Evaluation of the bactericidal effect of silver nanoparticles against Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA) strains isolated from mastitic milk of small ruminants and their surrounding environment in Aswan, Egypt

ARTICLE INFO In the recent years, emergence of infectious diseases caused by drug resistant pathogens had been increased; therefore there is an urgent need to search for new alternative and effective antimicrobial agents to overcome the drug resistance. In the present investigation, the study group consisted of 90 sheep and 90 goats with clinical evidences of mastitis in 17(18.89%) goats and 5 sheep (5.56%) that manifested swollen udder with or without systemic signs of illness related to mastitis. Standard bacteriology was performed on pretreatment milk samples from the 17 goats and 5 mastitic ewes as well as 60 soil samples and 60 pail water samples. The bacteria isolated were identified as Staphylococcus aureus 12 (70.6%) from goats and 5 (100%) from sheep. In addition, S. aureus could also be identified in 41 (68.3%) soil samples and 42 (70%) water samples. The current study aimed to explore the bactericidal effect of silver nanoparticles (AgNPs) on methicillinresistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) strains isolated from mastitic milk and their surrounding environment (water and soil) in Aswan, Egypt. AgNPs solution was synthesized by typical one-step synthesis protocol using soluble starch and was characterized using transmission electron microscopy and atomic absorption spectrophotometer. The minimum inhibitory concentration (MIC) and minimum Article history: Received: 19 October

. AgNPs can also be applied in cases of burn, traumatic wound dressings, diabetic ulcers, coating of catheters, dental works, medical devices and water filtration (Jain and Pradeep, 2005;Silver et al., 2006;Gong et al., 2007;Kim et al., 2007;Thomas et al., 2007;Rai et al., 2009;Martinez-Gutierrez et al., 2010;Dosoky et al., 2015;Ibrahim et al., 2020). In the present study, the bactericidal effect of silver nanoparticles against MRSA and MSSA strains isolated from mastitic milk of small ruminants and their surrounding environment in Aswan was investigated.

Materials and methods 2.1. Animals and samples Animals:
A total of 90 sheep and 90 goats were subjected for clinical examination where 17(18.89%) goats and 5 (5.56%) sheep showed obvious signs of clinical mastitis including swollen udder with or without systemic signs of illness related to mastitis based on the criteria described by Dirksen et al. (1993). Samples: a. Milk Milk samples were collected from 17 goats and 5 sheep mastitic milk samples out of 90 sheep and 90 goats. About 10 ml of milk were hygienically drawn from each quarter into a sterile 50 ml falcon tubes. Milk sampling was done according to the recommendation of National Mastitis Council (1999), immediately transported to the laboratory in ice-cooled containers and analyzed within 24 to 48 h after collection.

Phenotypic identification of Methicillin Resistant S. aureus (MRSA)
The positive S. aureus samples were re-streaked on mannitol salt agar (MSA, Oxoid) supplemented with 6 mg/L of oxacillin (Sigma, St. Louis, Mo.) for selective isolation of MRSA. A sample was positive for MRSA if one or more colonies were identified and one representative colony was selected from each sample for further testing. The isolates were further confirmed as MRSA by using DrySpot Staphytect Plus latex agglutination test kit (Oxoid, United Kingdom).

Inoculum preparation for minimal inhibitory concentrations (MIC):
Preparation of bacterial suspension: Colonies from MRSA and MSSA isolates were transferred to 5 ml nutrient broth and incubated at 37 o C for 17 h. Tenfold serial dilution were prepared using sterile nutrient broth as a diluent, by mixing 9 ml nutrient broth and 1 ml bacterial suspension and 1ml was transferred from the first tube to the second tube till 10 -7 concentrations and 1 ml quantity from each dilution was spread on plate count agar and incubated at 37 o C for 24 h. About 4-5 colonies from plate count agar contain 20-300 colonies were transferred to 5 ml nutrient broth then the broth culture was incubated at 35-37ºC, until it achieved turbidity of the 0.5 McFarland standards. The turbidity of broth culture was adjusted with sterile broth to obtain turbidity comparable to that of the 0.5 McFarland standards. This suspension was containing about 1-2x10 6 CFU / ml.

