INTRODUCTION:

Staphylococcus aureus secretes a phospholipase specific to phosphatidylinositol as an abnormal virulence factor in demonstrating more activity at acidic pH values than other enzymes in this class.

Candida is one of the predominating reasons of fungal disease global. Most yeast not causes to infectious commensals or counting humans; but, if the barriers of the mucous membrane are broken or the immune structure is weaken they can attack and cause disease, known as an opportunistic infection¹.

Candida tropicalis has emerged as one of the most important Candida species in Tropical Countries. It is a common pathogen in neutropenic hosts in whom it may spread through the blood stream to peripheral organs and represent a second most virulent of Candida with frequent o invasiveness 66%^{2, 3}.

The sturdiness of yeast cell is deem to be linked because action of PL. removed pests enzymes in yeast, for their suspected to strives to roles phospholipids turnover in the cell membrane⁴.The most famous enzymes include phospholipase A1, A2, B, and lysophospholipase. PLB catalyzes hydrolytic cleavage of both (sn-1and sn-2) acylester bonds of glycerophospholips and is detect in microorganisms⁵ plants and animal tissues. phospholipid remove acylation enzyme categorized from yeast by molecular consternation fit to the type of PLB while, biological significance and optimum pH molecular weight, substrate specificity effects of metal ions, as enzymatic properties vary commonly reliant on the yeast kinds. Lately, Phospholipase B1, B2 and B3 genes of Saccheromyces cervisiae are classified^6,7. From another point of view, Ma⁸indicated small development periphery in the Logarithmic phase, raise endpoint cell volume with disturbance of the PLB gene (PLB^8,9, an indication that PLB action should to be complex in determining the cell stiffness of this mould. C.albecans, contains 2genes of P.L. B1 and B2). Disturbance of ca PLB, compared to the iso genic wild type P. arental^{10, 11} therefore, PLB action are diagnosed for example a likely virulence agent with Candida albeicans, and the marks show enzymatic features of PLB vary according to the yeast types ¹¹. We thus additional filtered, differentiated P.L.B commencing C. tropicalis to improved knowledge the properties and the enzymatic and biological impact to this yeast. Oral cavity potentially serves as a reservoir for some pathogens that can cause systemic infection ¹².

The goal of the current study is to estimate the existence of Staph. aureus and Candida spp. and in sub gingival plaque and oral cavity of individuals with periodontal disease, to identify Staphylococcus aureus and its relationship to the secretion of interleukins interleukin-6 and interleukin-12.

MATERIALS AND METHODS:

Blood samples and cotton swabs were collected from patients with periodontal and oral sinus infections, from 25 patients, swabs were taken from the oral cavity and under the periodontal sinuses and after biological tests were performed, Staphylococcus spp and Candida spp. were isolated from samples. Chemicals and Molecular weight Marker proteins used we study was obtained by the Sigma Chemical Co.

Human ELISA kits - interleukin-6 and interleukin-12 (USA) were used to serum analysis using the biofilm was removed above the gingival and the area was relatively isolated using a sterile cotton pad and a very strong suction device. From below the gum, biofilm samples were obtained with a 7/8 graspic curettage. Patients had previously rinsed their oral cavities with sterile distilled water. Using a sterile swab, samples were taken through the oral cavity (buccal mucosa, tongue and mucosa for eugenics). The material collected from the subgingival and oral biofilms was soaked in sterile PBS (phosphate solution, pH 7.2), and stored at (4°C) until further processing. Samples were immediately sent to the laboratory. Direct microscopy studies were performed on samples to test the microbiological method by Gram staining. To isolate Staphylococcus spp., Samples were grown on a selective and differential medium of salt agar. Broth is a liquid selective culture medium for Staphylococcus spp. which has been used to increase method sensitivity. Samples were cultured and incubated at 37° C for 24-48 hr. Suspicious colonies was identified by Gram staining, catalase testing, and coagulation testing. A portion of the sample was grown on a chromogenic differential solid medium (CHRO-Magar Company, Paris, France) to isolate the yeast species, and incubated at 37°C for one week, with their evolution observed daily when green yeast was observed in CHRO-Magar Candida.

