic and non-pathogenic. In this study we aim to characterize the biofilm forming capabilities of the uropathogenic E.coli strain RRL-36, isolated from the urine of a pyelonephritic patient in order to provide an effective insight into its pathogenicity and the mechanism by which the biofilm formation occurs.

Materials and Methods

Bacterial strains utilized: A lyophilized culture of Escherichia coli RRL-36 and Escherichia coli ATCC 25922 was used in this study in a continuous culture.

Biofilm Assay: Biofilm growth with the addition of D-Mannose, in M63 medium at 0%, 0.1%, 0.2%, 0.5%, 1%, 5%. The same concentrations of mannose were considered in LB media. The biofilm formation was monitored by using sterile, non-treated 96-well flat-bottom microtiter plates as described (Pratt et al, 1998). The medium was added as controls in the wells. The wells were inoculated with non-pathogenic E.coli 25922 and with the strain under study RRL-36 without shaking at 37oC for 24hr, 48hr and 72hr. After the incubation period, the plates for each strain were washed 3 times with phosphate buffered saline (PBS) solution (1mM Na2HPO4, 2.7 mM KCl, and 13.7 mM NaCl at pH 7.4) to remove unbound cells. The cells attached to the wall of each well were stained for 20 min with 300µl of 0.1% crystal violet in water and washed again with the PBS solution to remove unbound crystal violet. Bound cells were quantified by adding 300µl of acetone/ethanol (20:80, v\v) and measuring OD540 with a microplate reader. For comparison of biofilm formation among strains in each medium, the total growth was monitored by measuring OD600 in each well, and then normalizing the biofilm growth with its total cell growth values (Rodrigues et al, 2009). The biofilm formation was also accesses by the microtiter biofilm formation assay and subsequent staining using 0.1% crystal violet.

Cell adhesion assays: Cells were made up to a final concentration of 108 cells mL-1 in M63 medium and the assays were carried out in a covered and sterile glass slide. Ten microliters of the suspended cells were further diluted in 290 mL of M63 medium containing 1% D-Mannose. The adhesion rate was calculated every minute by observing the number of attached cells. The number of deposited bacteria was determined by calculating the bacteria adhesion rate coefficient, k, using the equation k = J/C0, where J, is the observed bacteria deposition rate and C0, is the bacterial bulk concentration.

Microbial Adhesion to Hydrocarbon Test: This test is carried out to determine the relative level of hydrophicility (i.e. the fraction of the cells partitioned into the aqueous phase) and hydrophobicity (i.e. the fraction of the total cells partitioned into the hydrocarbon phase) of the strains under study by the MATH test. The cells were resuspended to an OD600 of 0.5 in M63 medium alone or M63 medium supplemented with 1% D-Mannose. The cell suspensions were divided into 4 mL aliquots and 1 mL of glycerol was added to each of these aliquots. The OD600 of the aqueous phase was measured after vigorous vortexing for 30 seconds, followed by a 30 min rest period to allow for phase separation. The MATH value was calculated from the change in the OD600 as follows:

MATH(%) = ((OD600 after treatment) x100)/ (OD600 before treatment)

Carbohydrate Extraction: The carbohydrate is extracted from the catheter containing biofilm and nutrient media. 10ml of the biofilm with the nutrient media is taken and 0.06ml formaldehyde (36.5%) is added for 1 hour at 4oC. 4ml 1N NaOH is added for 3hrs at 4oC and it is centrifuged at 10,000G for 20 min at 4oC, then it is filtered through 0.2 µm membrane at 25oC, then it is purified with dialysis membrane (3500D) at 4oC for 24hrs (Liu et al, 2002). The extracted carbohydrate is concentrated by sucrose concentration technique.

Carbohydrate Quantification: The carbohydrate is extracted from the catheter containing biofilm and nutrient media. 10ml of the biofilm with the nutrient media is taken and 0.06ml formaldehyde (36.5%) is added for 1 hour at 4oC. 4ml 1N NaOH is added for 3hrs at 4oC and it is centrifuged at 20000G for 20 min at 4oC, then it is filtered through 0.2 µm membrane at 25oC, then it is purified with dialysis membrane (3500D) at 4oC for 24hrs (Liu et al, 2002). The extracted carbohydrate was concentrated by sucrose concentration technique.

