تاثیر ضد باکتریایی نانو ذرات نقره همراه با آنتی بیوتیک‏های مهار کننده سنتز پروتئین بر استافیلوکوکوس اورئوس جداشده از موارد ورم پستان گاو

Introduction

Staphylococcus aureus is an opportunistic pathogen in dairy ruminants where it is found in healthy carriage and can be a major cause of mastitis (1). Mastitis in dairy cattle is a persistent, inflammatory reaction of the udder tissue. Fatal mammary gland infection is common potential (2). This disease costs the US dairy industry about 1.7 to 2 billion dollar each year (3). Reported cure rates for S. aureus mastitis vary considerably. The probability of cure depends on cow, pathogen, and treatment factors (4). Various mastitis control programs have been used to combat the problem but have not always been efficient (5). In most countries, antibiotic resistance is common extremely. Combination therapy with penicillin and gentamicin may be used to treat serious infections; its use is controversial because of the high risk of damage to the kidneys (6).

Nanoparticles are sized between 1-100 nm (7). High surface area to volume ratios and unique chemicophysical properties of various nanomaterials are believed to contribute to effective antimicrobial activities (8). Metal nanoparticles, which have a high specific surface area and a high fraction of surface atoms, have been studied extensively due to their unique physicochemical characteristics such as catalytic activity, optical properties, electronic properties, magnetic properties, and antimicrobial activity (9). Antimicrobial mechanisms of nanomaterials include:

1- photocatalytic production of reactive oxygen species (ROS) that damage cellular and viral components,

2-compromising the bacterial cell wall/membrane,

3-interruption of energy transduction, and 4- inhibition of enzyme activity and DNA synthesis (10-11). Silver nanoparticles (AgNPs) have recently been synthesized and shown to exhibit antimicrobial activity against several species of bacteria including S. aureus (12). The antimicrobial activity of silver particles is influenced by the dimension of the particles, with smaller particles showing greater antimicrobial effect. Bacteria treated by metals including silver do not acquire resistance to the metals (13). The MIC (minimum inhibitory concentration) of AgNPs is one order lower than the one of silver ions (14). Therefore, the bactericidal metals have advantages over the conventional antibiotics which often cause the selection of antibiotic-resistant microorganism. The use of modern technology and the therapeutic properties of silver nanoparticles have requirements that had already been proven and it seems necessary. In the present study, the effect of silver nanoparticles has been investigated along with antibiotics of operation on protein bacterial synthesis.

Material and methods

Sampling

For detection of clinical and subclinical mastitis, California mastitis test (CMT) carried out. Three hundred and eleven milk samples were collected from the cow farms of Tabriz and Urmia, Iran. The breasts of cows were washed and disinfected by 70% alcohol and then were dried using sterile cotton and disposable towel. First milking of the teat was discarded and 10 ml of milk were collected in a sterile BHI bottle. All samples were kept at 4°C and transported immediately to the lab for latter examination.

Isolation and identification of S. aureus

Each milk sample (100 μl) was cultured on the surface of mannitol salt agar (Merck, Germany) and was incubated at 37°C for 24 h. Colonies suspected as S. aureus were selected and transferred to 5% sheep blood agar (Difco, USA). Gram stain, culture characteristics, and coagulase test using fresh rabbit plasma (tube method) were used for the presumptive identification of all isolates (15). Out of the 311 bovine mastitis milk samples 72 isolates of S. aureus were studied.

Molecular diagnosis of S. aureus

S. aureusDNA extracted from the 24-hour culture of S. aureus in BHI medium according to DNA purification kit (Fermentas, Germany) manufacturer protocol. To accurately identify of S. aureus, nuc gene was amplified by PCR (16). The primers of
F (5'-GCGATTGATGGTGATACGGGT-3') and R (5'-AGCCAAGCCTTGACGAACTAAAGC-3') were used for nuc amplification. PCR reaction was taken using the PCR kit (CinnaGen PCR master kit) in a final volume of 25µl, containing 12.5µl of master mix (with 2X concentration), 0.4 mM of each primer and 2 µl of DNA sample. Volume of the mixture with deionized distilled water adjusted to 25 µl. For negative control, sterile water was used instead of DNA and the extracted DNA of S. aureus (ATCC 29213) used as a positive control. DNA replication with pattern of initial denaturation temperature of 94°C for 3 min, 35 thermal cycles each consisting of denaturation at 94°C temperature for 1 minute, binding stage at 55°C for 30 seconds, and along step at 72°C for 1.5 min were performed. The final step to complete the reaction at 72°C for 3.5 min was performed. A PCR product was obtained with equal size of all S. aureus isolates. The size of PCR products was determined using 1.2% garose gel electrophoresis and marker of GeneRuler^TM 100 bp DNA ladder (Frementas, Germany).

