BACTERICIDAL EFFICIENCY OF SILVER NANOPARTICLES AGAINST METHICILLIN-RESISTANCE (MRSA) AND METHICILLIN-SUSCEPTIBLE STAPHYLOCOCCUS AUREUS (MSSA) STRAINS ISOLATED FROM MILK AND ITS SURROUNDING MILKING ENVIRONMENT

BACTERICIDAL EFFICIENCY OF SILVER NANOPARTICLES AGAINST METHICILLIN-RESISTANCE (MRSA) AND METHICILLIN-SUSCEPTIBLE STAPHYLOCOCCUS AUREUS (MSSA) STRAINS ISOLATED FROM MILK AND ITS SURROUNDING MILKING ENVIRONMENT

SABER KOTB^* and MOHAMMED SAYED^**

^*Department of Animal Hygiene, Faculty of Veterinary Medicine, Assiut University, 71526 Assiut, Egypt.

E-mails: saberkotb@yahoo.com; saber.kotab@vet.au.edu.eg

^**Department of Food Hygiene, Faculty of Veterinary Medicine, Assiut University, 71526 Assiut, Egypt.

E-mails: dr.mohammedsayed@yahoo.com; dr.mohammedsayed@vet.au.edu.eg

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is a critically important human pathogen that is also an emerging concern in veterinary medicine and animal agriculture. It is present in a wide range of animal species; both are the cause of infection and also in healthy carriers. Many studies of MRSA carriage and infection in livestock animals and human contacts from 1993 to 2008 were listed in the review of David and Daum (2010).

Isolation of MRSA in animals was first recorded in 1972, when it was detected in the milk of mastitic cows (Devriese et al., 1972). It has been increasingly frequent reports of MRSA infections in animals in recent years, including 2outbreaks of infection in veterinary teaching hospitals in North America (Sequin et al., 1999; Lee, 2003; Goni et al., 2004; Weese et al., 2004; O’Mahony et al., 2005; Rich       et al., 2005).

Recently, the increasing number of drug-resistant bacteria has become a major challenge endangering human health. Staph. aureus infections have assumed new public health importance due to emerging multiple antibiotic resistant strains, particularly MRSA and its epidemic clones, increasingly being found in communities and hospitals (Moran et al., 2006; Pesavento et al., 2007). The emergence of pathogenic microorganisms resistant to commonly used antibiotics is a worldwide concern of the 21^st century. One of the most important bacteria in this regard is Staph. aureus, in particular its methicillin-resistant strains (David et al., 2008). Furthermore, investigators have found that over the last 10 years MRSA strains have overtaken and replaced methicillin-susceptible Staphylococcus aureus (MSSA) strains as the leading cause of staphylococcal infections, which in turn have become more prevalent (O’Hara et al., 2012).

Remarkable progress in research and development of metalnanoparticles (NPs) was noticed; that nanotechnology has dynamically developed as an important field of modern research with potential effects in electronic and medicine. Silver nanoparticles (Ag-NPs) have a number of applications from electronics and catalysis to biology, pharmaceutical and medical diagnosis and therapy. The antibacterial activity of silver ions is well known, however, the antibacterial activity of elementary silver, in the form of NPs has been developed (Glomm, 2005; Chan, 2006; Boisselier and Astruc, 2009). Ag-NPs have been also demonstrated as an effective biocide against drug-resistant strains (Kvítek et al., 2008; Jones and Hoek, 2010; Lara et al., 2010; Ansari et al., 2011).

Researches on Ag-NPs as a possible antimicrobial agent have received (Silver, 2003; Baker et al., 2005; Lee et al., 2008). It has been known that silver and its compounds have strong bacteriostatic and bactericidal effects against bacteria, fungi and virus since ancient times (Cho et al., 2005; Lok et al., 2006). In comparison with other metals, Ag-NP exhibits higher toxicity to microorganisms while it exhibits lower toxicity to mammalian cells (Zhao and Stevens, 1998). Hence, Ag-NPs have been applied to a wide range of healthcare products, such as burn dressings, water purification systems and medical devices (Thomas et al., 2007).

From the interesting point of the bactericidal effect of Ag-NPs, this study was planned to explore the efficiency of bactericidal effect of Ag-NPs against MRSA and MSSA strains that were previously isolated from milk and its surrounding animal milking environment.

MATERIALS and METHODS

Materials:

Silver nitrate (Ag NO₃) crystal (ACS Ag NO₃ F.W. 169.87 Gamma laboratory chemicals, assay: 99%).

Cultures:

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) strains were previously isolated and obtained from milk and its surrounding animal milking environment in the study of Sayed and Kotb (2011), from 60 dairy cattle houses including 30 for cows and 30 for buffalos.

