Applied and Environmental Microbiology Screening for pharmaceutically important exopolysaccharide producing streptococciand partial optimization for EPS production

Kanchankumar P.Patil, Bhushan L. Chaudhari* and Sudhir B. Chincholkar

School of Life Sciences, North Maharashtra University, PO Box: 80, Jalgaon 425001,India

Current Trends in Biotechnology and Pharmacy , Volume 3 (3) July - 2009

*For Correspondence -   blchaudhari@hotmail.com

Abstract

Capsular exopolysaccharide, hyaluronic acid produced by Streptococcus equi subsp. zooepidemicus, carries high importance in pharmaceutical as well as biomedical field. In this direction, isolation and screening for exopolysaccharide producingstreptococcusfrom nasopharynx of horses from Maharashtra and its neighbouring place was carried out. Out of 70 samples, none was observed to be Streptococcus equi subsp. zooepidemicus while, exopolysaccharide producing BM2 was isolated which showed close association with Streptococcus dysgalactiae with potential to produce hyaluronic acid. BM2 was compared with Streptococcus equi subsp. zooepidemicus (ATCC 43079), Streptococcus equi subsp. zooepidemicus (MTCC 3523) which produced exopolysaccharide 0.052, 0.050 and 0.097 g/l respectively. Streptococcus equi subsp. zooepidemicus (MTCC 3523) gave higher productivity; hence primary optimization studies were carried out to achieve maximum its growth and exopolysaccharide production.

Keywords: Exopolysaccharide, Horses, Mastitis, Streptococci, Streptococcus dysgalactiae

Introduction

Polymeric materials are gaining importance due to their extensive applications in pharmaceutical and medical sectors especially in diverse bio-medical fields like tissue engineering, implantation of medical devices and artificial organs, prostheses, ophthalmology, dentistry, bone repair etc (10). Among the polymers, Hyaluronic acid (HA) is a high-value biopolymer with a wide variety of medical, cosmetic and pharmaceutical applications (14). Commercially, HA is produced by the fermentation process using microbes like S. equi subsp. zooepidemicus. Being chemically identical to mammalian HA, streptococcal HA is non-immunogenic and hence regarded as a viable substitute for that from other sources. S. equi subsp. zooepidemicus is pathogenic Gram-positive bacteria which produce HA to protect against phagocytosis during infection. It is a catalase-negative, facultative anaerobe but is also aerotolerant microorganism (6). S. equi subsp. zooepidemicus belongs to the β-hemolytic Group C streptococci, which is mostly an opportunistic pathogen of non-human animal species, including important domestic animals like horses, cows, pigs, sheep, and dogs. Thus it is a pathogen of veterinary concern. It may be found in the nasopharynx, on the tonsils, in the respiratory tract, and on the genital mucous membranes of healthy horses and cattle. S. equi subsp. zooepidemicus is an important cause of respiratory tract infections in foals, young horses and is associated in uterine infections in mares. It is also a well-known cause of mastitis in cows, mares and is the most frequently isolated opportunistic pathogen of horses (16). β-haemolytic and Lancefield’s group C streptococci can be differentiated on the basis of fermentation of sugars like sorbitol and trehalose. S. equi subsp. zooepidemicus generally ferments sorbitol, but not trehalose (9, 15). The other streptococci like S. pyogenes, S. faecalis, S. dysgalactiae, S. equi and S. equisimilis belonging to Lancefield group A and C can also be used to produce an exopolysaccharide (EPS); especially hyaluronic acid (11). In this study, search for S. equi subsp. zooepidemicus was carried out in some part of Indian region and the EPS production by strain S. equi subsp. zooepidemicus (MTCC 3523) was analysed.

Materials and methods

Sampling. Sampling was done from Raver a rural place in northern side of Maharashtra state, eco-sensitive hilly area of Matheran in Maharashtra state and Ratlam in Madhya Pradesh (India) using sterile transportation swabs with Amies charcoal media (HiMedia, India). Sampling was carried out by nasopharyngeal swabs from 18 horses at Raver, 8 horses and 25 different ponies at Matheran and 21 horses from Ratlam. At Raver, sampling was done twice, in April and November which was summer (~ 40˚C) and winter (~ 28˚C). While sampling at Matheran was done in late winter in February (~ 18˚C) while sampling in Ratlam was done in summer i.e., in April (~45˚C). Milk samples of 1 cow and 3 different buffaloes suffering from bovine mastitis were also obtained from Yawal a rural place close to Raver. Samples were transported from place of collection to laboratory by keeping it in cool boxes.   

