Antibacterial Activity Of Bee Venom Against Multidrug Resistance Staphylococcus Aureus From Milk Of Cow And Buffaloes

Bacteriological study includes bacteria isolated from Buffaloes milk, and Cow at Wassit Province was done in Technical institute. 120 milk samples were collected randomly from different places in Wassit Province included 60 samples from Buffaloes milk and 60 samples from Cow milk during the period October to December 2018. A bacteriological study was conducted for isolation and identi(cid:977)ication S. aureus by morphological and biochemical tests. The results showed that (20%) of Buffaloes milk and (13.33%) in Cow’s milk (13.33 %) of Cow milk contaminated with S. aureus . Anti-biogram pattern of S. aureus was carried out by using a diffusion method from an antibiotic saturated disk, Results showed more effect in S. aureus isolated from Buffaloes milk was COT in ef(cid:977)iciency ratio ( 78.33 % ), CLR in ef(cid:977)i-ciency ratio ( 61 % ), SPX in ef(cid:977)iciency ratio ( 51.66 % ), and L in ef(cid:977)iciency ratio ( 15 % ). Also the results appeared more effective in S. aureus isolated from Cow milk was COT in ef(cid:977)iciency ratio ( 56.66 % ), CLR in ef(cid:977)iciency ratio ( 35 % ), SPX in ef(cid:977)iciency ratio ( 30 % ), and L in ef(cid:977)iciency ratio ( 8.33 % ), while S. aureus appeared resistant in all milk samples from Buffaloes and Cow for antibiotic OX (100%), also MIC to staphylococcal ( 0.70 - 3.10 (cid:22) g/ml ) While MBC concentration was (0.12-0.101 (cid:22) g/ml).


INTRODUCTION
Milk is a good medium for growth of various microorganisms that are responsible for changing taste and smell as well as milk composition. The main cause of milk contamination with bacterial pathogens is the treatment method, the workers hygene and the unhealthy environment. The presence of bacterial pathogens in milk can cause sig-ni icant health risks to the people because the milk contains high nutritional value and complex chemical composition (Cappuccino and Sherman, 2005;Al-Safar et al., 2018).
When the milk is exposed to different levels of the temperatures, or stored at temperatures ranging from 37C • -42 C • noted the presence of the toxins of S.aureus in it (Altekruse et al., 1994).
Warming the milk to the normal temperature of the conventional cooking may kill S.aureus, but their toxins remain effective, because toxins are more resistant to high heat in food (Balaban and Rasooly, 2000;Bergdoll, 1983). The symptoms of food poisoning in the S.aureus include sudden symptoms in digestive system. In addition, the toxins produced by Staphylococcus bacteria play vital role in damaged cells of host (Bhatia and Zahoor, 2007;Bonfoh et al., 2003).
Many studies have indicated a signi icant similarity among isolated of S.aureus from bovine spongiform   encephalitis with species isolated from food poisoning (Choi et al., 2015).
The rate of infection is still in high level, particularly in India, due to increase temperature of climate and humidity (Dingwell et al., 2003).
Infection of the cattle with mastitis led to signi icant economic losses and also led to a decrease in milk production as well as treatment cost. Where economic losses in England 187 million Euros annually, and in America, 180 million dollars annually (Ekici et al., 2004) Bee Venom is a natural complex material consisting of two major compound are peptides and proteins with active biochemical substance such as melittin, histamine, and dopamine (Han et al., 2013). Phospholipase A and Hyaluronidase in venom play important role in analyze Hyaluronic acid to simple unite, so use the bee venom against gram positive S.aureus mention by (Han et al., 2016).

Samples
120 Milk samples from cow and buffalo at variety place in Wasit province included (60 milk buffalo with 60 milk samples from cow) during period October to December 2018 by using sterile collection containers.

Isolation and identi ication of S. aureus
Isolation of S.aureus was adopted (Hegazi et al., 2014). Pepton water medium was used in which 10 ml of the homogeneity milk samples were taken with 90 ml of sterile Pepton water overnight at temperature of laboratory. Then samples inoculated at the selective medium MSA agar (Himedia /India) at 35C • for 18 hrs. Isolates of S.aureus determined based on morphological speci ications and biochemical tests. Bacterial smears were carried out of the isolates on clean, free grease glass slides, and then stained by Gram stain, the isolates were Gram positive and arranged in irregular clusters similar to the grape cluster. Biochemical tests were performed based on (Min et al., 2013) as shown after being compared with turbidity of McFarland Table 2.

Antibiotics susceptibility test
Antibiotics susceptibility test of the S.aureus was performed in a diffusion method from the antibioticsaturated disc in the Muller-Hinton Agar (Himedia /India) Lawn method by using ive antibiotics discs as shown in (Table 3) were then ixed and incubated at 37 C • for 18-24 hrs (Moroni et al., 2005).

Collect and Puri ication solution of Bee Venom
Bee venom was collected from Iraqi bees in farm that is located in Wasit by utilising electro-stimulate apparatus inside the hive, that generate pulses in voltage at maximum 27V at 1-2 seconds, all venom produced by bee accumulate on Flat glass then dry by exposure to air for 5-10 min with sharp scalpel then collected by sterile tube and stored at a refrigerator until usage. Prepared solution of Bee Venom (BV) use for the detect activity of bee venom against S.aureus dissolved 250 mg from bee venom in 1ml of distilled water (Nelson and Stephen, 2003) then added solution of BV in centrifuge apparatus approximately 10-12min. at 14,000 x g then worked serial dilution for next test according to (Normanno et al., 2007).

