INTRODUCTION:

Infectious diseases of bacterial etiology remain the most relevant problems of modern medicine and occupy one of the main places among all infectious pathologies. A deterioration of the overall immunological responsiveness of the population, changes in the ecological conditions of life of the population in recent decades have led to changes in the structure and nature of the pathogenesis of infectious diseases^1-4. Despite the improvement of methods for treating bacterial infections, development of new treatment methods is still relevant^{5, 6}. One of the characteristic features of the current course of a number of bacterial infections is the atypical clinical pattern: hypo- or hyperergic variants, a natural consequence of which is the development of severe complications such as acute sepsis, secondary septic processes, as well as the development of chronic forms of infections, and a high share of bacteria carriers^7-9.

The interaction of microorganisms with leukocytes is one of the key elements in the pathogenesis of infectious diseases. In addition to assessing the state of the immune system, it is important to characterize the pathogen’s resistance to phagocytosis mechanisms. According to modern concepts, the main cause of the low sensitivity of bacteria to the protective effect of the immune system is their adaptive ability to produce substances that inhibit the production and/or activity of immunity factors^{9, 11, 12}. Bacteria excrete factors inhibiting complement, lysozyme, and interferon activation^{10, 13}, which impacts their ability to survive in the macro-organism conditions and their resistance to phagocytosis. Adaptability of microorganisms allows them to effectively protect themselves from the damaging effect of free radicals^{12, 14}. Numerous attempts to develop new groups of antibacterial chemotherapeutic agents have undoubtedly achieved a certain success. But the effect of each new group of preparations is temporary, does not cover financial costs, and leads to a new round of genetic mutation of microorganisms^{13, 15-17}.

Not always effective, and often unsatisfactory results of treatment of patients with severe forms of infectious diseases force researchers and clinicians to expand the range of forecasting methods and develop new methods of therapy. Directed regulatory control of microbe defense mechanisms, based on the principles of molecular biology, is one of the most promising directions of scientific research. The interdisciplinary approach should be based first of all on the development of new methods for impacting microbes with simultaneous recovery of the functional activity of the antimicrobial immunity.

Its aim is to investigate the main persistent characteristics of Staphylococcus aureus strains in lacunar tonsillitis and odontogenic phlegmon and to develop methods for suppressing the defensive potential of pathogenic agents.

MATERIALS AND METHODS:

In this study, we used 251 strains of Staphylococcus aureus, the pathogenic agents of local and generalized infections (Table 1). The strains were isolated from different sources by inoculation on microflora (smear from the tonsils, wound fluid, blood). The isolation of bacteria from the material under investigation and their identification were carried out by conventional bacteriological methods. Bacteria were stored in test tubes with a semi-liquid agar at 6 °C and were reinoculated every month. Bacterial suspensions in 0.9 % NaCl solution with an optical density of A₅₁₀ 0.2, which corresponds to a concentration of 1.5×10⁹ cells/ml, were used for the study.

Table 1. Distribution of Staphylococcus aureus strains by the source.

Source	Number of strains
tonsils	115
wound fluid	104
blood	32
Total:	251

Identification of persistence factors of Staphylococcus aureus strains

Identification of anti-lysozyme activity. Anti-lysozyme activity (ALA) of bacteria was studied by the method of O.V. Bukharin⁷. Lysozyme diluted at 1~7 μg/ml was added to 1.5 % nutrient agar and poured into Petri dishes. After solidifying and drying, the daily culture of the strain under study was placed on the medium's surface. The dishes were incubated for 24 hours at 37 °C. The grown-up colonies were killed with chloroform vapors (for 20~25 minutes), and then a layer of nutrient agar (3~4 ml) with 0.2 ml of 1-mil suspension of Micrococcus lutens var. Lysodeiticus N2665 was poured onto the surface and incubated for 24 hours at 37 °C. The experiment was evaluated by the growth of Micrococcus lutens. The test culture grows around the colonies of those strains that neutralize lysozyme introduced into the medium. The following criteria were used to estimate ALA: a low level of ALA was at 1~2 μg/ml, an average level was at 3~5 μg/ml, and a high level was at more than 5 μg/ml.