Isolation and screening of bacterial strains. Isolation was carried out by streak plate method using β-Streptococcus selective agar medium (HiMedia, India) containing (g/l): Peptone 1.0, Meat extract 0.6, Yeast extract 0.5, L-lysine 0.02, Sodium chloride 6.0, Disodium hydrogen phosphate 2 and agar 15 with final pH 7.3. After autoclaving, medium was cooled at 45-50˚C and sterile defibrinated sheep blood was added at the concentration 7-10%. Isolates were cultivated on slants of streptococcus agar containing (g/l): Glucose 20, Pancreatic digest of casein 20, K₂HPO₄ 2, MgSO₄.7H₂O 0.1, agar 15 and final pH was adjusted at 6.8 at 25˚C. Cultures which showed β-haemolysis were isolated and further characterised byGram staining and biochemical characters like catalase test, sorbitol and trehalose utilization were sequentially carried out only when preceding test was found affirmative. Sugar fermentation test was carried out using phenol red broth containing sorbitol or trehalose (10 g/l). Simultaneously standard strains S. equi subsp. zooepidemicus (ATCC 43079) and S. equi subsp. zooepidemicus (MTCC 3523) were obtained fromMicrobiologics, USA and Microbial Type Culture Collection & Gene Bank (MTCC),India, respectively.

Biochemical characterization and genomic analysis of isolate. Identification of EPS producer isolate BM2 was carried out by biochemical characterization using HiStrep biochemical test kit (HiMedia, India) which subsequently was confirmed by 16S rRNA gene sequence analysis and phylogenetic studies. Universal primers 16F27N (5’-CCAGAGTTTGATCMTGGCTCAG-3’) and 16R1525XP (5’-TTCTGCAGTCTAGAAGGAGGTGTWTCCAGCC-3’) (5) were used for the amplification of 16S rRNA gene of the BM2.

The sequence of BM2 has been deposited at National Centre for Biotechnology Information (GenBank), http://www.ncbi.nih.gov under the accession number FJ238093.

Production and estimation of EPS

Inoculum. The inoculum was prepared in a 250-ml shake flask with 50 ml of Todd Hewitt Broth comprising (g/l): Brain heart infusion 500, Peptic digest of animal tissue 20, Dextrose 2, Sodium chloride 2, Disodium phosphate 0.40, Sodium carbonate 2.50, pH 7.8 and incubated at 37^oC for 12 h.

Cultivation conditions. The shake flask experiments were performed in 500 ml Erlenmeyer flask with a working volume of 100 ml, and the agitation was maintained at 120 rpm. The fermentation medium contained (g/l): Casein enzyme hydrolysate 20, NaCl 2, MgSO₄·7H₂O 1.5, K₂HPO₄ 2.5 and pH adjusted to 7.0. Glucose solution 5 g/l was autoclaved separately and added to it after cooling. This medium was inoculated with 1% (v/v) inoculum. Culture flasks were incubated at 37^oC for 24 h.

Cell biomass. Samples were centrifuged at 8000×g for 5min. The weight of cell biomass was measured after repeated washing of the cell pellets with distilled water and drying at 60 °C for 24h.

Analysis of EPS. Fermented broth was diluted with an equal volume of 0.1% w/v SDS and incubated at room temperature for 10min to free capsular EPS which was filtered through 0.45µm membrane as per method of Blank et. al (4). Filtrate was subjected to determine mucopolysaccharides by a turbidimetric assay (8). Briefly, 200 µl of the filtrate was mixed with 200 µl of 0.1M acetic acid (pH 6) and 400µl of 2.5% w/v cetyltrimethyl-ammoniun bromide (CTAB) in 0.5M NaOH. The mixture was incubated for 20min at room temperature and A₅₉₅ was recorded. A standard curve was established using a 0.18 g/l EPS stock solution prepared using standard HA of microbial origin (Focuschem, China).

