Novel Study Antimicrobial and Biocompatibility Effect of Magnesium Silver Alloys 1% on Bovine Bacterial Species

MgAg 1% alloys inhibit bacterial growth during the dry-off period. MgAg1% silver and magnesium amount was determined and the supernatant was used for the detection of antibacterial tests against S. aureus and E. coli and Geobacillus stearothermophilus var. calidolacits. The antibacterial effect of AgNO3 solution and degradation medium of MgAg1% sticks were evaluated. The bouillon dilution test showed a 5-fold reduction in bacterial colonies. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) calculations were used to test the antibacterial activity. The Brilliant Black Reduction Test (BRT-MRL screening test) showed inhibition of Geobacillus stearothermophilus bacteria at AgNO3 concentrations 0.01 mmol/l and 3 ml of degradation medium. Results were satisfying, concerning biocompatibility, degradation, and antibacterial effects.


Introduction
Mastitis is the infammation of the mammary glands, which occur primarily due to intramammary bacterial infection [1].Bovine mastitis remains the largest hazard in the global dairy industry since it causes major economic loss due to the reduction in milk yield.Most cases of subclinical mastitis are caused by contagious mastitis bacteria such as Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (Str.agalactiae), whereas other environmental pathogens like Str. uberis and Escherichia coli (E.coli) are also considered to cause chronic mastitis [2].Local antibiotic treatments of the mammary glands are the most common treatment method for mastitis [3].However, sometimes antibiotics show poor responses and these difer for diferent pathogens causing mastitis [4].Each usage of antibiotics causes the risk to develop antibiotic resistance in pathogenic and commensal bacteria.Terefore, alternatives to antibiotic compounds have to be investigated.A biodegradable magnesium silver alloy (MgAg 1%) could be a promising tool to treat bovine mastitis.Magnesium (Mg 2+ ) is an attractive material for biodegradable implants because of its low thrombogenicity, well-known biocompatibility, and lightweight [5].Te in vivo corrosion of Mg 2+ -based implants involves the formation of soluble nontoxic oxide which is excreted via the urine [6].Silver (Ag + ) is a broad-spectrum antibiotic with antiseptic and antiinfammatory properties that are not yet associated with bacterial resistance [7].Also, its ability to immediately destroy microorganisms by disturbing the bacterial cell membrane function and stopping cellular respiration recognizes it as an important element [8].It is considered efective at low concentrations in addition to its therapeutic activity [9].Tis study aimed to investigate the ability of MgAg1% alloy to inhibit bacterial growth during the dry-of period.We hypothesize that MgAg1% alloy is efective in controlling bacterial growth during the dry-of period.

Preparation of Magnesium-Silver 1% (MgAg1%) Stick and
Silver Nitrate.Te MgAg1% alloy was made from 99% magnesium and 1% silver with a weight of 10-70 mg, length of 1 cm, and a diameter of 0.2 cm (Institute of Materials Science, Leibniz University, Hannover, Germany) (Figure 1).Tese dimensions mentioned are for the original samples as received, and alloys which were used of samples with diferent dimensions and consequently diferent weight (10 to 70 mg) were cut from the original samples to be used in the diferent tests.Silver nitrate was purchased from Merck, Darmstadt, Germany.Te MgAg1% sticks were incubated in 5 ml NaCl solution in a water bath for 15 days and the supernatant was used.

Determination of the Silver Concentrations in the Degradation Medium.
Te amount of silver in the degradation medium of MgAg1% was measured using the kit NANOCOLOR Silver 3 test using a photometric procedure determination.Ag + could be measured between 0.20 and 3.00 mg/l Ag + , as silver ions, react with an indicator to form a blue dye.In brief, for the determination of Ag + concentrations in the degradation medium, the following was done: A stock solution of 1 mmol/l AgNO 3 was prepared, and a calibration standard curve of 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.8 mmol/l was prepared (Figure 2).Sterile MgAg 1% sticks were incubated in diferent volumes of the medium (1, 3, and 10 ml) or NaCl without adding any other supplements, in addition to one tube of the medium as the negative control.Tey were incubated in a water bath at 37 °C with a shaking process for 25 days.Te test was applied according to the manual manuscript with slight modifcations.28 µl NANOCOLOR Silver 3 was added per well of a 96-well plate.10 µl of the NANOCOLOR Silver 3 R2 was added.Ten, 80 µl of the tested sample was added and all the reagents were shaken.Afterward, 10 µl NANOCOLOR Silver 3 R3 was added per well, then it was mixed again and incubated for 10 min.Te samples were in duplicate numbers.Te measurements were performed on days 5, 10, 15, and 25 using a photometer with a wavelength of 620 nm.

