MATERIALS AND METHODS

Study compound: Sodium silicate complex (Na_8.2Si_4.4H_9.7O_17.6 M.Wt. 563.4 mol^-1) manufactured using a pyrosynthesis reaction was supplied by Cisne Enterprises Inc. (Odessa, TX). The total concentration of silicates (212.73 mg mL^-1) were quantified by the ammonium molybdate assay at 450 nm described previously (Townsend et al., 2010a).

Bacterial growth conditions: Gram-negative bacteria E. coli K-12 (ATCC 700926), E. coli O157:H7 (ATCC 25922) E. coli O157:H7 (ATCC 35150) and Salmonella enterica serovar Typhimurium LT2 (ATCC 15277) and Gram-positive bacteria were Enterococcus faecalis (ATCC 19433), Staphylococcus aureus (ATCC 12600), S. aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 19615) were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Antibiotic resistant strain of E. coli, S. aureus, Pseudomonas aeruginosa and Enterobacter cloacae fluorescens (Table 1) isolated from clinical subjects were a kind gift of Dr. Irene Lopez Lozoya (International Laboratory References and Services, Torreon, Mexico). Recommended media and growth conditions (Table 2) were used to prepare stock cultures in dimethyl sulfoxide and stored at -80°C.

Antibacterial activity and MIC₉₀: A broth microdilution method for susceptibility testing of antibacterial agents as recommended by the Clinical and Laboratory Standards Institute was used to determine the antimicrobial activity of SSC (Mansouri et al., 2011; Iroha et al., 2011).

Table 1:	Antibiotic resistance in bacterial strains isolated from clinical subjects

Table 2:	Standard media and growth conditions used for different bacterial strains tested

Briefly, a culture of the test organism was grown at 35°C with aeration in sterile cation-adjusted Mueller-Hinton broth (Becton-Dickinson, Sparks, MD) until slightly turbid and the turbidity was then adjusted to a 0.5 McFarland standard (equivalent to approximately 10⁸ colony-forming units (CFU) mL^-1. To improve cell yield in the Enterococcus faecalis and Streptococcus pyogenes cultures, the Cation-adjusted Mueller-Hinton broth was supplemented with 2.5 mg mL^-1 of yeast extract (Becton-Dickinson). Appropriate dilutions of SSC were prepared in sterile Cation-adjusted Mueller-Hinton broth such that the final concentrations of the product were 0.0 (Control) 3.2, 6.6l, 10.6, 13.2, 21.3, 26.6, 42.5, 53.2, 63.8, 106.4 and 212.7 μg mL^-1. Appropriate volumes of the standardized cell suspension were added to the dilutions of the product to achieve a final cell density of 5x10⁵ CFU mL^-1. The dilutions were then aliquoted into the wells of a 96-well microtiter plate (with a volume of 0.2 mL^-1 well) and incubated at 35°C for 16-20 h along with no cell controls. The culture turbidity (A₅₉₀) was measured by using a microtiter plate reader (Biotek, Winooski, VT) and used to calculate the Percent inhibition in growth using the following formula:

The concentration of the sodium silicate that inhibited the growth of bacteria by 90% or more was designated as MIC₉₀.

Determination of acute oral toxicity and LD₅₀: To determine potential suitability of sodium silicate complex in food safety applications, Acute oral toxicity potential of the sodium silicate complex was evaluated in female (nulliparous and non-pregnant) Sprague-Dawley albino rats (Vijayabalaji et al., 2010; Singh et al., 2012). The animal experiments were in accordance with Environmental protection agency health effects test guidelines (EPA, 2002) and conformed to Guide for the Care and Use of Laboratory Animals (NRC, 2011) and were approved by the Institutional Animal Care and Use Committee. Young, adult female (nulliparous and non-pregnant) Sprague-Dawley Albino rats with starting weight of 178-184 g were acclimated to laboratory conditions (22±3°C; RH-30-70%, 12 h dark/light cycle) for 5 days. Rats without any abnormality or pathological change were used in the study (Wiam et al., 2005). The animals were fed ad libitum with water and a commercial rodent diet Formulab # 5008 (PMI Feeds Inc. Saint Louis, MO) except for approximately 16 h before dosing. Three animals were randomly selected and an individual dose was calculated for each animal based on its fasted body weight and administered by gavage at a volume of 5.82 mL kg^-1 (5209 mg kg^-1). Clinical/behavioral signs of toxicity were made at least three times on the day of dosing (day 0) and at least once thereafter got 14 days. Individual body weights were recorded prior to dosing on days 7 and 14. On day 14 after dosing each animal was euthanized by an overdose of CO₂, all animals were subjected to gross necropsy and all abnormalities were recorded.