Identification of anti-complement activity. The anti-complement activity (ACA) of bacteria was studied by the method of O.V. Bukharin⁷. The daily culture of the strain was placed on the surface of 1.5 % nutrient agar. The dishes were incubated for 24 hours at 37 °C. The grown-up colonies were killed with chloroform vapors (for 20~25 minutes), and then a layer of nutrient agar (3~4 ml) with complement (50 u/ml, 25 u/ml, and 12.5 u/ml) was poured onto the surface. The dishes were incubated for 24 hours. Then, the third layer containing 0.1 ml of 1-mil suspension of daily agar culture of E.coli 212 was poured into the dishes and incubated for 24 hours at 37 °C. The experiment was evaluated by the growth of E. coli 212. The test culture grows around the colonies of those strains that neutralize the complement introduced into the medium.

Identification of anti-interferon activity. The anti-interferon activity (AIA) of bacteria was studied by the method of O.V. Bukharin⁷. The Intercid preparation (Russia) representing the antibacterial component of interferon, diluted at the ratio of 1\60~1\40, was added to 1.5 % nutrient agar and poured into Petri dishes. After solidifying and drying, the daily culture of the strain under study was placed on the medium's surface. The dishes were incubated for 24 hours at 37 °C. The grown-up colonies were killed with chloroform vapors (for 20~25 minutes), and then a layer of nutrient agar (3~4 ml) with 0.2 ml of 1-mil daily agar culture of Corynebacterium xerosis 181 was poured onto the surface and incubated for 24 hours at 37 °C. The experiment was evaluated by the growth of Corynebacterium xerosis 181. The test culture grows around the colonies of those strains that neutralize interferon introduced into the medium.

Identification of the catalase activity of Staphylococcus aureus strains. The catalase activity of Staphylococcus aureus strains was identified by the iodometric method. 1 ml of freshly prepared 0.0125M H₂O₂ solution standardized to the optical density of 0.2 cu was added to 0.2 ml of Staphylococcus aureus suspension and incubated for 10 min at room temperature. The reaction of H₂O₂ decomposition by the bacterial catalase was stopped by adding 20 μl of 2N solution of HCl. Then 0.5 ml of freshly prepared 0.025M KI solution was added, mixed thoroughly, and Staphylococcus aureus cells were pelleted by centrifugation for 15 minutes at 3000g. Next, the optical density was measured no later than 10 minutes after centrifugation. The bacterial catalase activity was evaluated on a calibration curve obtained using a commercial catalase preparation (Sigma, the USA).

In a number of experiments, bacterial cultures were treated with the fermented papaya preparation, and then the catalase activity of the microorganisms was evaluated. The treatment was performed in the following way: 1 ml of 0.9 % herbal preparation solution (100 mg/ml) was added to 1 ml of cell suspension (1.5x10⁹ cells/ml) and incubated at 37 °C for 1 hour. Then, the bacteria were washed three times with 10-fold volume of NaCl and used for further analyses. Bacterial cultures untreated with the preparation were used for the control purposes.

Identification of the phagocytic activity of neutrophils in ill and healthy donors.

The phagocytic activity of neutrophils was evaluated by the standard method for Staphylococcus aureus bacteria isolated from each patient. For this purpose, 1 ml of neutrophil suspension was mixed with 1 ml of bacterial suspension (10⁷ cells) in Hanks solution (pH 7.4). The mixture was incubated with stirring for 30 minutes at 37 °C. Smears on glass were prepared, fixed, and stained by the Romanovsky–Giemsa method. In smears, we counted the number of phagocytic macrophages per 100 cells. The data obtained were expressed as the phagocytic index and phagocytic number.

Identification of the effectiveness of intracellular killing of bacteria by neutrophils. The intracellular killing effectiveness was evaluated as follows: After the phagocytic response was set up (see above), the suspension was centrifuged for 10 minutes at 1500g, the precipitate was taken up; a three-fold volume of H₂O was isolated, and the suspension was plated onto a Petri dish with meat-peptone agar. The number of bacteria survived after phagocytosis was determined by the number of colonies in the sectors in 24 hours.