Synthesis of silver nanoparticles (Ag-NPs):
Stable AgNPs less than 100 nm were synthesized according to (Vigneshwaran et al., 2006). Briefly, 1gm of soluble starch was added to 100 ml of deionized water and heated till complete dissolution and cooling and 0.08 gm silver nitrate (AgNo3) was added to 1ml of deionized water in dark glass bottle. Soluble starch solution was mixed with silver nitrate solution. This mixture was put in dark glass bottle, wrapped in aluminum foil and kept in autoclave at 121 o C for 5 minutes. Clear yellow color solution indicated to the formation of AgNPs. Graphite Furnace Atomic Absorption (Model 210VGP) was used for determining the concentration of AgNPs stock solution. The size of AgNPs was measured by transmission electron microscopy (TEM) (JEOL-JEM-100CX) at Electron Microscopy Unit, Assiut University, Egypt.

Determination of minimum inhibitory concentration (MIC)
MIC technique was performed according to CLSI (2009) using successive serial 2 fold dilution technique. The original concentration of stock AgNPs solution was 100 µg / ml. The MIC technique was done in triplicate to confirm the MIC value for the tested organism.

MIC technique
Sterile capped test tubes were numbered from 1 to 6, one positive (MHB+ bacterial inoculation) and one negative (MHB+ AgNPs) tube. Two ml Muller Hinton Broth (MHB, Oxiod) were added to each tube, then 2 ml from AgNPs stock was added to the first tube and to the negative tube. The first tube was shacked and 2ml were transferred to the second tube. The contents of the second tube were mixed and then transferred 2ml to the third tube. Dilutions were continued in this manner till tube 6 and finally 2 ml were discarded. Finally, 0.1 ml of bacterial inoculum was added to all 6 tubes and to positive tube. All tubes were incubated at 37 o C for 24 h.

Determination of minimum bactericidal concentration (MBC)
Five grams / L of sodium thiosulfate (Na2S2O3) were added to Ag-NPs to halt the antimicrobial reaction between Ag-NPs and bacteria as described in the European quality standards (Nen, 1997). MBC was determined by subculturing the content of all positive MIC tubes on mannitol salt agar (MSA) plates and incubated at 37 o C for 24h. The MBC was determined as the lowest concentrations of silver nanoparticles that killed 100% of bacteria. Disinfection activity of AgNPs was measured by MBC/MIC ratio.

Statistical analysis:
MIC and MBC tests were performed in triplicate, and the results were expressed as the mean ± the standard errors of the mean. SPSS was used to compare these results. Table (

Discussion
In this study, clinical examination of a total of 90 goats and 90 sheep revealed the presence of clinical signs suggesting mastitis in 17 (18.89%) goats and 5 (5.56%) sheep. Results illustrated in table (1) reveled isolation of Staph.aureus from 12/17 (70.6%) of goat milk and in 5/5 (100%) of sheep milk, which is considered the most pathogenic microorganism for humans and animals. In this study, S. aureus was also present in 41/60(68.3%) soil samples and in 42/60 (70%) water samples. Interestingly, such findings refer to the importance of soil and water as potential sources of infection to susceptible goats and sheep. MRSA was isolated from 5/17 (29.4%) ,3/5 (60%), 21/60 (35%) and21/60 (35%) of goat milk, sheep milk, soil and water samples respectively. Data presented in

Conclusion
Results of the current study revealed that the effect of AgNPs on MRSA and MSSA did not vary substantially. The bactericidal effect of Ag-NPs against MRSA was higher than that of the commercial antibiotics

Conflict of interest statement
No conflicts of interest.