Refreshment of the cultured strains:

The cultured strains were harvested in tryptic soy broth (TSB, Difco), centrifuged (1000 g / 15 min) and suspended in saline. Bacterial viability was estimated through pour plate on tryptic soy agar (TSA, Difco), confirming populations over 10⁷cfu / ml according to Mazzola et al. (2009).

Inoculum preparation for minimal inhibitory concentrations (MIC):

Inocula were obtained from an overnight agar culture of the test organism, in which, the inoculum for MIC test was prepared by taking at least 3 to 5 well isolated colonies of the same morphology from an agar plate culture. The top of each colony was touched with a sterile loop and the growth was transferred into a tube containing 4 to 5 ml normal saline. The broth culture was incubated at 35-37ºC, until it achieved turbidity of the 0.5 McFarland standard. This suspension was containing about 1-2x10⁶cfu/ ml. The turbidity of the actively growing broth culture was adjusted with sterile broth to obtain turbidity comparable to that of the 0.5 McFarland standard.

Preparation of the 0.5 McFarland standard:

A 0.5 McFarland standard was prepared as described in NCCLS (1997). 1% V/V solution of sulfuric acid was prepared by adding 1 ml of concentrated sulfuric acid to 99 ml of dist. water and mixed well. 1.175% W/V solution of barium chloride was prepared by dissolving 2.35 g of dehydrated barium chloride (BaCl₂.H₂O) in 200 ml of distilled water with constant stirring. For making the turbidity standard, 0.5 ml of the barium chloride solution was added to 99.5 ml sulfuric acid solution1% and mixed well. The standard was distributed into screw cap tubes of the same size and with the same volume as those used in growing the broth cultures. The tubes were sealed tightly to prevent losses by evaporation and stored protected from light at room temperature. The turbidity standard was vigorously agitated on a vortex mixer before use. Standards may be stored for up to 6 months, and then they should be discarded.

Preparation of silver nanoparticles (Ag-NPs):

Stable Ag-NPs<100 nm were synthesized according to Vigneshwaran et al. (2006), where 1 g soluble starch was added to 100 ml de-ionized water and heated till complete dissolution, 1 ml of 100 mMaq silver nitrate (Ag NO₃) solution was added and stirred well. This mixture was put into dark glass bottle and autoclaved at 121ºC for 5 min. Formation of clear yellow solution indicates the synthesis of Ag-NPs. The size of Ag-NPs was measured by Transmission Electron Microscopy (TEM) Model JEOL-JEM-100CX II in the Electron Microscopy Unit, Assiut University, Egypt; while, the total concentration of Ag-NP stock was measured by Graphite Furnace Atomic Absorption Model 210VGP in the Faculty of Science, Assiut University, Egypt.

MIC and MBC determination:

MIC technique: applying MIC technique was performed according to CLSI (2009) using successive serial 2-fold dilution technique. The original concentration of Ag-NP was 100µg/ ml. The MIC is the lowest concentration of Ag-NPs that inhibit microbial growth. The MIC technique was done in triplicate to confirm the MIC value for each tested organism.

Minimal bactericidal concentration (MBC): MBC was determined after performing MIC technique. Aliquots of 50 µl from all positive MIC tubes; Ag-NPs was quenched by adding 5 g/L sodium thiosulfate (Na₂S₂O₃) to stop the antimicrobial reaction between Ag-NPs and bacteria as described in the European quality standards (Nen, 1997). Tube has no bacterial growth was planted in Muller Hinton agar(DIFCO, Becton Dickinson, USA) free from Ag-NPs then incubated at 37ºC for 24 hr. The MBC endpoint is defined as the lowest concentration of antimicrobial agent that kills 100% of the initial bacterial population.

Statistical analysis:

MIC and MBC tests were performed in triplicate, and the results were expressed as the mean ± the standard errors of the mean. Student’s‘t’ test was used to compare these results. P values lower than 0.05 were considered significant.

RESULTS

Figure 1: Transmission Electron Microscopy (TEM) image of Ag-NPs with spherical shapes (sizes ranged from 12.4 to 31.1 nm).

Table 1: MIC and MBC and MBC/MIC ratio of Ag-NPs tested against MRSA and MSSA.

^a: non-significant correlation (p>0.05).

Figure 2: Percentages of MRSA isolates showing values of MIC and MBC.

Figure 3: Percentages of MSSA isolates showing values of MIC and MBC.