Growth curve. Growth pattern of selected streptococcal strain was determined by incubating at 37^oC, 120rpm for 30h in above mentioned fermentation medium. Samples were drawn at the interval of 2 h to determine cell growth and EPS produced. Cell growth was monitored turbidometrically by spectrophotometer at 595nm. Culture samples were diluted in water to an absorbance of less than 1 before measurement which was later multiplied by the dilution factor.

Partial optimization of medium ingredients for EPS production

Carbon source. Six carbohydrates viz. glucose, sucrose, starch, galactose, fructose and lactose were evaluated. Fermentation medium contained (g/l); Carbon source 5, casein enzyme hydrolysate 20, NaCl 2, MgSO₄.7H₂O 1.5 and K₂HPO₄ 2.5. Selected carbon source taken was starting from 0.5% to 2.5% with addition up of 0.5% each time to find out optimum concentration.

Nitrogen source. Five different organic nitrogen sources viz. tryptone, peptone, soyapeptone, yeast extract and meat extract were evaluated. Fermentation medium was similar as above except the nitrogen source. Thus, the medium composition was (g/l); nitrogen source 20, NaCl 2, MgSO₄.7H₂O 1.5 and K₂HPO₄ 2.5. Glucose (1.5%) was used as a carbon source. Concentration of selected nitrogen source was optimized by assessing its concentrations starting from 1.5% to 5% with addition of 0.5% each time to find out optimum concentration.

Metal. Five different metal salts were checked, viz. MgSO₄.7H₂O (Mg), MnSO₄.H₂O (Mn), CuSO₄.5H₂O (Cu), ZnSO₄.7H₂O (Zn) and FeSO₄.7H₂O (Fe). Fermentation medium was similar as above and each metal salt was added in separate flasks at an equimolar concentration. Thus, the concentrations were (g/l) Soyapeptone 45, NaCl 2, metal salt 1mM and K₂HPO₄ 2.5. Glucose (1.5%) as carbon source and soyapeptone (4.5%) as nitrogen source were used. Concentration of selected metal salt was optimized by assessing its different concentrations (1 – 9 mM).

Phosphate source. Four different inorganic phosphate salts were assessed viz. K₂HPO₄ (Dipotssium), KH₂PO₄ (Potassium), Na₂HPO₄.2H₂O (Disodium) and NaH₂PO₄.2H₂O (Sodium). Each phosphate source was added in medium separately in flasks at an equimolar concentration. The medium ingredient concentrations were Soyapeptone 4.5%, NaCl 0.2%, and MgSO₄.7H₂O 1mM. Glucose (1.5%) was used as a carbon source. Concentration of selected inorganic phosphate salt used was 10 - 110mM for optimization.

pH. To determine the best pH for EPS production, culture was grown in the fermentation medium having six different pH values as 6, 6.5, 7, 7.5, 8, and 8.5 containing glucose 1.5%, soyapeptone 4.5%, NaCl 0.2%, MgSO₄.7H₂O 1mM and K₂HPO₄ 30 mM.

Results

β-haemolytic cocci were characterized by using Gram’s nature, catalase test,  ability to utilize sorbitol and trehalose for differentiating S. equi subsp. zooepidemicus from other strains where failure of any test lead to non performance of subsequent further tests. Out of 20 isolates from Raver, 7 were β-haemolytic and 1 was α haemolytic. But these 7 β-haemolytic isolates did not exhibit further characters of S. equi subsp. zooepidemicus i.e. cocci in shape, Gram positive, catalase negative, sorbitol fermentation reaction positive and trehalose fermentation reaction negative (data not shown). Similarly, out of 33 isolates from horses and ponies at Matheran, 23 were β-haemolytic which did not exhibit further desired characters (data not shown). Some horses at Ratlam used for local transport were also found to be free from the infection of S. equi subsp. zooepidemicus as none of the isolates out of 21 matched it (data not shown) .  Apart from horses, milk samples from 3 different domestic milking buffaloes and 1 cow showing mastitis symptoms were collected and further screened. Though isolates BM1, BM2, BM3 from buffaloes milk and CM1 from cow milk did not match biochemical characters of S. equi subsp. zooepidemicus (data not shown), BM2 was further studied because of its glossy and mucoid appearance on agar plates.