Protein quantification: Cultures of every 24 hrs are taken till 120 hrs to estimate the proteins developed in the biofilm by pinching the catheter tubes, which would expel out the biofilm from the tube lumen. 1ml of the cell paste was collected on ice and centrifuged at 12000G for 10 min. Further the paste was washed with PBS thrice followed by 5.84% sorbitol thrice. It was resuspended in U9 Lysate buffer (PMSF 2µg/l(m/v), IPG Buffer 0.5%(V/V), DTT 65Mmol/L, CHAPS 4%(m/v), thiourea 2 mol/l, urea 7 mol/l) containing 50µg/ml RNase and 200µg/ml DNase. The biofilm samples were all lysed by sonication on ice using 6, 10 second bursts at 4 W and was centrifuged at 14000G for 30 min at 4oC (Chen et al, 2009).

Electron Microscopy: Visual confirmation of the presence of biofilms on the catheters was obtained by scanning electron microscopy. Sections of catheters (2cm in length), taken from the region just below the retention balloon, were plunged into liquid nitrogen-cooled propane and then transferred to liquid nitrogen. Cross sections were produced by freeze-fracturing and freeze-dried for 24 h at 280°C. These samples were then mounted on aluminum stubs, sputtered with gold, and examined in a JEOL LSM5200 scanning electron microscope. To observe the nature of the biofilm surfaces, sections (approximately 1 cm long) from the region just below the retention balloon were cut longitudinally into halves. They were fixed in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h and then washed overnight in the phosphate buffer before being postfixed in Millonig’s phosphate-buffered osmium tetroxide (1.0%) for 1 h. The samples were dehydrated in a graded series of aqueous ethanol solutions (30 to 100%) and then critical point dried by using liquid carbon dioxide. Finally, the samples were mounted on aluminum stubs sputtered with gold and examined in the scanning electron microscope

GC-MS: The extraction of the biofilm exopolysaccharide for compositional analysis was adapted from the procedures described by (Hebbar et al.1992) and (Liu et al, 2002). E. coli K12 wild type, grown for 24 h at 37oC without shaking, were diluted 1:100 and incubated with 25 g of glass wool in 500 ml of either LB or M63 supplemented with 1%D-mannose for 72 h. After 72 h, all contents were harvested at 5000g for 15 min at 48C. The glass wool and cells were washed three times with 500 ml of 100 mM NaCl. The cells attached to the glass wool were then resuspended in 100 ml of 100 mM NaCl, at which time, 0.6 ml of formaldehyde 37% solution were added and the mixture was gently mixed for 1 h at 48C. Then, 40 ml of 1 M NaOH was added and gently mixed for 3 h at 48C. The mixture was centrifuged at 20,000g for 20 min at 48C. The supernatant was filtered through a 0.22 mm membrane and boiled for 15 min to inactivate any polysaccharide degrading enzymes, after which 1 M NaCl was added. The pH was adjusted to 7.25 with concentrated HCl and two aliquots of ethanol (200 ml) were added to induce overnight precipitation at 48C. The precipitate was recovered by centrifugation at 3000g for 15 min. Polysaccharides were then dehydrated in an alcohol series (60, 70, 80, 95% ethanol). The polysaccharides were then resuspended in 30 ml sterile distilled water and dialyzed with a 3500 Da MWCO dialysis bag for 2 days with water changes every 3 h. The bulk aqueous solution conductivity was measured between every change. At the end of the dialysis, the contents of the dialysis bag were sucrose concentrated and send to VIT Central Instrumentation Facility for GC-MS analysis of the extracted EPS. The analysis consisted of trifluoroacetic acid hydrolysis of the polysaccharides and quantification of the following monosaccharides: L-fucose, D-galactosamine, D-glucosamine, D-galactose, D-glucose, and D-mannose.

Results

The biofilm forming capability of the cultures was accessed in both the media and it was found that the bacteria produced the maximum biofilm formation by adhering to the glass, when compared to the rubber Foley catheters. It was also noticed that the maximum yield of the biofilm formed was up to 72 hours, after which the amount of biofilm formed started to decrease. Upon supplementation of D- Mannose, it was noticed that the amount of biofilm formed gradually arose up to 72 hours after which there was a constant reduction in the formation. Maximum growth was noticed at 0.5% supplementation of D-Mannose. While at all the other concentrations although there was growth at 24 h, this was inhibited upon further incubation. The lyophilized cultures of Escherichia coli RRL-36 were revived in M63 minimal media and in LB broth. The cultures were then confirmed by plating them on EMB agar plates and used for further characterization of the cultures (Fig 1, 2 & 3).

The content of Carbohydrate after incubating the cultures for 72 hours, decreased (Fig 5). The concentration was measured by using phenol-sulphuric acid method using dextrose as the standard an R2 value of .99 was obtained, to estimate the total carbohydrate content at 490nm O.D. in a plate reader and the data was extrapolated. The experiment was repeated 6 times to obtain an accurate value of the amount of carbohydrate formed.

