The Effect of MRET Activated Water on Staphylococcal Infection in vivo in Animal Model (on the cells of immune system) and in vitro on the Culture of Staphylococcus aureus Wood-46


I. The Effect of MRET Water on Staphylococcal Infection in vivo in Animal Model

The research was conducted at Kiyv National Shevchenko University, Ukraine on 400 mice. The mice-male of line BALB in the age of 11 – 13 weeks and of the weight 18 – 21 grams were used. In the process of investigation all mice were divided in three groups. Prior to the inoculation of Staphylococcus aureus Wood-46 culture one group of mice consumed MRET water during 4 weeks (Group 1), another group consumed MRET water during 2 weeks (Group 2), the control group consumed non-activated ordinary distilled water. During the following 2 weeks of experiment the first two groups continued to consume MRET water and the control group consumed ordinary distilled water. The first line of experiments was conducted on 225 mice in order to analyze the persistence of pathogen in homogenate of kidneys of mice comparing 5 groups of mice (two Group 1 and two Group 2 on 15 minutes and 30 minutes MRET activated water and Control). After preliminary experiments the optimal 30 minutes MRET activated water (distilled) was chosen for the main line of investigation.


RESULTS:


1. Significant protective properties of MRET water.

The significant protective properties of MRET water were confirmed by substantial decrease of Staphylococcus CFU (colony forming units) in homogenate of kidneys of mice on MRET water compare to control group of mice following the intra-peritoneal staphylococcal infection after the first 24 hours. The analysis of data in the beginning of experiments leads to the conclusion that significant decrease of pathogen’s colonies in homogenate of kidneys of mice on MRET water begins only after 24 hours following the inoculation of Staphylococcus culture. The results on 30 minutes activated water were much better than on 15 minutes activated water and all further experiments were conducted on 30 minute activated water.

 
2. The consumption of MRET water eliminated the death rate from 30% (control group) to 0% (MRET group) during the first 9 days of experiment.

There was no case of animal death in all investigated groups within the first 24 hours after intra-peritoneal inoculation of Staphylococcus culture, which is a pretty standard result. During the next 8 days 30% of animals died in control group which also is a standard result. But there were no death cases in both groups of mice that ingested MRET activated water and it is a remarkable result. Nevertheless, the main consequences of Staphylococcus infection do not manifest in death of animals as in case of oncology diseases. Staphylococcus virus affects the live systems and organs of the body. These pathogenic microorganisms cause inflammations, suppurations, abscesses, furuncles, quinsy, cepsical conditions, etc. That’s why a detailed investigation of the process of stimulation by MRET water of phagocytes and of lymphoid organs of immune system of mice infected with Staphylococcus aureus culture was conducted and is presented in this report.


3. The development of the local acute inflammation is essentially suppressed in case of ingestion of MRET activated water.

The local inflammation was induced with the help of the inoculation of Staphylococcus aureus culture into the hinder left paw. The ordinary inflammatory reaction was observed in the group of mice on non-activated water: the intensive reddening of the hinder left paw (Fig 1). Both groups of mice on MRET water did not develop any reddening the hinder left paw inoculated with Staphylococcus aureus culture (Fig 2). The results of this experiment confirm the fact of the substantial inhibition of inflammatory infection in case of the regular consumption of MRET water.

4. The consumption of MRET water stimulates the activity of phagocytic system and the level of natural resistance of animals to pathogenic microorganisms.

For the following series of experiments the inoculation of Staphylococcus aureus Wood-46 was conducted intra-peritoneal in dose LD30 in order to spread infection all over the body.

The phagocytic system is one of the main factors of natural non-specific cellular resistance to infections and inflammations. It is the first line of protection of an organism against penetration and reproduction of pathogenic microorganisms. The protective role of phagocytic cells is based on their capacity to identify, engulf and utilize the alien agents penetrated into internal environment of a macro-organism. Phagocytosis is the main mechanism of natural resistance especially at the first stage of contagious process; it is a regular part of formation of the specific immune response.