 Characterization of isolate BM2

BM2 culture was grown on streptococcus maintenance agar medium that showed off white round shaped small glossy colony with mucoid consistency. Cells were cocci and Gram positive.

Biochemical analysis

The isolate BM2 showed Voges Proskauer’s test, esculin test, L-pyrrolidonyl ß-naphthaylamine test, and o-nitrophenyl- ß-D-galactopyranoside test negative while arginine utilization was positive. Among sugars, it could utilize glucose, ribose, arabinose, sucrose and sorbitol; but not mannitol and raffinose. These results of biochemical tests of BM2 matched with S. dysgalactiae as per HiStrep biochemical test kit (HiMedia, India).

16S rRNA sequence of BM2:

Biochemical characterisation, 16S rRNA gene sequence analysis and phylogenetic position (Fig. 1) showed close association of BM2 (about 99% homology) with S. dysgalactiae.

The strain S. equi subsp. zooepidemicus (MTCC 3523) produced higher amount of EPS i.e. 0.097 ± 0.00g/l than other two strains S. equi subsp. zooepidemicus (ATCC 43079) 0.050 ± 0.01g/l and Streptococcus(BM2) 0.052 ± 0.01g/l. Hence EPS production from strain MTCC 3523 was further studied. Its growth curve revealed that logarithmic phase started from 4h and after 8h entered in to stationary phase which was observed till 30h. Similarly, capsular EPS production was observed to increase radically at 8h and stabilized with slight increase up to 30h (data not shown). The effect of the carbon sources on EPS production was studied (Fig 2a) where glucose was found to be the best carbon source with a production of 0.090g/l EPS subsequently 1.5% was the optimum glucose concentration  in the fermentation medium (Table 1a). While, the best nitrogen source for EPS production was found to be soyapeptone which resulted in maximum production (0.131g/l) (Fig. 2b). Higher EPS production was obtained at the 4.5% of soyapeptone (Table 1b). Whereas, fig 2c and table 1c shows that the metal salt MgSO₄.7H₂O when used at concentration 3mM enhanced the production of EPS to 0.376 g/l & 0.383 g/l respectively. While ZnSO₄.7H₂O and CuSO₄.5H₂O completely inhibited the growth of microorganism. Fig 2d and table 1d shows that use of an inorganic phosphate source K₂HPO₄ at the concentration 30mM gave higher production of EPS. Fig 3 shows that pH 7.5 is the optimum pH for higher production of EPS that yielded 0.443 g/l.

Discussion

A total of 74 samples from nasopharyngeal mucosa of horses and ponies were investigated with objective to isolate EPS producing bacteria especially S. equi subsp. zooepidemicus. Isolated cultures were 20, 33 and 21 from Raver, Matheran and Ratlam respectively where horse population was high. Though, S. equi subsp. zooepidemicus is an important pathogen of horse being associated with respiratory tract infections of foals and with uterine infections in mares (1). Unexpectedly the nasopharyngeal flora of horses in these regions was found to be free from infection by this pathogen although particular animals were mucus excreting. Regions selected for isolation were having different climatic and geographical conditions, and the animals selected were also from diverse hygienic conditions. But β-haemolytic, S. equi subsp. zooepidemicus of group C causing strangles in horses was not observed which could not be answered and needs to be explored further. Perhaps mucus might be due to other microbial infection. Association of S. equi subsp. zooepidemicus with mastitis in dairy cattle like cows, goat, sheep and buffaloes is also reported (12), but instead of this organism, S. dysgalactiae was found in one of the milk samples of dairy animals showing symptoms of mastitis. This isolate S. dysgalactiae was compared with S. equi subsp. zooepidemicus ATCC 43079 and S. equi subsp. zooepidemicus MTCC 3523 strain for its EPS productivity, where MTCC 3523 proved better than others.