Determination of the Magnesium and Calcium
Concentrations in the Degradation Media.Te Mg 2+ and Ca concentrations in the degradation media were measured by the test kit NANOCOLOR hardness 20 using a photometric procedure.Te test is based on measuring the total hardness of the medium, which is primarily maintained by Mg 2+ and Ca but is also determined by barium and strontium ions.Te color intensity depends on the total hardness of the surrounding medium.Te calcium content can be obtained from the measured values of total hardness, and the Mg content can be calculated.
A stock solution of MgCl2 was prepared (10 mmol/l), and a calibrations standard curve of 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 mmol/l was prepared (Figure 3).Te samples were prepared from the degradation media by incubating MgAg1% sticks in diferent volumes of 10, 3, and 1 ml of medium or NaCl.One tube of pure medium or pure NaCl served as a negative control.Subsequently, 6 µl of tested samples were added in each well of a 96-well plate, and then 180 µl of NANOFIX hardness 20 R2 was added per sample and 6 µl of hardness 20 R3 solution was added per sample.It was mixed by pipetting up and down.Te samples were in duplicate numbers.Te samples were measured at 570 nm.
Samples of higher concentrations above the calibration curve were diluted.Te measurements were performed on days 5, 10, 15, and 25.  interpreted by assessing the change in the color of the indicator present in the test medium.Penicillin G served as a positive control and milk as a negative control.MgAg1% sticks were incubated with 1, 3, and 10 ml of Dulbecco's MEM medium (DMEM), and the concentration of Mg 2+ and Ag + was noted in each volume.In addition, diferent concentrations of AgNO 3 solution (control, 0.0001, 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 mmol/l) were incubated with DMEM medium in test tubes.All tubes were incubated in a water bath for 5 days, then 100 µl of each sample was added per well and incubated for 5-6 hours at 65 °C.

Statistical Analysis.
Data are presented as mean-± standard deviation (SD).Statistical analysis was carried out by two-way analysis of variance.Calculations were performed with GraphPad Prism ® 5.03.P values < 0.05 were considered statistically signifcant.

Te Concentration of Magnesium and Silver.
Te amount of magnesium and silver in MgAg1% degradation media with NaCl was about 0.8 mmol/l and 0.3 mmol/l after 15 days of incubation, respectively (Figure 4).

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
Te MIC and MBC confrmed the qualitative antibacterial efcacy of the MgAg1% sticks, and the results are presented in Table 1.Te minimum inhibitory concentration (MIC) of MgAg1% sticks was quantitatively investigated using the turbidity method.Results showed that a normal microbial growth inhibition zone was present.Te antibacterial efect of various concentrations of MgAg1% relative to the tested pathogenic bacteria was highly signifcant.MIC and MBC ranged between 0.2 and 0.35 mL.Results show that the inhibition ratio of microbial strains increased to the maximum level of 620 nm added before reaching the limit, where MgAg1% inhibited growth.S. aureus obtained the highest inhibitory efect at concentrations of 0.2 mL and 0.35 mL on MIC and MBC.

Bouillon Dilution Test.
Te number of bacterial colonies of E. coli without incubation with MgAg1% stick was 1299, while after incubation with MgAg1% degradation medium after 15 days bacterial colonies decreased to 269.In addition, the number of bacterial colonies of S. aureus was 1194, while after incubation with MgAg1% degradation medium bacterial colonies decreased to 177.Tus, the MgAg1% sticks had an antibacterial activity with 5 times fewer colonies with bacterial dilutions 100 × 10 6 and 210 × 10 6 KbE/ml (Figure 5).