Statistical analysis: For antibacterial assays, experiments were replicated twice for each concentration and a minimum of six replicates were set up for each concentration and used to calculate the average mean and standard deviation. Data were subjected to analysis of variance and differences between means were regarded to be statistically significant when p<0.05.

RESULTS

Antibacterial effect on gram negative bacteria: Sodium silicate complex was effective in inhibiting the growth in all Gram negative bacteria tested (Fig. 1). In the control strains, used to test the validity of the antibacterial assays, E. coli K-12 (ATCC 700926) and E. coli (ATCC 25922) the MIC₉₀ was determined to be 21.3 and 26.6 μg mL^-1, respectively (Table 2). The MIC₉₀ values for both E. coli O157:H7 (ATCC 35150) and S. enterica (ATCC 15277) were determined to be 21.3 μg mL^-1 (Table 3).

Antibacterial effect on gram positive bacteria: Sodium silicate complex at 53.2 and 42.5 μg mL^-1 inhibited the growth of E. faecalis (ATCC 19433) and S. aureus (ATCC 12600), respectively by more than 90% (Table 4). The MIC₉₀ for another strain of S. aureus (ATCC 25923) was determined to be 26.6 μg mL^-1 (Table 4). Among all Gram positive bacteria, the MIC₉₀ for S. pyogenes (ATCC 19615) was the lowest and was determined to be 10.6 μg mL^-1 (Table 4). Overall, sodium silicate was effective in inhibiting the growth in all Gram-positive bacteria tested (Fig. 2).

Table 3:	MIC90 of SSC against tested Gram-negative bacterial strains

Table 4:	MIC90 of SSC against tested Gram-positive bacterial strains

Fig. 1:	Inhibitory effect of SSC against tested Gram-negative bacterial strains

Fig. 2:	Inhibitory effect of SSC against tested Gram-positive bacterial strains

Table 5:	MIC90 of SSC against clinically isolated multi-drug resistant bacterial strains

Antibacterial effect on antibiotic resistant bacteria: Sodium silicate complex inhibited the growth of all four strains of bacteria resistant to different antibiotics (Table 3) (Fig. 3). The MIC₉₀ for E. cloacae (212.7 μg mL^-1) was highest for all the bacteria tested. The MIC₉₀ for antibiotic resistant S. aureus was 106.4 μg mL^-1 (Table 5) which was higher than the MIC₉₀ for two other strains of S. aureus (Table 5). The MIC₉₀ for the drug resistant strain of E. coli was also higher than the non-drug resistant strains and was determined to be 42.5 μg mL^-1. The complex was also effective in inhibiting the growth of multi-drug resistant P. aeruginosa and the MIC₉₀ was calculated to be 42.5 μg mL^-1 (Table 5).

Acute oral toxicity and LD₅₀: There was no mortality observed during the study. The body weight gain was not affected by the administration of sodium silicate complex (Table 6).

Fig. 3:	Inhibitory effect of SSC against clinically isolated multi-drug resistant bacterial strains

Table 6:	Evaluation of acute oral toxicity effect of SSC at 5209 mg kg-1 by oral-gavaging in female albino Sprague-Dawley rats
NOA: No observable effects

All animals appeared normal for the duration of the study with no abnormal clinical signs or behavior (Table 6). The gross necropsy at the termination of the termination of the study revealed no observable abnormalities. The LD₅₀ was determined to be greater than 5000 mg kg^-1.

CONCLUSIONS

SSC exhibited antimicrobial activity against many Gram positive and Gram negative bacteria. The MIC₉₀ values ranged from 21.3-26.6 μg mL^-1 for Gram negative bacteria and 10.6-53.2 μg mL^-1 for Gram positive pathogens. In addition, SSC was also effective in inhibiting the growth of clinically isolated multi-drug resistant strains of bacteria (MIC₉₀ range: 42.5-212.7 μg mL^-1). A LD₅₀ for oral toxicity suggests that the SSC may have potential applications in reducing clinical infections and microbial contamination of food and water.

ACKNOWLEDGMENTS

We would wish to thank CISNE Enterprises Inc. (Odessa, TX) for manufacturing the SSC; Dr. Derek Jeter (Texas Tech University, Lubbock, TX) for assistance with the antibacterial assays, Dr. Irene Lopez Lozoya (International Laboratory References and Services, Torreon, Mexico) for providing us with the multi-drug resistant strains and Stillmedow Inc. (Sugarland, TX) for their assistance with the animal experiments.