Chemicals:

Sigma Company has been brought in all attended phospholipids, phenyl-Sepharos CL-4B, Hiload 16 column |60 Superdex 200 pg from Pharmacia Biotech, and all other chemicals used were of the more pure grades obtainable.

Strain and Medium:

Isolates of bacteria and fungi were collected from patients with infected mouth and a number of morphological &biochemical test were carried out, even by Vitech Compact system, then, the more stable and highly Phospholipase producer was used, and it is kept me laboratory in YEMED Media (0.3% malt Extract, 0.3% yeast extract, 2% glucose and 0.5% peptone) having 2% agar¹³.

Preparation of cell- free extract:

Staph. aureus and Candida tropicalis was cultured aerobically in YEMPD Media at 24 ºC at 72 hr, then reaped by centrifuge tool at 10 000 xg, 10 minute, 4 ºC. The harvested cells were re-suspended in Tris –acetate (0.1 M), EDTA (1 µM) and glycerol (10%,w\v ), pH 7.5, and centrifuged (10 000 xg at 10 minute, 4 ºC). Cell pellets are again suspended in 40 m L of similar buffer, cooled in ice prior to sonic in MSE Sonprep 150W ultrasonic apiary. The uninterrupted cells and organism's cell wall were distant by centrifugation (40 000 XG, 30 minute at, 4ºC) and the floating is decanted and stored at 4ºC¹⁴.

Phospholipase B activity test:

The alkaline pH standard reaction mixture consists to 0.8 ml Tris HCl enzyme solution 50µM (PH 8.0) comprising 1.6 µM PC, 0.25% (wt.) triton x-100, and 0.1 ml enzyme explanation in case of metallic molecules. To typical PH, the return fusion involved of 0.8ml of 50 µM glycine –HCl buffer (pH 3.0) having 1.6 µM (PC)0.25% Triton –X100, 0.1 ml of enzyme elucidation. Complete fat in the response liquid are mined by the technique ¹⁵, unit enzyme motion was distinct as the measure of enzyme necessary to create 1µmol 0f free F.A per mint at 30 ºC.

Test the effectiveness of the enzyme transferees:

The PLB enzyme is incubated using a mixture above the standard solution at 30 °C interaction, bar. At suitable periods the response yields were extracted by the technique¹⁵. The reaction yields were divided to a tinny –coat platter in progress method chloroform –methanol-water (66:26:4 v/v/v). PL is discovered with Hanes–Isherwood reagent, what contains of 1.5% NH4moly date, 4% per HCL3and 1.2 M Hydrochloric Acid.

Protein purification and characterization:

Purification of the crude PLB was performed by ammonium sulfate sedimentation at 4ºC, unless specified otherwise. The crude enzyme extract was precipitin by treatment with 20% ammonium sulfate (NH₄).2SO₄(300mg/ml) plus cell-free extract (30 ml) and 80% (w/v) (NH4).2SO₄ (423mg/mL) plus cell-free extract (30 ML) at various saturation levels and was left to stand for 4h prior to centrifugation (40 000 xg, 30 min, 4ºC). All the collected precipitates were resuspended in Tris-acetate buffer at (0.1 M ,6 mL, pH 7.5) and dialyzed overnight against 20 Mm Tris –acetate buffer (200 mL, PH 7.5) to remove residual (NH₄)₂SO₄. Then the clarity of the purified PLB enzyme was specified by (SDS-PAGE). All the concentrated fractions were subjected to protein and PLB activity assays to establish the fraction with the most PLB activity.

Determination of PLB MW:

(SDS-PAGE) (TGX-Fast cast, Bio-Rad) was made with a 5%loading, 12% unraveling gel toward limit M.wt. for the distilled Staph. aureus PLC, B and C. tropicalis PLB¹⁷. A 50 ml buffer specimen (0.05% C₁₉H₁₀Br₄O₅S blue, 5% b-C₂H₆OS, 10% C₂H₈O₃ and 1% SDS in o.25 M Tris- Hydrochloric acid buffer, PH 6.8) was other to 100 ml aliquot of protein samples in append off tubes, and the solution was boiled in a hot water bath at 5 minute, let in room heat then electrophoreses. The protein bands were imagined by marking with C₅₄H₄₄N₃NaO₇S₂ blue G so inside in D.W. immediate. The m. w. of the distilled PLB was indomitable by comparing its band to those of marker proteins (standard protein markers, 25-225 kDa Sigma, USA). Further assay ways. The protein level of every purification rung stayed assess at 280 nm.