The effect of fermented papaya preparation on phagocytic responses was estimated by treating bacterial cultures according to the following procedure: 1 ml of 0.9 % herbal preparation solution (100 mg/ml) was added to 1 ml of cell suspension (1.5x10⁹ cells/ml) and incubated at 37 °C for 1 hour. Bacteria were washed three times with 10-fold volume of NaCl and used for further analyses: evaluation of the phagocytic activity and efficacy of intracellular killing of bacteria by leukocytes of ill and healthy donors. The control experiments were conducted with bacteria untreated with herbal preparations.

Clinical methods for studying the efficacy of patient treatment with the fermented papaya preparation

Characteristics of patient groups

Four groups of patients were observed for the purpose of studying the efficacy of treatment with the fermented papaya preparation (Table 2):

Group 1 included 15 patients with moderately severe lacunar tonsillitis of streptococcal-staphylococcal etiology, who received local treatment with herbal preparations combined with conventional detoxification and antibiotic therapy (Herbal Preparation 1 Group).

Group 2 included 15 patients with lacunar tonsillitis of streptococcal-staphylococcal etiology, who received conventional therapy (Control 1 Group).

Group 3 included 15 patients with moderately severe odontogenic phlegmons of staphylococcal genesis, who received local therapy with herbal preparation, combined with traditional detoxification and antibiotic therapy (Herbal Preparation 2 Group).

Group 4 included 15 patients with odontogenic phlegmons of staphylococcus genesis, who received conventional therapy (Control 2 Group).

Preparation administration method: The fermented papaya preparation was applied topically twice a day for a week along with conventional therapy. In the group of tonsillitis patients, the preparation was applied topically onto the tonsils. In the group of phlegmon patients, the preparation was applied topically under the bandage. The preparation acceptability was assessed by the absence of local and systemic responses in patients during the treatment and after a longer period.

Clinical methods for assessing the efficacy of the herbal preparation in patients with pyoinflammatory bacterial diseases: Clinically, in lacunar tonsillitis patients of the Herbal Preparation 1 and Control 1 groups, the inflammatory process in tonsil tissues was estimated by the following criteria: the size of the tonsils, contents of the lacunae, swelling of the arches and uvula, hyperemia of the tonsils and pharynx. In the clinical assessment of the patient's condition, general symptoms of intoxication and subjective feelings of the patient, such as temperature response, severity of pain syndrome, and general well-being, were taken into account. The clinical efficacy of treatment was evaluated on a 3-point grading scale by the severity of signs of the inflammatory process in tonsil tissues. The effect of preparations was considered pronounced when the signs of local tonsil inflammation reduced to 4~5 points, moderate when to 6 points, and absent when to 7~8 points.

Clinically, in patients of the Herbal Preparation 2 and Control 2 groups, the course of the wound process was evaluated by the following criteria: the size and dynamics of the infiltrate changes by days, periods of suppuration, severity of the edema, exudation, and the time of granule appearance. The infiltrate size was estimated by determining its area as follows: the infiltrate projection was considered as a circle or ellipse and calculated by the formulas: S=πR² and S=πab, where R is the circle radius, a and b are the major and minor semi-axes of the ellipse, respectively. The data were expressed in cm². In the clinical assessment of the patient's condition, general symptoms of intoxication and subjective feelings of the patient, such as temperature response, severity of pain syndrome, and general well-being, were taken into account. The clinical efficacy of therapy was evaluated on a 3-point grading scale by the severity of signs of the inflammation in soft tissues. The effect of preparations was considered pronounced when the signs of local inflammation in the soft tissues were reduced to 4~5 points, moderate when to 6 points, and absent when to 7~8 points.

RESULTS:

The problem of infectious bacterial diseases is relevant all over the world, since despite active development of chemotherapeutic groups of antibiotics, the share of patients remains the same^{5,
15, 18, 19}. The emergence of bacteria strains highly resistant to any modern antibiotic preparations used makes it necessary to develop a comprehensive therapy with fundamentally new microbe targets. And the new developments should be based on treating microbial cells as a superorganism^{11,
12, 14, 20}, which has the highest potential of genetic variability.

