Porcine mucin

Murine peritoneal sepsis models with A. baumannii ATCC 17978, P. aeruginosa PaO1 or E. coli ATCC 25922 strains were established by i.p. inoculation of the bacteria^{44 (link)}. Briefly, mice were inoculated with 0.5 mL of the bacterial suspensions, which were incubated for 20–24 h in LB at 37 °C and mixed in a 1:1 ration with a saline solution containing 10% (w/v) porcine mucin (Sigma, Spain). The minimal bacterial lethal dose 100 (MLD100), LD50 and LD0 were determined by inoculating various groups of mice (6 mice per group) with decreasing amounts of A. baumannii ATCC 17978, P. aeruginosa PaO1 and E. coli ATCC 25922 strains inocula from 8.5 to 2.3, 9.11 to 3.3, 9.13 to 3.47 log CFU/mL, respectively, and monitoring the survival of the mice for 7 days.

Thymol (L33300) was procured from the El-Gomhouria pharmaceutical company, Zeitoun, Cairo.
Eudragit RL-30D^® and Eudragit RS-30D^® were kindly donated by Evonik Rohm GmbH, Pharma Polymers, Germany. Kolliphor^® P188 (P-188), acetone, porcine mucin, and Mesocell (methyl cellulose) were purchased from Sigma-Aldrich Chemical Co., St. Louis, MO, USA. Methanol was purchased from Fisher Scientific, Leicestershire, UK. Cetyl trimethyl ammonium bromide (CTAB) was procured from Loba Chemie, Mumbai, India. Other chemicals were of analytical grade.

Akkermansia muciniphila (ATCC BAA-835, Guangdong Microbial Culture Collection Center) was cultured anaerobically in brain-heart-infusion (BHI) broth (BD Bioscience, San Jose, CA) supplemented with 0.5% porcine mucin (Sigma–Aldrich) and 0.05% cysteine (Sigma–Aldrich) at 37°C for 2–3 days [70 (link)]. Before Akkermansia transplantation, all rats were fed adaptively according to the above conditions for 1 week. After that, the rats were randomly divided into three groups: VDN (n = 6), VDN+ hk-AKK (n = 6) and VDN + AKK (n = 6). The way that VDN was used to induce vascular calcification in rats was the same as the first modelling method, in which rats received vitamin D3 (300,000 IU/kg, Sigma Aldrich) by intramuscular injection and nicotine (25 mg/kg, Merck Millipore) by gavage at the same time and then nicotine by gavage again 9 h later. Starting the day after modelling, on a daily basis, rats in the VDN + AKK, VDN + hk-AKK and VDN groups received intragastric administration of 5×10⁹ cfu/200 μL active Akkermansia, heat-killed Akkermansia of an equal volume or isovolumetric sterile PBS (1×) each time for 8 consecutive days. In the VDN + hk-AKK group, Akkermansia were heat killed at 121°C under 225-kPa pressure for 30 min.

Rotigotine was purchased from Shanghai PI Chemicals Ltd., Shanghai, China and Sigma-Aldrich, St. Louis, MO, USA. Chitosan, Sodium Tripolyphosphate (TPP) and Porcine mucin were purchased from Sigma–Aldrich, St. Louis, MO, USA. Glacial acetic acid, methanol, and ethanol was purchased from Biotek Abadi Sdn, Bhd, Selangor, Malaysia. All other reagents used were of analytical grade.

Eight- to twelve-week-old female BALB/c mice were purchased from Institute of Biochemistry, Life Science Center (Vilnius University, Vilnius). The animals were maintained and used in accordance with the recommendations of the directive 2010/63/EU of the European Parliament and of the Council of 22 September, 2010 on the protection of animals used for scientific purpose. Study was performed under permission of Lithuanian State Food and Veterinary Service No. G2-72.
A sepsis model was established as described previously (Huang et al., 2014 (link)). Briefly, A. baumannii cultures were grown in LB medium for 18 h at 37°C and adjusted to the designated concentrations with PBS according to the OD₆₀₀ values based on previously determined concentrations by seeding serial dilutions and counting CFUs. Samples were prepared by mixing the bacterial suspension with 5% of porcine mucin (w/v; Sigma-Aldrich). Mice were injected intraperitoneally with 0.5 mL of the sample. CFUs corresponding the bacterial loads were determined by plating sequential dilutions on LB plates. Bacterial colonies obtained from animal sources were confirmed by PCR with A. baumannii-specific primers (Supplementary Table S2).

The N-acetylglucosamine concentration in the reaction mixture and the chitinase activity were determined by previously established Di-nitrosalicylic acid (DNS) method [20] . Chitinase activity was assayed here by estimating reducing sugars by measuring the OD at 540 nm. Varied concentration of purified rabbit [19] (link) and porcine mucin (Sigma, St. Louis, MO) were incubated with 50 µg/ml purified ChiA2 in phosphate buffer pH 7.4 for 3 h at 37°C. The reaction was stopped by adding DNS solution. The mixture was boiled at 100°C for 10 min and cooled by keeping it in ice immediately after boiling. The amount of reducing sugar was then estimated spectrophotometrically. The specific activity of the enzyme was calculated by measuring the amount of GlcNAc produced in µmole/mg of protein/min. The velocity of each reaction was calculated by measuring the amount of GlcNAc produced in µmole/ml of reaction mixture/min. The Michaelis Menten constant K_m and the maximum velocity V_max were calculated from a Lineweaver Burk plot. The catalytic constant K_cat and the catalytic efficiency (K_m/K_cat) were also determined. Detailed methods are provided in File S1.