RESULTS:

Isolation and Identification of bacteria from patients samples:

Twenty-five isolates of Staphylococcus spp. And Candida spp isolates with clinically diagnosed of oral cavity and under the periodontal sinuses were collected aseptically from sinuses infection were collected, and 25 healthy controls from hospitals in Baghdad included: Teaching Laboratories in Medical City, Education Baghdad Hospital, and Al- Harery Hospital in Medical City for two months.

Staphylococcus aureus were isolated from 25 individuals 16 (64%) that most frequently isolated specie, and Candida tropicalis were 5 (20%), and Streptococcus sp. was 3 (12%), and other bacteria 1 (4%) of at both sites of the oral cavity of patients with oral infections as shown in Table 1.

Table1: Percentage of causative pathogens isolated from oral infections

Microorganisms	Number	Percentage
Staphylococcus aureus	16	64 %
Streptococcus sp.	3	12 %
C. tropicalis	5	20 %
Other bacteria	1	4 %
Total	25	100 %

The result shows the effect of bacterial inflammation on the multidirectional secretion of interleukin (IL-10, IL-6) has been studied in patients with oral cavity infections. Blood Samples and wash swabs were taken from 25 healthy patients and 25 patients. The supernatant fractions of IL-10 and IL-6 were examined by commercial ELISA.

The mean of serum concentration of IL-10 ± S.E was (67.04 ± 10.93) pg/ml in the patients group while it was (2.3 ± 0.65) pg/ml in the control group. Mean of IL-10 level was significantly high in the patients than in the controls (P≤0.000), while mean of serum concentration IL-6 ± S.E was 4.10 ± 0.83 pg/ml in the patient group and 1.15 ± 0.57 pg/ml in the control. Mean of IL-10 level was significantly higher in patients than controls (P< 0.0026) as shown in (Table 2).

Table 2: Level of Interleukins in patients and control

Interleukins

(Mean ± SD)

Control

(Mean ± SD) Patients

Maximum

Minimum

IL-10(pg/ml)

P< 0.00

2.3± 0.65

67.04± 10.93

10-6 (pg/ml)

F=5.07

P< 0.0026

1.15± 0.57

4.10± 0.83

5.8

2.5

Based on the results, the mean serum concentration of IL-10 + S.E was 67.04 ± 10.93 pg / ml in the patient and 2.3 ± 0.65pg / ml in the control. The level serum IL-10 was significantly greater in the of patient from control (P 0.000), as shown in (Figure-1).

Figure 1: Levels of IL-10 with oral cavity infections patients compare with control

Based on the results, the mean serum concentration of IL-10 was 4.10 ± 0.83pg / ml in the group of patient and 1.15 ± 0.57pg / ml in the control. The mean serum IL-10 was significantly upper in the patient from control (P< 0.000), as shown in (Figure 2).

Figure 2: Levels of IL-6 with oral cavity infections patients compare with control

Purification of phospholipase B:

Aerobically, in YEMPD medium Staphylococcus aureus and C. tropis was cultured at 24 ° C. The cell density was in the middle to maximum 72 hours of growth. Activity was measured PLB and PLC, which was launched in the broth culture in the stages of growth phase knight increased PLB activity commensurate with the cell thickness through incubation in 72 hours, did not lead more nurseries to increase the activity of PLB in broth culture, then tried to filter the enzyme since 72 hours. The concentration of the broth media, ammonium sulfate hashed. It was purified PLB more. The ablution route is in (Table 3).