The phagocytosis resistance ability of strains isolated from various sources (blood, smears from tonsils, wound fluid) was investigated at the preliminary stage. It was found that the effectiveness of intracellular killing is significantly lower in the case of S. aureus strains taken from blood (Table 3).

Table 3. Effectiveness of intracellular killing of Staphylococcus aureus strains taken from blood, swabs from tonsils, and wound fluid with phagocytes of healthy individuals.

Strains	Effectiveness of intracellular killing, %
Staphylococcus aureus strains isolated from tonsil smears, n=115	65.0±5.5
Staphylococcus aureus strains isolated from wound fluid, n=104	46.0±6.5
Staphylococcus aureus strains isolated from blood, n=32	15.5±7.0

To find the causes of high resistance to phagocytosis, we investigated the main persistent characteristics of strains. 62.5 % (157 cultures) of 251 studied strains had anti-lysozyme activity (ALA). The highest number of strains with anti-lysozyme activity was detected among cultures isolated from the wound fluid: 83 % (74 strains). A smaller number of bacterial cultures, 46.9 % (57 strains), isolated from a throat smear also had ALA. 43.8 % (14 strains) of strains isolated from blood had ALA. In all groups of strains, the most frequent are cultures with medium ALA indices. No correlation was revealed between the ALA degree of the studied strains and their resistance to intracellular killing (k=0.46).

Taking into account the data on the direct correlation of anti-interferon activity (AIA) with the virulence of bacteria⁷, we studied S.aures strains isolated from various sources to identify this persistence factor. 37.8 % (95 cultures) out of 251 studied strains had AIA. Staphylococcus aureus strains isolated from lacunar and follicular tonsillitis patients had this activity the most frequently: 58 strains (51 %). A bacteriological examination of patients with surgical infections of soft tissues detected 40 AI-active strains (44 %). 10 strains (31.3 %) isolated from blood had this persistence factor. No correlation was revealed between the degree of AIA intensity of the studied strains and their resistance to intracellular killing (k=0.51).

According to publications, Staphylococcus aureus strains are capable of inactivating complement⁷. 34 % of the S. aureus strains studied by us had ACA. Strains isolated from wound fluid and throat had nearly the same frequency of such activity (35.5 % and 34 %, respectively). Only 13.6 % of S.aureus bacterial cultures isolated from blood had the ability to inactivate complement. As in the study of ALA, most strains had average activity indices (25 u/ml) in the study of ACA. No correlation was revealed between the degree of ACA intensity of the strains studied and their resistance to intracellular killing (k=0.54). Thus, the dependence of the resistance of staphylococci strains on intracellular killing is not associated with such factors of persistence as anti-lysozyme, anti-interferon, and anti-complement activity.

The primary bactericidal factors released by phagocytes are hydrogen peroxide and products of its free radical decomposition, such as hypochlorite and hydroxyl radicals. Staphylococci adapt to survive in an environment with a high hydrogen peroxide concentration by inducing early response genes for oxidative damage¹⁴. Protein products of these genes are, among others, catalase enzyme decomposing hydrogen peroxide to neutral products (water and molecular oxygen) and superoxide dismutase enzyme decomposing superoxide anion radical to molecular oxygen.

It can be assumed that one of the efficient defenses of bacterial cells in the phagocytosis process is the synthesis of antioxidant enzymes. A qualitative estimate of the catalase activity of bacteria showed that the highest enzyme activity was inherent in strains isolated from blood (4.0~5.1 u\20 mil), while strains taken from wound fluid and pharynx had similar catalase activity (2.2~2.5 u\20 mil and 2.3~2.7 u\20 mil, accordingly) (Figure 1).

Fig. 1. Catalase activity of S.aureus strains isolated from different sources (u/106 bacteria).

When assessing the effectiveness of intracellular killing of bacteria with phagocytes, it was found that strains actively producing catalase, on average, in 40 % of cases are not destroyed in phagosomes, which leads to the persistence of the infectious agent in the body. The correlation ratio between the catalase activity of strains and their ability to colony formation after phagocytosis was equal to 0.9.