Bactericidal assay
Bacteria are collected at absorption A600=0.5 and added to a buffer phosphate solution (PBS) pH = 7.2 at (10cfu/ml) followed by incubation of different serial dilution of bee venom solution with bacterial samples at 25C • for 1800 sec. The subsequence of dilution is then taken and grown on the blood agar (Himedia /India) plates for 18 hrs. At 37C • to calculate the numbers of remaining bacteria according to (Novoslavskij et al., 2018).

Minimum Inhibitory Concentrations (MIC) assay
To determine MIC for BV towards bacteria, method of micro-dilution broth used the concentration of cells adjust to (1x10cell/ml) in phosphate buffer solution at pH = 7.0 for bee venom dissolved in phosphate buffer solution at pH = 6.0 before dilution, then (10 µl) solution of BV is added to (190 µl) of diluted bacteria, and incubate for 18-20 hrs. At 35-37 C • MIC read as the less BV concentration inhibits growth of bacteria, and determines a clear optical by ELISA reader (Human reader HS /Germany) depending on (Payne and Wood, 1974).

Minimum Bactericidal Concentrations (MBC) assay
To determine MBC for BV Versus antibiotic resistant bacteria, depend on following method was adopted by adding 0.1 ml of the minimum inhibitory concentration mixture, which showed no bacterial growth, inoculated in liquid medium brain heart infusion broth (Himedia/India) and incubated for 2 days at 37 C • Thus, the value of the MBC is less concentration of bee venom that requires reduction of 99.9% from the accumulation of live bacteria according to (Presscott et al., 2002).

RESULTS AND DISCUSSION
The results showed that the percentage of positive isolates of the S.aureus under study of buffalo milk is (20%), and cow's milk is (13.33%) (Figure 1). Table 1 explains the consequences of biological tests for the diagnosis S.aureus isolates from milk of cow and buffalo. Table 2 depicts categories and concentration of antibiotics that were adopted for antibiotic susceptibility test against S. aureus isolates.
The milk is sterile in buffalo and cow that does not suffer from mastitis, but if it is infected with mastitis, it will result in the presence of large numbers of Gram positive bacteria, such as S.aureus in milk when it is out of the udder (Singh and Prakash, 2008). Milk is not only a food source, but suitable media to appear staphylococcal bacteria that can cause milk damage, leading to foodborne diseases and the spread of gastrointestinal diseases in hot places (Soomro et al., 2003).
The lack of hygiene of milk collection containers, cleanliness of workers to collect milk, and atypical storage of milk increase the presence of Grampositive bacteria and Gram-negative bacteria in milk containers (D.H and S.A, 2006). The percentage of S.aureus isolates in buffalo milk was 20% slightly higher than conducted by (D.H and S.A, 2006;Wikler, 2006) which was 18.18 % and 17.39 % respectively, while the percentage of S.aureus isolates in cow's milk was 13.33%, which is almost consistent with (Wayne, 2012) which was 12.8%.
The results of Antibiogram showed sensitivity of the S.aureus; it isolates against antibiotics in different levels which was (78.33%) of Co-Trimoxazol, 61% of Clarithromycin, (51.66%) of Spar loxacin, and 15% of Lincomycin. While all S.aureus iso- lates showed resistance to the antibiotic Oxacilin (100%). The ability of S.aureus isolates to resist antibiotics due to the possession of a number of resistance mechanisms, like production of largespectrum Beta-lactamase enzymes that may occur because of genetic mutations in the sequences genes that are encoded of this protein Beta-lactamase enzymes analyze the Beta-lactam antibiotics and transformed into ineffective compounds in addition to ef lux pump mechanism, change metabolic pathways and change in the target location is recognized by the antibiotic also randomly using of antibiotics or the use of insuf icient concentrations to kill bacteria, which increase ability of bacteria to resist antibiotics.
The results of bactericidal effect of (BV) concentration treatment for 30 min against MRSA in our study showed bacterial viability decrease in > 90% at BV concentration among (1.25-12.25µg/ml) and no bacteria survived incubation with more than (12.5 µg/ml) concentration of BV that results suficient agreement with the study of (Jenkins et al., 2011), while disagree with another study (Seo et al., 2012) that was BV reduced bacteria viability at concentration (0.85), also minimum inhibitory concentration to staphylococcal bacteria approximately among (0.70 -3.10 µg/ml ) This shows that bee venom contains anti-bacterial molecules targeted MRSA isolates agree with study achieved (Jenkins et al., 2011), and not agree with study (Seo et al., 2012) because concentration that inhibits growth were ranged between (0.17-0.85 µg/ml). BV a natural peptides substances and most important component that plays an active role in its effectiveness is phospholipase and melittin, where the two components work together to kill a wide range of bacteria such as the strains of MRSA (Jenkins et al., 2011), while results showed MBC concentration against MRSA strain were rang between (0.12-0.101 µg/ml) As expected higher (BV) MIC concentration as compared to the MBC concentration were observed in our study that indicates the higher concentration of bee venom kill the strain of MRSA that results opposite to result from the study (Seo et al., 2012).

CONCLUSIONS
S.aureus was a contaminated sample of milk collected from infected animals with mastitis. Percentage of its presence in buffalo milk reached 20% and in the milk of cows was 13.33%. The most effective antibiotic against the bacteria was Co-Trimoxazol in 78.33% of buffalo isolates and 56.66% of bovine isolates. All bacterial isolates were resistant to Oxacilin 100%. All those lead to making the milk that dis-tributed in the market carrier for the disease and, Bee venom can inhibit the growth and survival of MRSA, and thus can be used as a supplement alternative agent at limited concentrations.