Table 3: the purification of phospholipase B and C from Staphylococcus aureus and Candida tropicalis

Fraction	Dimen sions (ml)	Total activity (U/ml)	Total protein (mg/ml)	Sp. activity (u/mg)	Purifi cation	Harvest %
Crude extract	100	6.500	3.22	1.50	1.00	100
60-80% (NH₄)So₄Purification	6	4.47	1.19	3.75	2.00	67.50
Dialysis	6	3.10	0.82	3.76	2.50	48.23

Molecular mass:

On SDS-PAGE, the finishing tester traveled as abroad sole band at a site conformingnear 50 kDa as in (Figure-3).

Figure 3: SDS -PAGE analysis of the purified phospholipase from Staphylococcus aureus

Level 1; Standard protein/ Level 2; purified PL protein (50 kDa)

PH and temperature monuments:

Figure (4) revealed results of PH targets were diagnosed for the (F.A) released in PL. volume of F.A secreted through the enzyme was estimated at PH 1.5 in excreting the quantity of fatty acids created in the absence of the enzyme. From 2.0 to 9.0.PL indicated2optimum pHs at pH 3.0 and pH7.5. Byan pH level enzyme was inactive since 5.0 to 6.5. The best temperature to PLB activity is 40 °C at measured pH 3.0, however activity were totally absent with incubation up 80 ° C.

Figure 4: pH effect on enzyme action

Kinetics of enzyme reaction

Our results showed that the hydrolysis response was linear at pH 3.0 and 30 min of incubation. By pH 8.0 the response curve were observed in being there Al 3+, when the PLB was powerfully activated by Al3 + at basic pH as in Figure 5. At pH 8.0 after a certain delay the hydrolysis reaction increased and a typical sigmoid Figure 6.

Figure 5: Effect of time of incubation on PLB activity at different pH (4.0, 8.0)

Figure 6: Effect of various concentration of ALCl3 on PL activity

Aceyltransferase activity:

The distilled PLB of Staphylococcus aureus and C. tropicalis was incubated by Lysol-PC, different Rf value was generated (0.28, 0.22 and 0.20) at pH 3.0,8.0 and 7.0 respectively (Table 4.) PLBs from microbes, animal cells 22 were described to change Lysol-PC near PC with different and slight motion.

Table 4: The retention factor (Rf) value of Staphylococcus aureus and C. tropicalis purified (PL) at different pH

Ph	Rf
3	0.28
8	0.22
7	0.20

DISCUSSION:

In this study, explain that live invasive Staphylococcus sp. Bacteria Candida sp. Bacteria induce Interleukins secretion. The level of IL-10 increased significantly is similar to that found by ¹⁸that indicated to much higher than that of the control group in women with Preeclampsia and this result is similar to that of ¹⁹, was indicated to a Among prostate cancer patients and pre-operative healthy individuals in the levels of interleukins (IL-1, IL-2, IL-3, IL-5), the results showed a significant increase in cytokines compared to controls where the relationship was negative besides here statistically important variances between levels of cytokines in the patients and healthy people. This increases agreed with the result by ²⁰indicated points of pro, anti-inflammatory (INT-γ and TNF – α) with UTI patients compared to healthy individuals. Similar cytokine mechanisms may operate in sepsis in both neonates and adults.

Our results showed that Staphylococcus aureus and C. heterogeneity of the sugar fraction bound of covalent to the protein in the enzyme (glycol form) was purified, and high-molecular-weight proteins were identified in other types of bacteria and yeast and known high-glycosylated forms. Yeast may be important in protecting enzyme activity by proteolysis enzymes through the secret by progression in to the medium ¹⁰.

Show study that the Ca2+-dependent PKCα binds to S. aureus-containing oral cavity and that α-hemolysin, release by S. aureus, enhance this renew of PKCα to phagosomal membranes. well, the presence of PKCα prevents the association of the autophagic protein LC3. ¹⁷.

Lastly, PLB, C of bacteria and yeast enzymes looks to be a uncommon enzyme, because a only protein comprises three catalytic actions, lysophospholipase activity, phospholipase B activity and lysophospholipase/ transacylase activity, and 2 optimum pH average. The single enzymatic possessions will develop more clearly through investigating of the protein by three-dimensional structure ¹⁷.

