Isolation, Screening and Characterization of Hyaluronidase Producing Bacteria

1. Introduction

      Hyaluronidase (hyase) is a general term initially introduced by Karl Meyer in 1940 to describe enzymes that are able to break down primarily hyaluronic acid (HA) [1]. The degradation of HA produces a disaccharide product. The enzyme acts as an adjuvant, accelerate and increase absorption and dispersion of injected drugs, e.g. antibiotics, to promote resorption of excess fluids and improve the effectiveness of local anaesthesia and to diminish pain due to subcutaneous or intramuscular injection of fluids [2], for hypo-dermoclysis and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents [3]. The enzyme is used as a spreading factor in several medical fields viz. orthopaedia, surgery, ophthalmology [4], dermatology, dentistry [5], oncology [6], gynecology [7] and internal medicine [8]. Bacterial hyaluronate lyases were reported to be virulence factors that facilitate the spreading of bacteria in host tissues by degradation of hyaluronan [9]. Hyase facilitates diffusion of antiviral drugs, dyes and toxins [10]. Based upon the medical, physiological, biological and commercial importance of hyases, authors have screened and isolated a newly promising bacterial strain with higher yield followed by its characteri-zation employing detailed taxonomic studies [11]. Turbidity reduction assay [12, 13] method and determination of hydrolyzed zone of HA were employed for isolation and screening of hyase producing microorganisms.

2. Materials and methods

2.1. Primary screening

      The pathological specimens were collected from different pathological laboratories at Bhubaneswar, Orissa, India. The pathological samples were collected and transferred into sterile screw capped culture tubes containing Stuart's transport medium [14]. Each specimen collected (swab, sputum and blood) was immediately spread on sterile blood agar plates containing an antifungal agent (fluconazole 25 μ g/ml) to prevent fungal contamination and incubated at 37 °C for 24 h. A total of 20 isolates selected on the basis of colony size, shape and extent of hemolysis were screened for hyase activity. The isolates exhibiting good hemolytic zones were aseptically transferred into different 100 ml EM flasks containing 25 ml sterile nutrient broth and incubated at 37 °C for 24 h. Five ml culture broth was then withdrawn, centrifuged at 8000 rpm for 20 min. at 4 °C and clear supernatant collected was subjected to turbidity reduction assay [12, 13] (A₆₀₀ nm by UV-visible spectrophotometer; Systronics, Model-118) and determination of zone of hydrolysis.

2.2. Secondary screening

     The four isolates showing good hyase production were further screened along with reference strain Streptococcus mitis MTCC*2695 (IMTECH, Culture Collection and Gene Bank, Chandigarh, India) by shake flask fermentation method. The selected isolates were sub-cultured onto nutrient agar slants, incubated at 37 °C for 24 h. The growth contents of each slant was transferred into 50 ml of nutrient broth in 250 ml Erlenmeyer flask. The flasks were incubated at 37 °C for 48 h on rotary shaker (Ilshin Lab Co., Korea, Model BBT-1) (150 rpm). The fermentation broth of each flask was centrifuged at 4 °C (8000 rpm) and the clear supernatant was used for the enzyme assay. The experiments were conducted in triplicate and the average values were taken into consideration. The results are recorded in Table 2.

2.3. Hyaluronidase assay

      Hyaluronidase activity was measured spec-trophotometrically by turbidity reduction assay using HA sodium salt (Sigma Aldrich, USA) as a substrate. The enzymatic assay is based on Dorfmans method [15] in which the enzymatic reduction in turbidity, resulting when 1 ml of HA at 70 μg/ml was incubated with 1 ml of enzyme sample in the presence of 0.05 M sodium phosphate buffer with 0.05 M NaCl (pH 7.0). After incubation of the mixture for 30 min., 2.5 ml of acidified protein solution (1% w/v) bovine serum albumin fraction-V (BSA) in 0.5 M sodium acetate buffer, (pH 3.1) was added and incubated at 37 ºC for 10 min. and reduction in turbidity was read by measuring the absorbance at 600 nm.

       One unit of enzyme activity was defined as the amount of enzyme that causes a reduction in turbidity, measured spectropho-tometrically at 600 nm (A₆₀₀) in 30 min. at 37 ºC, at pH 7.0 under specified assay conditions similar to that caused by one unit of an international standard.

T*-Reduction in turbidity of broth cultures after 30 min. at 37 °C (A₆₀₀ nm) (Uninoculated culture broth as blank). -Increase in turbidity (A₆₀₀ nm) after 30 min. at 37 °C.

2.4. Turbidity reduction assay

      To one ml of substrate [containing 0.25 ml (0.04 %) hyaluronic acid, 0.5 ml distilled water and 0.25 ml of acidified bovine serum albumin (BSA) fraction V (1% w/v) in 0.5 M sodium acetate buffer (pH 3.1)] 0.5 ml of the supernatant (diluted 1:2 in saline) of an 18-24 h broth culture of isolated microorganisms was added, mixed and incubated at 37 °C for 30 min. At the end of incubation time the tubes were cooled in ice bath. To the above mixture 0.1 ml of acetic acid (2 N) was added to precipitate the remaining HA [12, 13]. Tubes containing sterile broth or broth from inactive cultures became turbid while tube containing broth from hyaluronidase producing organism remained clear on addition of the acid. The isolates exhibiting reduction in turbidity is given in Table 1.