Brilliant Black Reduction (BRT-MRL Screening Test).
B. stearothermophilus bacteria shifted from brilliant black color to yellow or colorless, when treated with concentrations of AgNO 3 equal to or below 0.03 mmol/L.While the color of the bacteria shifted to blue color at a volume higher than 3 ml of degradation medium of MgAg1% sticks.Positive results were seen in 0.03 up to 1 mmol/l of AgNO 3 solution (Figure 6).

Discussion
In the present study, MgAg1% alloys are used for killing the pathogens responsible for causing mastitis.A combination of magnesium and silver was chosen due to its distinct properties and negligible side efects.Mg 2+ serves as a biodegradable material, and Ag + has a good antimicrobial efect.Depending on the concentration of Ag + used, it can act as a bacteriostatic or bactericidal [10].Te use of antimicrobial therapy afects the human consumption of dairy products due to the presence of antibiotic residues in milk, it also increases labor and veterinary costs.As stated by Tilton, the soluble form of silver is most toxic to bacteria, therefore silver sticks are degraded in the solution of NaCl and used in the present study 7. Some clinical scenarios among humans that consume infected dairy products have public health importance, such as streptococcal sore throat, food poisoning due to S. aureus, tuberculosis, diphtheria, and brucellosis [11].Antibiotic infusions into the mammary glands are the most common treatment method for mastitis [3,12].
Results showed that the number of colonies of E. coli and S. aureus incubated with diferent volumes of MgAg1% in NaCl, decreased 5 times compared to the control.Te concentration of silver which induced the antibacterial efect was 0.2-0.3mmol/L which was measured after 15 days of incubation.Another study by Starodub and Trevors also showed the same result that E. coli strain R1 cultures treated with 0.3, 0.5, and 1 mM of AgNO 3 reduced the fnal cell biomass [9].Among the three concentrations used 0.5, and 1 mM showed a good antibacterial efect even after the incubation period was extended to 48 hours.Te result of another study conducted by Kyun et al. showed a decrease in the bacterial count of S. aureus and E. coli due to silver ions [10].
Te results obtained from the BRT test showed that B. stearothermophilus shifted from brilliant black color to yellow or colorless at a concentration equal to or more than 0.03 mmol/, which indicates that concentrations of AgNO 3 solution starting from 1 up to 0.002 mmol/l have an antibacterial efect.Moreover, diferent volumes of degradation medium of MgAg1% shifted the blue color to yellow or colorless at volumes equal to or below 3 ml.Te concentration of Ag + in the solutions which showed an antibacterial efect was 0.02-1 mmol/l.Jansen et al. and Schierholz et al. stated that the efect of Ag + is continuous and long-lasting due to the oligodynamic efect of elementary Ag + [8,13].Moreover, Bowswald et al. stated that the ability of many bacteria to produce a bioflm is reduced, and the likelihood of bacterial colonization is decreased when Ag + is introduced [14].Many studies stated the antibacterial activity of nonsteroidal anti-infammatory 4 Veterinary Medicine International drugs, local anaesthetics, phenothiazine such as chlorpromazine, levomepromazine, promethazine, trifuoperazine, methdilazine and thioridazine, antidepressants, antiplatelet, and statins [13].Several studies have explored a possible protective efect of statins in decreasing the morbidity and mortality of many infectious diseases [15].Various nonantibiotic agents exhibit antimicrobial activity via multiple and diferent mechanisms of action [14,16].Te present  Tere is a signifcant increase of silver in degradation medium after 25 days of incubation, ( * p < 0.05, n � 6).(b) Mean silver concentration (mmol/l) in degradation medium after incubation of MgAg1% sticks in 1, 3, or 10 ml medium.Tere is a signifcant increase of silver in degradation medium after 25 days of incubation, ( * p < 0.05, n � 6).Veterinary Medicine International study could be a useful aid for the treatment of mastitis.MgAg1% sticks can be useful as an alternative or adjunct to antibiotics for the treatment of mastitis at the dry-of period [17].