Thus, the increased resistance of Staphylococcus aureus isolated from blood is due to their high catalase activity. The anti-oxidant status of bacteria along with the immune parameters of the patient are of high prognostic importance for the prediction of the infection process severity and selection of an effective treatment. One of the most optimal ways to control catalase microorganisms is to select preparations capable of inhibiting catalase.

The pronounced antimicrobial effect of many medicines based on herbal raw materials is widely known²¹.

Table 4. Influence on the activity of the bacterial catalase of plant extracts.

No.	Plant extract	Preparation effect (% of inhibition)	% of surviving bacteria after treatment with the preparation (cells/ml)*
1	Papaya tea	8.5±0.5	5х10⁵
2	Papaya flowers	7.0±1.0	5х10⁵
3	Papaya leaves	9.0±0.5	5х10⁵
4	Papaya syrup	6.0±0.5	5х10⁵
5	NONI powder	7.0±0.5	5х10⁵
6	Pure NONI Extract	11.0±1.0	10⁵
7	NONI (capsules)	15.0±0.5	10⁵
8	NONI puree	13.0±1.0	10⁵
9	NONI juice	14.5±0.5	10⁵
10	Bignay	21.0±1.0	5х10⁴
11	Fermented papaya	35.0±1.0	10⁴
12	Control	0	5х10⁶

* - S. aureus 242 strain with catalase activity of 5.1±0.1 u/20 mil bacteria was chosen as the phagocytosis object.

Assuming that one of the mechanisms of the antibacterial effect of herbal preparations can be inhibition of antioxidant enzymes of microorganisms, we considered it expedient to search for preparations capable of reducing bacterial catalase activity. We incubated S. aureus suspension with each preparation (Table 4) and evaluated catalase activity in washed bacterial cells by iodometric and chemiluminescent methods. The best inhibition of catalase activity was achieved when treating with fermented papaya preparations (Table 4).

Simultaneously, we determined the effectiveness of intracellular killing of staphylococci by neutrophils based on the ability of absorbed bacteria to colony formation. Preincubation of bacteria with herbal preparations for 1 hour had no bacteriostatic or bactericidal effect (data not shown); however, it significantly reduced the initial catalase activity of staphylococci (Table 4). The presented data show that the “fermented papaya preparation” (non-commercial name) (NBSCo. Ltd, Japan) (registration certificate No. 003117.И.392.07.2011) had the best effect, reducing the catalase activity of S.aureus by 35 %. Simultaneously with catalase inhibition, the intracellular killing of strain 241 staphylococcus increased significantly, by 500 times (the catalase activity of strain 241 was 4.9 u/20 mil of bacteria) (the number of surviving bacteria after phagocytosis was between 5x10⁶ cells/ml in the control group and 10⁴ cells\ml).

Thus, the “fermented papaya preparation” can be considered as an indirect antibiotic weakening the adaptive capabilities of staphylococci.

Clinical laboratory study of the effectiveness of therapy of acute tonsillitis patients with the BioRex preparation: Patients in the Herbal Preparation 1 and Control 1 groups were admitted to the hospital under emergency circumstances. The age of the disease since the first clinical signs appeared was 3–4 days. At admission, all patients had enlarged tonsils (grade 3 in 86.7 % of cases), edema and hyperemia of the arch and uvula (intensive in 100 % of cases). 73.4 % of patients had caseous plugs; 26.7 % of patients had mucopurulent lacunae. The severity of intoxication and pain syndrome were intense in 100 % of patients; the body temperature of 66.6 % of patients was 37.5–38.0 °C. Patients in both groups had a laboratory picture of the inflammation process: 25±6ESR; 12.4±0.8 leukocytes; the leukocyte formula was shifted to the left. No differences in laboratory blood indices (the number of erythrocytes, leukocytes, the hemoglobin level, etc.) and urine indices (protein, specific gravity) were observed between patients in the Herbal Preparation 1 and Control 1 groups upon admission and in the dynamics of the infectious process.

The clinical efficacy of comprehensive treatment with “fermented papaya preparation” was evaluated according to a three-point grading scale. The effect was considered excellent if the signs of tonsil inflammation reduced to 3~4 points, good if they reduced to 6 points, and satisfactory if to 7~8 points.

Fig. 2. Clinical efficacy of topical therapy with fermented papaya preparation of lacunar tonsillitis patients, in points.