2.5. Determination of hydrolyzed zone of hyaluronidase

      Each isolate from slant was transferred into conical flask containing 25 ml sterile nutrient broth, incubated at 37 °C for 24 h. Five ml broth was then drawn, centrifuged at 8000 rpm for 20 min. at 4 °C and clear supernatant was collected. Then 25 μl of clear supernatant of each selected (20) isolates were aseptically added into sterile wells of 20 different molten nutrient agar plates containing 1 ml of substrate hyaluronic acid at a concentration of 10 mg/ml. The plates were then incubated at 37 °C for 24 h. The hydrolyzed zone (diameter in mm) of isolates was recorded and is given in Table 1. Thus four isolates showing good hydrolyzed zone were subjected for secondary screening.

      The isolate showing maximum hyase activity was subjected for identification following detailed taxonomic studies [11].

 Table 2. Hyaluronidase production of selected isolates by shake flask method.

2.6. Identification of the promising isolate

      The micromorphology studies include the shape of the cells, formation of spores, test for motility and gram staining were done. The cultural characters were studied by inoculating the organisms in different media viz. nutrient agar medium, blood agar medium, trypticase soy agar medium, Streptococcus selective agar medium and recording the growth pattern. The physiological and biochemical tests [11, 16, 17] were carried out by inoculating the isolate into the prescribed media. The tests included growth in air, growth under anaerobic condition, growth at different temperatures, growth at pH 9.6 (pH tolerance), sodium chloride tolerance, bile esculin agar, optocin test, alpha haemolysis, beta haemolysis, arginine dihydrolase test, decarboxylase test (Moeller's method), carbohydrate metabolism (acid-gas production) test, ONPG, lysine decarboxylase, ornithine decarboxylase, phenylalanine deamination, methyl red, indole, malonate, hydrogen sulphide production test, b-galactosidase production test, urease test, indole production test, nitrate reduction test, citrate utilization test, test for acid from esculin, arabinose, xylose, adonitol, rhamnose, cellobiose, melibiose, saccharose, raffinose, trehalose, glucose, lactose and Voges-Proskauer (acetoin production) test (VP) for identification of Streptococcus species.

3. Results

      The strains S_II9 (0.324 OD, 38 mm) followed by S_I7 (0.239 OD, 36 mm), S_IV19 (0.173 OD, 29 mm) and S_III13 (0.164 OD, 22 mm) showed highest reduction in turbidity (A₆₀₀ nm) and hydrolyzed zone, respectively, were selected for secondary screening by shake flask fermentation method.

      Four promising isolates which showed good hyase activity were further screened for their enzymatic activity by shake flask method and the increase in enzyme yield was compared to the reference strain S. mitis MTCC*2695. The data recorded in Table 2, indicated that isolate S_II9 (Dental caries specimen (patient with bleeding gums and inflammation) from P.G. Dept. Microbiology, OUAT, Bhubaneswar) exhibited maximum hyase activity (117 U/ml) while the reference S. mitis MTCC*2695 exhibited (106 U/ml) after 48 h. The promising isolate S_II9 (117 U/ml) was subjected to detailed taxonomic studies.

      The morphological and cultural charac-teristics of the isolate S_II 9 are appended herewith. The isolate grew as small spheres or ovoids, occuring in pairs or chains. It was nonmotile, nonsporing and gram positive. The growth of the isolate was restricted at 10 ºC while growth observed at 45 ºC (Optimum 37 ºC). The isolate showed no growth at pH 9.6 and 6.5% sodium chloride. An abundant, smooth, scanty circular, semitransparent growth was observed on nutrient agar medium. In case of nutrient broth, moderate turbid growth was observed.

Results of the biochemical studies revealed that the isolate S_II9 utilized different carbon sources and showed acid and gas production as indicated in Table 3. The isolate SII9 also utilized inulin, ribose, salicin, trehalose, glucose, sucrose and malonate and produced acid while lactose, mannitol, raffinose, sorbitol, arabinose, xylose, adonitol, rhamnose, cellobiose, mellibiose and saccharose showed no acid production. The isolate utilized arginine, lysine and ornithine as nitrogen sources. The strain could reduce nitrate and also utilized citrate, glucose, lactose, trehalose and malonate during its growth. The isolate was H₂S, ONPG, indole, esculine and VP negative. The isolate did not show production of urease, phenylalanine deamination, b-galactosidase as indicated in Table 3. From the above morphological, cultural and biochemical tests, it is proposed that our isolate S_II9 can be characterized as a strain of S. equi with few characters differentiated from S. equi subsp. equisimilis and thus it was designated as S. equi SED 9.