The most common methodology applied in the studies of the functional activity of phagocytes is the examination of their phagocytic (engulfing of alien cells) and oxygen-dependent bactericidal activity. Phagocytic activity of neutrophils and macrophages is estimated based on Index of Phagocytosis (percentage of the phagocytes which engulfed test-bacteria) and on Phagocytic Number (average number of test-bacteria engulfed by one phagocyte). The cultures of Staphylococcus aureus and Latex are usually used as test-bacteria. The oxygen-dependent bactericidal activity of phagocytes is studied with the help of NBT-test: an oxygen-dependent reduction of Nitro Blue Tetrazolium into an insoluble Diformazan of Nitro Blue Tetrazolium derivative by phagocytes. With the help of NBT-test it is possible to distinguish the activated phagocytes from the non-activated ones.

MRET water stimulated the phagocytic capacities of neutrophils of a peripheral blood and peritoneal macrophages increasing their phagocytic activity, particularly Phagocytic Indexs (Fig 3A) and Phagocytic Number (Fig 3B). It also stimulated their oxygen-dependent bactericidal activity, particularly the increase of quantity of NBT-positive phagocytes (Fig 4).

These experiments confirmed the increase of effective potentials of phagocytes which constitute one of the main factors of natural protection of an organism and initiate the immune response.

The analysis of data in the beginning of experiments leads to the conclusion that significant intensification of phagocytic and bactericidal activity of macrophages and neutraphils of mice on MRET water begins only after 24 hours following the intra-peritoneal inoculation of Staphylococcus culture. At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water substantially increased compare to the control group. The differences in mean values of the parameters of functional activity of phagocytes of groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for Phagocytic Index and NBT-test). These results confirm the significant intensification of phagocytic and bactericidal activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short time (2 weeks in case of the animal mice model).


5. The consumption of MRET water substantially enhances the immune activity of lymphoid organs.

By the end of another series of experiments in both groups of mice on MRET water was observed substantial statistically significant (p<0.05) increase of the weight and the cellularity (quantity of cells) of a spleen and lymph nodes as well as the insignificant increase of weight and cellularity of thymus (Fig 5 and Fig 6).

These results confirm the fact of significant intensification of immune system response in animals on MRET water subject to Staphylococcus infection. The difference in studied parameters between the groups of mice on MRET water (4 weeks and 2 weeks of preventive consumption of MRET water) was insignificant which confirms quite fast beneficial effect of MRET water on the immune activity of lymphoid organs.

In the beginning of experiment the cellularity and the weight of lymphoid organs in MRET groups did not show the distinct tendency to modifications. It is reasonable to admit that the consumption of MRET water affects the weight and the cellularity of lymphoid organs only during the infection period.


CONCLUSIONS:

The consumption of MRET activated water significantly enhances the factors of natural resistance of the body which constitute the first line of protection of an organism against the penetration and reproduction of pathogenic microorganisms.

The analysis of data in the beginning of experiment leads to the conclusion that significant changes in all studied parameters of mice on MRET water (decrease of pathogen colonies in homogenate of kidneys, increase of the weight and the cellularity of lymphoid organs, intensification of the phagocytic and bactericidal activity of macrophages and neutraphils) begins only after 24 hours following the inoculation of Staphylococcus culture. Another words the consumption of MRET water increases the potentials of immune capacities of the body to counteract the infections without any changes in the vital parameters of immune organs and functions prior to the penetration of infectious pathogens in the body.

At the end of two weeks of experiment the mean values of studied parameters in both groups of mice on MRET water (preventive for 4 and 2 weeks respectively) significantly increased compare to the control group. The differences in mean values of the studied parameters of the groups of mice consuming MRET water compare to the control group of mice on non-activated water were statistically significant with p<0.05 (for most of the parameters). These results confirm the significant intensification of phagocytic activity and of immune system response following the consumption of MRET water.

The differences in mean values of studied parameters for the groups of mice on MRET water compare to each other were statistically insignificant, which confirms the similarity of the level of the beneficial effect of MRET water in both groups. This fact also confirms that the regular consumption of MRET water provides health benefits in rather short period of time (2 weeks in case of the animal mice model).