The results of the conducted studies showed that, at admission, the intensity of the signs of tonsil inflammation in patients of the Herbal Preparation 1 group was at 11.4±0.5; and in patients of the Control 1 group at 11.3±0.4 (Figure 2, Table 5). By the 3^rd–4^th day of herbal preparation therapy, swelling and hyperemia of the mucous membranes significantly reduced, and the size of the tonsils decreased. At the same time, it was noticed that soreness in swallowing and feeling of dry sore throat disappeared quickly. 93.3 % of patients had excellent results after the treatment. One patient had an allergic response in the form of urticarial rash on the skin (6.7 %). Thus, the topical application of the BioRex herbal preparation along with an antibacterial and detoxification treatment was followed by a more rapid and pronounced reduction of the inflammation process in tonsil tissues. The preparation was well accepted by patients and was effective in 93.3 % of cases (an excellent result).

Table 5. The main signs of lacunar tonsillitis patients in the dynamics of the infectious process (%).

Indicator

Patient of the Herbal Preparation 1 group

Patient of the Control 1 group

Indicator

Day at hospital

Patient objective examination data:

Severity of tonsillar hypertrophy:

Inapparent

Moderate

Copius

100

32.2

67.8

53.5

46.5

100

28.6

71.4

35.7

64.3

92.8

14.2-

Severity of the arch and uvula edema:

Imperceptible

Moderate

Pronounced

10.8

89.2

55.4

44.6

35.7

64.3

91.1

8.9

10.8

89.2

55.4

44.6

28.6

71.4

82.2

17.8

Painful condition:

Imperceptible

Moderate

Pronounced

10.8

89.2

55.4

44.6

35.7

64.3

91.1

8.9

10.8

89.2

55.4

44.6

28.6

71.4

82.2

17.8

Regional limfadenitis:

89.2

14.2

7.2

89.2

35.7

14.2

Table 6. The main signs of odontogenic perimaxillary phlegmon patients in the dynamics of the infectious process (%).

Indicator

Patient of the BioRex group

Patient of the BioRex Placebo group

Indicator

Day at hospital

Patient objective examination data:

Suppuration severity:

Inapparent

Moderate

Copius

100

32.2

67.8

53.5

46.5

100

28.6

71.4

35.7

64.3

92.8

14.2-

Infiltrate size:

Imperceptible

Moderate

Pronounced

10.8

89.2

55.4

44.6

35.7

64.3

91.1

8.9

10.8

89.2

55.4

44.6

28.6

71.4

82.2

17.8

Indicators of wound healing:

Removal of necrotic tissues from the wound

Granulation of the wound

Epithelialization of the wound

91.1

89.2

100

92.6

14.2

100

98.2

96.4

85.7

80.3

100

92.6

14.2

100

98.2

96.4

Regional limfadenitis:

89.2

14.2

7.2

89.2

35.7

14.2

After a course of treatment with “fermented papaya preparation” in combination with conventional antibacterial and detoxification therapy, an excellent result was noted in 86.7 % of patients and a good result was noted in 13.3 % of patients. Reliability of differences between the comparison groups on the 3^rd, 5^th, 7^th, and 14^th day was p<0.05.

DISCUSSION:

The main studies of the pathogen (staphylococci) control mechanisms have for a long time been mainly limited to the invention of various antimicrobial substances and impacts (antibacterial preparations, non-drug methods)^{7, 8}. One of the effective ways of staphylococci infection control can be selective inhibition of the induction of bacterial antioxidant enzymes, or selective suppression of persistence factors¹⁰.

However, the most objective sign of the phagocytic response effectiveness is the intracellular killing evaluation. The obtained data in the presented publication allowed revealing the dependence of the effect of preparation from the source of S. aureus strains. It was revealed that the studied preparations improve the efficiency of intracellular killing of staphylococci strains isolated from throat and wound fluid.

We found that the previously revealed dependence of staphylococci strain resistance to intracellular killing from the source of isolation is not associated with such persistence factors as anti-lysozyme, anti-interferon, and anti-complement activity, but revealed a direct correlation between catalase activity and the isolation rate after phagocytosis (k-0.9).