Conclusion
Te current study was successful in treating the infections by the potential efect of the alloy sticks, and further investigations were done in reducing the culling rates in the dairy industry and minimizing the harmful efects on humans.However, it may be of interest to try to achieve higher concentrations in further investigations in vivo.6 Veterinary Medicine International

Figure 4 :
Figure 4: (a) Mean silver concentration (mmol/l) in degradation medium after incubation of MgAg1% sticks in 1, 3, or 10 ml medium.Tere is a signifcant increase of silver in degradation medium after 25 days of incubation, ( * p < 0.05, n � 6).(b) Mean silver concentration (mmol/l) in degradation medium after incubation of MgAg1% sticks in 1, 3, or 10 ml medium.Tere is a signifcant increase of silver in degradation medium after 25 days of incubation, ( * p < 0.05, n � 6).

Figure 5 :
Figure 5: Bouillon dilution test: the number of the bacterial colonies of E. coli and S. aureus incubated with MgAg1% sticks were decreased 5 times compared to nonincubated ones.

Figure 6 :
Figure 6: BRT screening test: + wells indicates antibacterial activity, while −ve wells indicate negative results.Positive results were seen in 1, 0.3, 0.1, 0.03, and 0.01 mmol/l of AgNO 3 solution, 0.003 and 0.001 mmol/l of AgNO 3 solution showed a suspicious result, while 0.0003 and 0.0001 mmol/l of AgNO 3 solution showed a negative result. 1 ml and 3 ml of MgAg 1% degradation medium showed a positive result, and 10 ml of MgAg 1% degradation medium showed a negative result ( * p < 0.05, n � 6).
Antibacterial Activity 2.4.1.Determination MgAg 1% Sticks on MIC and MBC of the Pathogenic Bacteria.Te broth dilution method (31) used Staphylococcus aureus, and Escherichia coli, as indicator species to assess the MIC and MBC values of MgAg1% sticks.(a) Diferent mass amounts of MgAg1% stick concentrations were incubated in 5 mg/ml to 0.156 mg/ml with adjusted bacterial concentration (10 8 CFU/ml, 0.5 McFarland's standard) for 24 h at 37 °C.Te MIC endpoint is the lowest concentration of MgAg1%, where no visible growth is seen in the tubes.
(b) One ml of pathogenic organism inoculum (2 × 10 5 CFU/ml) was applied to tubes containing MgAg1% sticks growth media in a serial two-fold dilution.(c)Turbidity tubes were measured after 24 hours as a growth indicator.Te minimum inhibitory concentration (MIC) related to control is the lowest MgAg1% stick concentration that inhibits the organism's growth as detected by no visual turbidity.bouillondilution test was done by preparing a bacterial suspension by incubating about 2 E. coli and S. aureus colonies (GK) in 100 ml bufered peptone water at 37 °C for 24 hours on the shaker.Te bacterial dilution series was 770 × 10 5 , 910 × 10 5 , 100 × 10 6 , and 210 × 10 6 KbE/ml.Ten, 2 test tubes were flled with 5 ml of bacterial suspension, then degrading was added to one of the tubes, and the other 2Veterinary Medicine International one serves as a negative control.Later, the number of bacterial colonies was determined.Secondly, E. coli and S. aureus colonies were cultivated in Petri dishes.Afterward, the bacterial colonies were incubated with the degrading supernatants, and the number of bacterial colonies was determined.2.4.3.Brilliant Black Reduction Test.Te BRT was frst described by KRAACK and TOLLE (1967) (15).Tis test is used for detecting antimicrobial agents in milk.Te test medium consists of a mixture of nutrients, brilliant black indicator, bacteria B. stearothermophilus var.calidolactis C953, and other supplements.Te results are visually

Table 1 :
Efect of MgAg 1% sticks at diferent concentrations on the growth inhibition of S. aureus and E. coli using the turbidity method at 620 nm; values are means ± ?standard deviation(SD)of at least three experiment (n? > 3).Means with different letters within each coloum are significant at ? � 0.01 level and means without letters are not significant N.S � not significant.