The specific growth rate and volumetric EPS production rate by S. equi subsp. zooepidemicus were found to be less favourable in the chemically defined media (2) and Group A as well as Group C streptococci possess fastidious nutrient requirements with respect to organic nitrogen (3, 7). Hence organic sources for carbon and nitrogen were selected to optimize the growth of selected strain i.e. S. equi subsp. zooepidemicus (MTCC 3523) in which glucose as carbon source and soyapeptone as nitrogen source showed better increase in growth of organism and production of EPS. The enzyme hyaluronic acid synthase (HAS) involved in HA synthesis in streptococcus and mammals prefer magnesium ions while Chlorella virus HASs prefer manganese ions to stimulate HA synthesis (17). Similarly use of magnesium sulphate showed better results than the other metal salts in EPS production by MTCC 3523 strain. Large number of ATP molecules are consumed in various enzymatic reactions involved in HA biosynthesis pathway in streptococcus, hence source of phosphate is essential to synthesize ATPs. In this study, salt dipotassium hydrogen phosphate as a source of inorganic phosphate showed better results. Earlier Johns et. al (13) have reported 6.7 as optimum pH for both the rate of production and yield of hyaluronic acid (HA) by S. equi subsp. zooepidemicus from glucose medium while, our results showed optimum pH for better production of EPS was 7.5.

Conclusion

The results obtained suggested that the occurrence of equine pathogen S. equi subsp. zooepidemicus could be rare in

Maharashtra and its neighbouring region. These results can provide a base for further study on animal pathogens in the above said area. Secondly, the strain S. equi subsp. zooepidemicus (MTCC 3523) could be effectively used for fermentative production of HA that could be further exploited.

Acknowledgements

Authors are thankful to Dr. Yogesh Shouche, NCCS, Pune, India for 16S rRNA sequence analysis of isolate. Generous gift of HA sample by Focuschem Enterprises Ltd,China and kind help of Mr. Jake Chen is gratefully acknowledged.

References

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Figure 1. Phylogenetic position of BM2

(a) Selection of carbon source for higher yield of EPS

(b) Selection of nitrogen source for higher yield of EPS

(c) Selection of metal for higher yield of EPS

(d) Selection of inorganic phosphate source for higher yield of EPS
Figure 2.

Figure 3. Optimization of pH for higher yield of EPS

Table 1a. Optimization of glucose concentration for higher yield of EPS

Glucose (%)	Cells biomass (g/l)	EPS (g/l)
0.5	0.67 ±0.05	0.044 ±0.01
1.0	0.69 ±0.02	0.079 ±0.01
1.5	0.61 ±0.06	0.093 ±0.01
2.0	0.63 ±0.05	0.091 ±0.01
2.5	0.63 ±0.05	0.091 ±0.01

Table 1b. Optimization of soyapeptone concentration for higher yield of EPS

Soyapeptone (%)	Cells biomass (g/l)	EPS (g/l)
1.5	0.65 ±0.08	0.108 ±0.04
2.0	0.68 ±0.04	0.149 ±0.03
2.5	0.68 ±0.02	0.178 ±0.01
3.0	0.76 ±0.04	0.203 ±0.01
3.5	0.81 ±0.01	0.186 ±0.04
4.0	0.88 ±0.04	0.201 ±0.04
4.5	0.94 ±0.04	0.245 ±0.01
5.0	1.01 ±0.06	0.170 ±0.01

Table 1c. Optimization of MgSO₄.7H₂O concentration for higher yield of EPS

MgSO4.7H2O (mM)	Cells biomass (g/l)	EPS (g/l)
0	0.83 ±0.03	0.205 ±0.02
1	1.08 ±0.08	0.313 ±0.03
3	0.99 ±0.04	0.383 ±0.01
5	0.98 ±0.10	0.297 ±0.02
7	1.05 ±0.08	0.293 ±0.02
9	1.06 ±0.03	0.256 ±0.02

Table 1d. Optimization of K₂HPO₄ concentration for higher yield of EPS

K2HPO4 (mM)	Cells biomass (g/l)	EPS (g/l)
10	1.22 ±0.01	0.442 ±0.04
30	1.74 ±0.13	0.485 ±0.08
50	1.73 ±0.15	0.241 ±0.08
70	0	0
90	0	0
110	0	0