To test this hypothesis, we evaluated the effectiveness of intracellular killing of bacteria with different catalase activity. It was found that the survival of bacteria after phagocytosis and their ability to decompose hydrogen peroxide are correlated. Evaluation of the effectiveness of intracellular killing of bacteria by phagocytes showed that strains actively producing catalase, on average, in 40 % of cases are not destroyed in phagosomes, which leads to the persistence of the infectious agent in the body. The correlation ratio between the catalase activity of strains and their capability of forming colonies after phagocytosis was equal to 0.9.

Many herbal preparations are used with an antimicrobial purpose, but not always their effect is directly bactericidal; some plants reduce the microbes’ ability to form a biofilm and synthesize protective enzymes¹⁷. We set the objective to find a herbal preparation that would reduce the initial activity of bacterial catalase. For this purpose, we incubated S. aureus suspension with each preparation and evaluated the catalase activity in washed bacterial cells by iodometric and chemiluminescent methods. Simultaneously, we determined the efficacy of intracellular killing of staphylococci by neutrophils by the ability of absorbed bacteria to form colonies.

Preincubation of bacteria with different herbal preparations for one hour had no bacteriostatic or bactericidal effect (data not shown); however, it significantly reduced the initial catalase activity of staphylococci. The fermented papaya preparation had the best effect, reducing the catalase activity of S. aureus by 35 %. Simultaneously with the catalase inhibition, the intracellular killing of strain 241 staphylococci increased significantly, by 500 times (the number of bacteria surviving after phagocytosis was between 5x10⁶ cells/ml in the control group and 10⁴ cells/ml). The preparation effect was dose-dependent.

Thus, the papaya-based preparation can be considered as an indirect antibiotic weakening the adaptive capabilities of staphylococci.

Clinical efficacy of comprehensive treatment with the fermented papaya preparation was evaluated according to a three-point grading scale. By 3^rd–4^th day of treatment with the fermented papaya preparation, swelling and hyperemia of the mucous membranes significantly reduced, and the size of tonsils decreased. At the same time, it was noticed that soreness in swallowing and feeling of dry sore throat disappeared quickly.

By 2^nd–3^rd day of topical therapy of odontogenic phlegmon with fermented papaya preparation along with antibacterial and detoxification treatment, the purulence and edema intensity decreased significantly. In this case, a quicker removal of necrotic tissues from the wound and a reduction of the infiltrate size were noted (Figure 3 and Table 6). The validity of differences between the groups on the 3^rd, 5^th, 7^th, and 14^th days was p<0.05.

The clinical implementation of the developed methods will enable a significant reduction of the antibiotic pressure, improvement of the therapy efficacy through individual selection of a comprehensive treatment based on the characteristics of the patients’ microflora, which will result in shorter hospitalization periods, help avoid complications, and will have significant social and economic effects.

The scope of the objectives to achieve in the project is significantly expanded because the developed therapy methods in the presence of a clinical effect and the developed recommendations can be successfully applied against most infectious diseases of bacterial etiology.

SUMMARY:

1. When assessing the role of persistence factors (anti-lysozyme, anti-complement, anti-interferon, catalase) that affect the resistance of Staphylococcus aureus strains to phagocytosis with neutrophils, we found that the main protective factor of bacteria in the process of phagocytosis is catalase production.

2. The antioxidant herbal preparation, fermented papaya, improves the efficacy of intracellular killing of staphylococci with neutrophils in both ill and healthy individuals due to the ability to reduce bacterial catalase activity. Antibacterial properties of a herbal preparation are the basis for its effective application in local pyoinflammatory processes.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 14.06.2018 Modified on 11.08.2018

Research J. Pharm. and Tech 2018; 11(12): 5304-5312.

DOI: 10.5958/0974-360X.2018.00966.6

Age	Herbal Preparation 1 Group		Control 1 Group		Herbal Preparation 2 Group		Placebo 2 Group
Age	Male	Female	Male	Female	Male	Female	Male	Female
20–40	4	3	5	4	3	6	5	3
40–60	4	5	2	4	3	3	2	5
Total	8	7	7	8	6	9	7	8