The content of protein was estimated by using Bradford’s method considering BSA as a standard where an R2 value of .99 was obtained at 595 nm. The values obtained were from a plate reader. The experiment was repeated for about 6 times to standardize the data obtained. Here the protein content decreased after 72 hours but the markedly decreased after 96 hours (figure 4).

The Hydrophilicity assay was performed to determine by how much amount the cultures were hydrophilic or hydrophobic in nature. As the cultures became old the cultures became more hydrophobic in nature (figure 6). The level of decrease of hydrophilicity was almost constant.

Figure 1: The above figure shows that after 24 hours of incubation the biofilm formed by the ATCC 25922 strain of Escherichia coli has more amount of biofilm both comparatively in the M63 media and in the LB media than the RRL-36 strain under current study.

Figure 2: Represents that after 48 hours of incubation the ATCC 25922 culture biofilm increased in the M63 media and that of the LB media decreased to some extent. Both the RRL-36 cultures in the LB and M63 media showed marked increase in their biofilm content, but was relatively less than the ATCC 25922 cultures. At 0% mannose concentration the biofilm yield was more in both the cultures of LB media. The RRL-36 cultures formed higher amount of biofilm in the M63 media than in the LB media.

Figure 3: The RRL-36 strains showed maximum formation of biofilm in the M63 media than in the LB media. The biofilm formation was higher in the RRL-36 cultures than in the ATCC 25922 cultures after 72 h of incubation in the M63 media than in the LB media.

Figure 4: Biofilm formed decreased after 72 hours after 48 hours the protein content was stationary but after 96 hours the content decreased distinctly.

Figure 5: The carbohydrate content after 72 hours decreased and in the 120 hour culture the content decreased even more than in the 24 hour culture.

Figure 6: The level of hydrophilicity is observed to be the maximum in the 24 hour cultures and decreased almost at the same rate till 96 hours.

Figure 7: The number of cells adhering per minute to M63 medium.

Discussion

Biofilms have for long been one of the major defence mechanisms of the bacteria for their survival and multiplication in the host. It has been diversely classified as one of its virulence mechanisms, through which a clinical onset of infection has been reported to occur in the case of nasal infections, meningitis, Proteus mirabilis induced urinary tract infections and pulmonary infections. The formation of a biofilm by pathogenic bacteria requires the initial adhesion to a surface followed by the rapid multiplication and finally detachment from the initial adhesion site to form new microcosms. This is aided by the production of exopolysaccharides and the proteins of the host environments.

The weak Vander Waals force and the strong electrostatic repulsion form the basis of the adhesion process in the biofilm formation. Most bacteria differ in their hydrophilicity and their surface charges, which in turn influence their surface interactions. In this study it is noticed that the hydrophilicity of the uropathogenic strain under study was the maximum at 24h (62% hydrophilicity) and started to decrease upon further incubation. This hydrophilicity can be attributed to the presence of 1% D-Mannose in the media, which is synonymous to the uriniary milieu, where the protein present are highly glycosylated and thus frequently aid in the development of adhesive fimbriae, which aid in the development of attachement with the available surfaces especially for bacteria that are grown under static conditions (Rodriguez et al, 2009).

The high hydrophilicity of the bacteria as determined by the MATH test, also provides an indication, that the higher the MATH score, the less hydrophilic the bacterium is and the more the fimbriation bacteria contains. Also the presence of D – Mannose in the medium also decreases the hydrophilicity, thus making the surface of the bacterium less acidic.

During this study it was noticed that the bacterium had a higher affinity to bind to the glass surface when compared to the rubber Foley catheter surfaces, indicating the underplay of the repulsive electrostatic forces. The maximum biofilm formation was noticed at 72 h and the protein content and the carbohydrate content of the extracted biofilm also coincided with this result.

Conclusion

Thus, the biofilm which is formed by the Escherichia coli RRL-36 strain have been characterized on the basis of its rate of biofilm formation, Adhesivity, gradual change in carbohydrate and protein content and its hydrophilicity. Particularly, the activity of biofilm formation for this species of bacterium is found to be different from the model strain of Escherichia coli ATCC 25922, which showed a very steep increase of biofilm formation, after which the amount of biofilm decreased. On the other hand, the biofilm of the RRL-36 strain increased steadily till 72 h after which the amount decreased steeply. Thus, such properties of the biofilm having established, the possible methods to inhibit the same in catheters and other common routes of Urinary tract Infection can also be studied further for this specific strain.