Susceptibility test antibiotics

Bacterial samples were incubated in Mueller Hinton broth medium (Merck, Germany) and were cultured at 37°C for 24 h. After growth, the sampleswere compared with a turbidity tube of 0.5 McFarland (number of bacteria 10⁸-10⁹). The 48 wells of micro plate were considered with four wells each in one row. To determine the pattern of isolates resistance against Erythromycin, Gentamicin, Streptomycin and Doxycycline, Disk Agar Diffusion method was carried out on Muller Hinton Agar and results were reported as resistance percentage (17).

Measurement of minimum inhibitory concentration and minimum bactericidal concentration for silver nanoparticles

Eight dilutions (0, 25, 50, 60, 70, 80, 90 and 100 µg/ml) of silver nanoparticles with 97 nm in diameter (Malvern instrument, UK) were prepared using saline normal. In each well, 900 µl of bacterial suspension was added and then the 100 µl of different concentrations of silver nanoparticles were added in the wells. To establish the antimicrobial activity of silver nanoparticles on the bacterial growth, the minimum inhibitory concentration and minimum bactericidal concentration of silver nanoparticles were determined for S. aureus using optical density of the bacterial culture solution containing different concentration of silver nanoparticles after 24h. All of the experiments were triplicated, on three different days.

Measurement of MIC and MBC for silver nanoparticles along with antibiotics

To determine the MIC and MBC of silver nanoparticles in combination with antibiotics, 50 µl of each antibiotic and 50 µl of different concentrations of silver nanoparticles were added into each well containing 30 µl Hinton medium (Merck, Germany) then incubated for 24 h at 37°C (18). Finally, the rate of bacterial growth on culture plates containing bacterial suspensions with silver nanoparticles and various antibiotics were tested and the MIC and MBC of silver nanoparticles were determined along with antibiotics.

Results

Molecular Identification of S. aureus isolates

A total of 311 milk samples were cultured, 72 S. aureus isolates identified using cultural and biochemical tests. Specific molecular diagnosis carried out by nuc gene amplification. The primers amplified the expected size of 279 bp in 58 S. aureus isolates which 50 of them used in the next stages of the research. (Fig. 1)

Fig 1- electrophoresis of PCR products of nuc gene.

M: 100 bp DNA ladder (fermentas-Germany)

1: positive control (S. aureus ATCC 29213).

2: Negative control (reaction without DNA).

3-9: PCR products of the expected size of 279 bp.

Antibiotic Resistance

All of 50 isolates cultured in a concentration of 50µg/ml of the operative antibiotics separately. The resistance of S. aureus isolates against erythromycin, gentamicin, streptomycin and doxycycline were 100, 22, 100 and 8%, respectively. The MIC and MBC were determined along with silver nanoparticles only in resistant isolates.

Measurement of MIC and MBC

In this study, 50 isolates of S. aureus with eight different dilutions of silver nanoparticles were examined; the growth of four isolates was inhibited at 25 µg/ml concentration of silver nanoparticles, which was recorded as MBC. Eight percent of all isolates were sensitive to 25 µg/ml concentration of silver nanoparticles. Nighty eight percent of the samples at concentrations between 50-100 µg/ml were inhibited. The sensitive isolates to 25 µg/ml silver nanoparticles in next tests were excluded. The results of MIC and MBC for silver nanoparticles and antibiotics along with silver nanoparticles on 46 remained isolates are shown in Table 1. All tests performed in triplicate.

Table 1- Results of interaction of silver nanoparticles and antibiotics.

S: Synergistic, A: Antagonist, I: Inactive

*: In the samples that were susceptible to antibiotics, MIC and MBC of antibiotics along with silver nanoparticles were not investigated.