EXTRACTION OF ANTIBACTERIAL SUBSTANCES FROM ArtrhospiraplatensisBIOMASS AGAINST ANTIBIOTIC- RESISTANT Staphylococcus sp. ISOLATED FROM BOVINE MASTITIS

Mastitis is an inflammation of the udder which may be caused by bacteria and cause economic losses by produce harmful substances that result in inflammation, reduced milk production, and altered milk quality. Arthrospiraplatensis extract in two different solvents, water (W) and sodium acetate buffer (SAB), was tested for its antibacterial activity against 14 differents antibiotic-resistant Staphylococcus sp. isolated from bovine mastitis. Both Arthrospiraplatensis extract exhibited antimicrobial activity against all Staphylococcus sp., but SAB was most effective than W. The minimum inhibitory concentration of the A. platensis extracts at 100mg/ml in SAB was obtained in 29% of isolated and in W was 21%. More than 50% of inhibition was observed at 6 mg/ml in SAB and 12 mg/ml in W. Results suggest that Arthorspiraplatensis extracts in SAB has the potential to be evaluated as an alternative or adjunct to antibiotics as intramammary infusion to treat bovine mastitis.


INTRODUCTION
Bovine mastitis is a highly prevalent disease in dairy cattle, and one of the most important diseases affecting the world's dairy industry; it places a heavy economic burden on milk producers all over the world (Bennett et al., 1999). Worldwide, annual losses due to mastitis have been estimated to be approximately 35 billion US dollar. In the US, the annual costs of mastitis treatment have been estimated to be 1.5-2.0 billion US dollar (Wells et al., 1998).
Mastitis is defined as an inflammatory reaction of the parenchyma of the mammary gland that can be of an infectious, traumatic or toxic nature. Mastitis is characterized by physical, chemical and microbiology changes in the milk and by pathological changes in the glandular udder tissue. The diagnosis of mastitis is based on clinical signs, e.g. swelling of the udder, tender to the touch, fever, and depression. In many cases, a reduced milk production can be observed. Mastitis-causing pathogens include mycoplasms, fungi, yeasts, and bacteria (Radostits et al., 1994). These pathogens infect the udder generally through the ductuspapillaris, which is the only opening of the udder to the outside world.
Staphylococcus sp. is the predominant pathogen isolated from cases of mastitis and it causes one of the most common types of chronic mastitis.Antimicrobial are commonly used to trat clinical mastitis and occasionally cases of subclinical mastitites (Mc Dougall et al., 2014). Mc Dougall et al. (2014 relatated that resistance to some beta-lactam antimicrobials and trimethoprim/sulfamethoxazole were found in Staphylococcus sp.isolates from cases of bovine mastitis. Staphylococcal species isolated of goat milk were S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest somatic cell count (SCC) in milk and the highest prevalence of clinical udder alterations were associated with coagulasepositive S. aureus (Deinhofer and Pernthaner, 1995) Natural substances offer interesting pharmacological perspectives for antibacterial drug development with regard to broad spectrum antiviral properties and novel modes of action. Although extremely effective, antibiotics are able to induce resistance in bacteria. For 450 years, bacterial resistance has been the main factor responsible for the increase of morbidity, mortality and health care costs of bacterial infections (Abd El-Baky, et al, 2006) Cyanobacteriumis rich sources of structurally novel and biologically active metabolites. Secondary or primary metabolites produced by these organisms may be potential bioactive compounds of interests in the pharmaceutical industry.
A nontoxic cyanobacteriumArthrospira strain can be new source of natural antimicrobial (Shafar et al., 2013). Several studies have focused on physiological properties of some valuable antimicrobial compounds in these cyanobacteria. The search for cyanobacteria with antimicrobial activity hasgained importance in recent years due to growing worldwide concern about alarming increase in the rate of infection by antibiotic-resistant microorganisms.
The present work aimed to study the antimicrobial activity fromA. platensisextract disrupted with water (W) or sodium acetate buffer (SAB) against Staphylococcus sp. conventional antibiotic-resistantisolates of bovine mastitis.

Cell Disruption
Cell lyophilized was suspending in distilled water (W) or sodium acetate buffer (SAB) at concentration of 100mg/mland subsequently sonicatedfor 1 min pulse during10 minutes in an ice bath. After sonication, the sample is centrifuged at 2500xg at 4 ° C for 5 min. The supernatant was utilized for assessment of biological activities.

Determination of Minimum Inhibitory Concentration using plate microdilution
The minimal inhibitory concentration (MIC) was determined by the microdilution method described by NCCLS (2003) using 96-well standard microtiter plates. A series of dilutions with concentrations ranging from 100 to 3 mg/ml for extract was used in the experiment against every microorganism tested. Briefly, 50 μL of twofold serial dilutions of examined samples was added to 50 μL microbial suspension adjusted to yield approximately 1.0 × 10 5 CFU mL −1 . MIC was encountered as the lowest concentration of examined sample that inhibits the microbial growth after 24h incubation at 37 °C. Negative controls were included too. Bacterial growth is determined by measuring the absorbance at 630 nm.