II. The Effect of MRET Activation on the Process of Growth of Staphylococcal Culture in Nutrient Medium

The following experiments were designed to study the effect of MRET activation on the process of growth and development of Staphylococcus aureus Wood-46 culture in vitro in nutrient medium (MPA – meat-peptone agar). The bacterial culture was grown overnight to stationary phase and then platted on MPA in the form of suspension at different inoculation densities. The MPA with culture was MRET activated during the different periods of time (activation for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively) following the requirements of sterility. Petri dishes with activated and non-activated medium (MPA with culture) were covered with glass caps (aerobic environment) and placed in the thermostat for cultivation at a temperature of 37oC during 18-24 hours.

After the cultivation the morphological and tinctorial properties of cultures were observed and the numbers of colonies grown on MPA were counted. The bacteriostatic activity of MRET activated nutrient medium (MPA) was measured as an Index of Bacteriostatic Activity (IBA). An Index of Bacteriostatic Activity is defined as a coefficient of the inhibition of growth and reproduction of pathogens in bacteriostatic medium, particularly in MRET activated nutrient medium. It is calculated as reduction of the number of colonies (CFU – Colony Forming Units) in MRET activated medium related to the control samples not exposed to the activation:

IBA = (Ncontrol – Nact)/Ncontrol
where N – number of bacteria colonies (CFU) in Control (non-activated) and MRET activated nutrient medium, respectively.

In order to verify the sterility of experiments Petri dishes with nutrient medium (MPA) without staphylococcal culture were exposed to the process of activation and then were kept in the thermostat. No colonies of culture were observed that confirms the sterility of environment.


RESULTS:

Following the investigation the direct correlation between times of activation (tact), initial concentrations of staphylococcal culture (N0) and a number of colonies grown on MRET activated medium were observed. The results are presented below in the form of a series of photos of Petri dishes with the colonies grown on MPA surfaces and the following diagrams based on the data of these experiments (Fig 7 – 12).

In the process of investigation the effect of MRET activation on the growth of staphylococcal culture at rather small initial concentration of pathogens was analyzed. The data corresponding to higher initial concentrations N0 > 103 bacteria/ml were not analyzed due to the difficulties related to calculation of very high values of a number of colonies, despite the fact of the high bacteriostatic activity of MRET activated nutrient medium in case of high initial concentrations.

The highly significant bacteriostatic effect of 92 – 93% was observed after MRET activation for 30 minutes and more of cultures with initial concentration N0 = 103 bacteria/ml (Fig 7 and 8) and of 70 – 90% with initial concentration of N0 = 102 bacteria/ml (Fig 9 and 10). In case of cultures with low initial concentration N0 = 10 bacteria/ml the bacteriostatic activity in 15 minutes activated nutrient medium exceeded 93% and in 30 minutes activated nutrient medium was observed 100% inhibition of staphylococcal colonies (Fig 11 and 12).

Fig 7: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 103 bacteria/ml.

 

Fig 9: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 102 bacteria/ml.

 

Fig 11: The effect of time duration of MRET activation on the inhibition of growth of culture of Staphylococcus aureus Wood-46 with initial concentration of culture N0 = 10 bacteria/ml.

 
CONCLUSIONS:
  1. MRET activation of the water based nutrient medium with suspended staphylococcal culture leads to the origination of high bacteriostatic activity of nutrient medium which depends on the time duration of activation and initial concentration of culture cells.
  2. The bacteriostatic activity increases following the increase of time of activation (the times of activation up to 60 minutes were studied).
  3. The efficacy of bacteriostatic activity increases following the decrease of initial concentration of the suspension of staphylococcal culture. The process of MRET activation is most effective on culture suspensions with the concentration not more than 103 bacteria/ml.
  4. The results of investigation provide the evidence regarding the high efficacy of MRET activation on the inhibition of growth of colonies and reproduction of staphylococcal microorganisms in vitro. These results allow admitting that the process of MRET activation and the sterilization effect of MRET water can be applied in food industry and for water purification.