DISCUSSION

Due to the growing number of outbreaks of infection caused by MRSA and the development of antibiotic resistance, it becomes essential to investigate other antibacterial agent’salternative for treatment of MRSA infections. Among the range of compounds whose bactericidal activity is being investigated, Ag-NPs rise as a promising new antibacterial agent that could be helpful to confront drug resistant Staph. aureus. Furthermore, recently, the field nanotechnology represents a modern and innovative approach to develop new formulations based on metallic nanoparticles with antimicrobial properties.

From the aforementioned results in Table 1, it was found that the mean values of MIC and MBC of Ag-NP sagainst MRSA were 23.44±0.21 and 31.25±0.26 µg / ml, respectively. In the same time, it was observed (Table 1) that the mean values of MIC and MBC of Ag-NPs against MSSA were 11.33±0.14 and 13.28±0.17 µg / ml, respectively. Percentages of MRSA isolates showing values of MIC and MBC were illustrated in Figure 2, and for MSSA isolates in Figure 3.

The obtained findings nearly coincided with the findings reported by Martinez-Castanon et al. (2008), Kulkarni et al. (2014), who mentioned that Ag-NPs were inhibitory against Staph. aureus ATCC25923 and methicillin-resistant Staphylococcus at concentration of 16.67 and 15µg/ml, respectively. In the study of Ansari et al. (2011), who examined bacterial growth curve, MIC and MBC of Ag-NPs towards Staph. Aureus ATCC25923, MSSA and MRSA and found the lowest MIC and MBC of Ag-NPs to MRSA were 12.5 and 25 μg / ml, respectively, suggesting that Ag-NPs exhibit excellent bacteriostatic and bactericidal effect towards all clinical isolates tested regardless of their drug-resistant mechanisms.

On the other hand, the obtained findings were not coincided with Ayala-Nunez et al. (2009), who found that the MIC and MBC against MRSA and MSAA were 1.35 and 2.7mg/ml, respectively. Also, the obtained results were not agreed with Ghosh et al. (2013) who reported that the MIC value of Ag-NPs against Staph. aureus was 3.062 ng/ml. These difference results might be attributed to the difference in Ag-NPs sizes (Morones et al., 2005; Song et al., 2006). Generally, the smaller sized Ag-NPs have the larger surface area which could interact with bacterial cell membranes and then alter their permeability and respiration (Panacek et al., 2006).

The MBC/MIC ratio is a parameter that indicates the bactericidal capacity of Ag-NPs by relating bothvalues. In addition, the MBC/MIC ratio can reflect if the bacteria are susceptible, tolerant, or resistant to the Ag-NPs that are being challenged. The obtained data illustrated in Table (1) showed that Ag-NPs inhibited the bacterial growth of both of MRSA and MSSA showing a bactericidal rather than a bacteriostatic effect (MBC/MIC ratio ≤4). Clinically, bactericidal agents are favorable than bacteriostatic and if the pathogen is killed rather than inhibited, leading to faster combating of spreading of infection and reducing emergence of bacterial resistance (French, 2006).

From the statistical analysis of the present results, it was found that no significant difference between the effect of Ag-NPs on MRSA and MSSA (p> 0.05). This result indicated that bactericidal activity of Ag-NPs was not affected by the resistant mechanisms that responsible to differentiate between MRSA and MSSA strains. Moreover, bactericidal efficiency of Ag-NPs against MRSA was higher than data of commercial antibiotic. For instance, the MIC of gentamicin antibiotic against MRSA was 64 µg/ml (Ayala-Nunez et al., 2009).

Several research papers illustrated the mode of action of Ag-NPs. Apparently; Ag-NPshave nodirectinhibiting effect on the expression or the activity of the PBPs (penicillin-binding proteins) (β-lactamic resistance) (Palavecino, 2007). Silver ions are known to react with sulfhydryl groups on the cell wall to generate disulfide bridges, which would block respiration and cause cell death (Raffi et al., 2008) or cause protein denaturation (McDonnell, 2007). In addition, silver ions cancomplex with electron donor groups containing sulfur, oxygen or nitrogen that are normally present as phosphates or thiolson amino acids and nucleic acids (Starodub and Trevors, 1989). Thus, Ag-NPs would not bind to the specific proteins orstructures of the bacterial cell of both MRSA and MSSA but to a broad spectrum of targets that would include membrane and cytoplasmic proteins and genomicor plasmid DNA. Indeed, Ag-NPs have been found to attach to the surface of the cell membrane and disturb its function, penetrate bacteria and release silverions (Lok et al., 2006).

It can be concluded that Ag-NPs have potent bactericidal activity against MRSA as well as MSSA, supporting their potential use as antibacterial agents with a wide number of biomedical and therapeutic applications.

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