RESULTS AND DISCUSSION
The water (W) or sodium acetate buffer (SAB) extracts were tested for antibacterial activity against 14 different antibiotic-resistant Staphylococcus sp. isolated from bovine mastitis. The degree of activity was varied according to concentration and type of A.platensis extracts. The SAB extract showed the antibacterial activity with MIC at 100mg/ml against four different pathogens, corresponding to 29% of all Staphylococcus sp. analyzed.With decrease of cell extracts concentration to 50 mg/ml, the inhibition degree decrease and was not observed MIC in concentration among 50 -3 mg/ml. However, cell extracted at concentration of 12 mg/ml obtained more than 50% of inhibition in 14% of Staphylococcus sp and only one bacteria obtained inhibition higher than 50%. At 3 mg/ml of cell concentration, all Staphylococcus sp. was resistant (Table 1).
In relation to cell extracts with water (W), it was observed that there was an inhibition of 100% of the antibiotic-resistant Staphylococcus sp. isolated from bovine mastitisat a concentration of 100mg/ml, but only in 7% of the pathogens was observed MIC and 86%, IC50. At 50mg/ml, 50% of the Staphylococcus sp. obtained inhibition more than 50% (IC50), at 25mg/ml, only 3% of the Staphylococcus sp. obtained IC50 and in the others concentration (12, 6 and 3 mg/ml), the IC50 was less that 2% In the same way as observed with water extract, at 3 mg/ml, all Staphylococcus sp. was resistant (Table 1).  (Najdenski et al., 2013) This value is lower than our because the MIC was done with dry residues after centrifugation and freeze-dried, while our results was obtained after only centrifugation. Shafar et al. (2013) related that cold water extract, hot water extract and phosphate buffer extract of Arthrospira strain inhibited herpes simplex infectivity by 54.9%, 64.6%, and 99.8%, respectively, in a dose-dependent manner, obtained the higher inhibition using phosphate buffer, as observed in our work.
Many fresh water cyanobacteria have been recognized as potential source of antibacterial substances. It is evident that microorganisms, living in an environment where competition and predation are the maximum without physical-defence structure, defend themselves by production chemical to survive. There, the exploration of the cyanobacterium represents a promising strategy in the search for active compounds, while there is a need for new medicines, due to the appearance of resistance to available treatments in many microorganisms, specifically for antifungal, antiprotozoal, antibacterial and antiviral activities (Yasuhara-Bell et al., 2010).
Kaushi and Chauhan (2008) related that A. platensis extract concentrated using organic solvents such as hexane, ethyl acetate, dichloromethane exhibited different degree of antibacterial activity against one Gram-positive bacterium (Staphylococcusaureus) and four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiellapneumoniae). The results showed that the MICs of methanolic and dichloromethane extract were 128 μg/ml and 512 μg/ml against S. aureus. This value is above to obtainedin our work because we not used A. platensis extract concentrated.
The results concerning activities of the crude extracts are not surprising. Similarly, aqueous cyanobactria extracts of Arthrospira maxima, Nostoc, Synechocystis, Gloeothece sp, were found antagonistic against different pathogenic microorganisms (excepting Salmonella sp.) in a large studies (Guedes et al., 2011;Medina-Jaritz et al., 2011).
Phycobiliproteins (PBPs) (phycocyanin, allophycocyanin and phycoerythrin) are a family of coloured photosynthetic accessory pigments of cyanobacteria and red algae. C-PC from Spirulinaplatensis was identified as a potent agent against E. coli, Klebsiellapneumoniae, P. aeruginosa and S. aureus (Saradaet al., 2011). Besides, the PBPs of the two red microalgae (P. aerugineum and P. cruentum) were active against S. aureus andS. pyogenes. The growth inhibition activity towards antibiotic-resistant Staphylococcus sp. from A. platensisextractswere an interesting finding, due to a lack of reports on such activity in the literature.
Numerous green alga, such asDesmococcusolivaceous, Chlorococcumhumicola, Chlorella vulgaris, Ulvafasciata, Enteromorphaintestinalis, Chaetomorphaaerea were screened for antimicrobial activity in search of the new antimicrobial agents (Seenivasan et al., 2010;Uma et al., 2011), but in this study, for the first time, the antimicrobial activity of the sodium acetate buffer extract of A. platensis was investigated. In correlation with results obtained in experiments with other algae, we noticed that the SAB of A. platensis showed relatively antimicrobial activity The present study results revealed that the SAB extracts of A. platensis showed the antibacterial activity against antibiotic-resistant Staphylococcus sp. isolated from bovine mastitis. It suggests that in addition to the available drugs, alternatively we can use the extracts of A. platensis against bovine mastitis. The results of the present study suggest that the SAB extracts of A. platensismay be used to bovine mastitis.

CONCLUSION
In conclusion, the performed comparative study allows us to determine that sodium acetate buffer extracts of A. platensis is the most promising which show a relatively high level of antimicrobial potency and wider range of substances, active against the most antibioticresistant Staphylococcus sp. isolated from bovine mastitis.