Dear Yangqi Gu,

here is the method how to digest bacterial EPS at physiological temperature.

If You need more, you can contact me.

Sincerely Yours,

Dr. Bratko [email protected]

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ENZYME DIGESTION OF BACTERIAL EPS AT PHYSIOLOGICAL TEMPERATURES

Bacterial strains and growth conditions

Unless otherwise indicated, all reagents were purchased from Thermo-Fisher Scientific and were of the highest purity available. S. epidermidis strain NRS-101 was obtained from the Network on Antimicrobial Resistance in S. aureus (NARSA). All other strains were purchased from the American Type Culture Collection (ATCC). These include two P. aeruginosa strains, 700829 and 700888, both known biofilm production strains; two K. pneumoniae strains, 700603, a multi-drug resistant strain and 700831, a biofilm strain; two A. baumannii strains, BAA-1878 and BAA-1605, a multi-drug resistant strain; and one E. coli strain, 43894, a toxigenic O157:H7 serotype. S. epidermidis strain NRS101 was grown in brain-heart infusion media, K. pneumoniae strain 700603 was grown in Luria broth, and all other strains were grown in tryptic soy broth. Glycerol stocks of all strains were stored at −80°C. Biofilms grown for EPS purification were prepared by inoculating 20 ml of overnight culture into 400 ml of fresh media in a 1.5 L Fernbach flask to provide a large surface area for biofilm adherence. Biofilms were grown at 37°C without shaking for 4–5 days until a thick biofilm “sludge” was observed.

EPS extraction

After development of a mature biofilm, 60 µl of formaldehyde (36.5% solution) was added to each 10 ml of sludge to fix the cells and prevent cell lysis during subsequent steps. The formaldehyde-sludge mixture was incubated at room temperature in a chemical hood with gentle shaking (100 rpm) for 1 hour. Four ml of 1 M NaOH was added for each 10 ml of sludge and incubated at room temperature, with shaking, for 3 hours to extract EPS. Cell suspensions were then centrifuged (16,800×g) for 1 hour at 4°C. The supernatant containing soluble EPS was filtered through a 0.2 µm filter (Corning) and dialyzed against distilled water using a 12–14 kDa molecular weight cut-off (MWCO) membrane for 24 hours at 25°C.

ENZYMATIC TREATMENTS

Enzymes used in the study

Activity of the enzymes, listed in Table 1, on biofilm removal was evaluated for biofilm removal. The proteases were: (1) Savinase, (2) Everlase and (3) Porlazyme. The Amylases were: (4) Amyloglucosidase (AMG) and (5) Bacterial Amylo Novo (BAN). All enzymes used were purchased from Novozymes (Ltd) South Africa. Proteases were diluted in 0.1 M Phosphate buffer, pH 8.3. Bacterial Amylo Novo (BAN) was dissolved in 0.2 M Tris-maleate, pH 7.0; and Amyloglucosidase (AMG) was diluted in Phosphate buffer, pH 5.

Degradation of biofilm EPS

Following protein and carbohydrate analysis, 1 ml of suspended EPS was added into 50 ml centrifuge tubes containing the protease or amylase enzymes diluted in specific buffer solutions. The samples were incubated at 26°C and aliquots were taken at 15 min intervals. For the protease activity, 300 µl of sample was transferred to micro plates and analyzed via the Bradford assay, while the amylase activity was analyzed using the Anthrone assay.

TABLE 1

Enzyme: Optimal pH Optimal

Temperature (oC)

Savinase Protease Novozyme Genetically modified B. clausii pH 8 - 11 Temp. 15 - 75oC Everlase Protease Novozyme Genetically modified B. clausii pH 8 - 11 Temp 15 - 75 oC Polarzyme Protease Novozyme Genetically modified Bacillus spp pH 9-11Temp20-40oC BAN Alphamylase Novozyme Bacillus amyloliquefaciens pH6 - 7Temp. 20 - 60 oC AMG Glucoamylase Novozyme Aspergillus niger pH4 - 5 Temp. 20 - 60 oC

Microtiter assay for efficacy of biofilm removal

The Microtiter assay was performed according to Pitts et al. (2003) with the following modifications; 200 µl of standardized bacterial suspension was added to the wells of a polystyrene microtiter plate (Lasec, S.A.) and incubated at 26°C with shaking at 100rpm for 48 h. Biofilm formation was monitored periodically by visual inspection. After incubation, the supernatant was discarded and plates were washed three times with 200 µl sterile distilled water to remove non adherent bacterial cells. To each well, 1 U/ml and 2 U/ml of proteases and amylases were added. A well without enzymes was used as control. Plates were incubated for 1 h at 26°C. Following incubation, plates were emptied and washed twice with sterile distilled water. The remaining cells were fixed with 200 µl of 95% ethanol for 15 min and allowed to dry. Crystal violet solution (200 µl) was added into each well for 30 min. Plates were washed five times with sterile distilled water. Wells were washed with 30% glacial acetic acid (200 µl) (Merck, S.A.). Plates were read at 595 nm using a Multiskan Ascent ELISA plate reader (Termo Labsystems). The experiment was performed in duplicate. The micro titer screening method was used to quantitatively measure the removal efficacy of proteases and amylases on biofilms of P. fluorescens.

A measure of efficacy called Percentage Reduction by Pitts et al. (2003) was used to evaluate the efficacy of these enzymes.

Percentage reduction = [(C –B) – (T – B))/ (C – B)] × 100%

Where: B denotes, the average absorbance per well for blank (no biofilm, no treatment); C denotes the average absorbance per well for control wells (biofilm, no treatment) and T denotes the average absorbance per well for treated wells (biofilm and treatment

Some EPS (esp. pseudomonad alginic-acid based biofilms) are disaggregated with EDTA.

Following