ACKNOWLEDGMENT:

This works was sponsored by Biology Department /College of Science, Mustansiriyah University (www.uomustansiriyah.edu. iq) Baghdad, Iraq.

REFERENCES:

1. Bander, K. I., & Hamad, T. A. Prevalence of Vaginal Candidiasis among women and Diagnosis of Candida species from vaginal infection in Kirkuk city. Tikrit J. of Pure Sci., 20(4), 5-15(2018).‏

2. Colombo, A. L., de Almeida, J. N., Slavin, M. A., Chen, S. C., & Sorrell, T. C. Candida and invasive mould diseases in non-neutropenic critically ill patients and patients with haematological cancer. The Lan. Infec. Dis., 17(11), e344-e356(2017).

3. Kaur, R., Dhakad, M. S., Goyal, R., & Kumar, R. Emergence of non-albicans Candida species and antifungal resistance in intensive care unit patients. Asian Pacific J. of Tro. Biom., 6(5), 455-460(2016).‏

4. Shaker, D. S., Ishak, R. A., Ghoneim, A., and Elhuoni, M. A. Nanoemulsion: A review on mechanisms for the transdermal delivery of hydrophobic and hydrophilic drugs. Scientia Pharmaceutica, 87(3), 17(2019).‏

5. Bolen, A. L., Naren, A. P., Yarlagadda, S., Beranova-Giorgianni, S., Chen, L., Norman, D., Tigyi, G. The phospholipase A1 activity of lysophospholipase AI links platelet activation to LPA production during blood coagulation. Journal of lipid research, 52(5), 958-970.‏(2011).

6. Ma, Z., & Turk, J. The molecular biology of the group VIA Ca2+-independent phospholipase A2(2001).‏

7. Ishiguro, S., Kawai-Oda, A., Ueda, J., Nishida, I., & Okada, K. the defective in anther dehiscence1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmine acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. The Plant Cell, 13(10), 2191-2209(2001).‏

8. Wainszelbaum, M., isola, E., wilkowsky, S., cannata, J., florin-christensen, J., & florin-christensen, M. Lysosomal phospholipase A1 in Trypanosomacruzi: an enzyme with a possible role in the pathogenesis of Chagas’ disease. Biochemical J., 355(3), 765-770. (2001).‏

9. Fujino, S., Akiyama, D., Akaboshi, S., Fujita, T., Watanabe, Y., &Tamai, Y. Purification and characterization of phospholipase B from Candida utilis. Bioscience, biotechnology and biochemistry, 70(2), 377-386(2006)..‏

10. Feldman, M., Sionov, R. V., Mechoulam, R., & Steinberg, D. Anti-Biofilm Activity of Cannabidiol against Candida albicans. Microorganisms, 9(2), 441(2021).‏

11. Zuza-Alves, D. L., Silva-Rocha, W. P., & Chaves, G. M. An update on Candida tropicalis based on basic and clinical approaches.Frontiers in microbiology, 8, 1927(2017).

12. Kong, E. F. Clinical and Therapeutic Implications of Biofilm-Associated Fungal-Bacterial Interactions: Candida albicans and Staphylococcus aureus (Doctoral dissertation, University of Maryland, Baltimore(2017).‏‏

13. Collee, J. G.; Fracer, A. G.; Marmion, B. P. and Simmon, A. Practical Medical Microbiology (Mackie and MacCartenyeds). 14^th edition. Churchill Livingstone, Singapore(1996).

14. Abdul, F. R. Evaluation of some Virulence Factors, Hemagglutination and Agglutination of Antigens of Acinetobacter baumannii Isolated From Clinical Samples. Journal of Global Pharm. Technology; 10 (03):200-208.(2018).

15. Oishi, H., Morimoto, T., Watanabe, Y., &Tamai, Y. Purification and characterization of phospholipase B from Kluyveromyceslactis, and cloning of phospholipase B gene. Bioscience, biotechnology, and biochemistry, 63(1), 83-90(1999).‏

16. Naglik, J. R., Rodgers, C. A., Shirlaw, P. J., Dobbie, J. L., Fernandes-Naglik, L. L., Greenspan, D., Challacombe, S. J. Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections. The Journal of infectious diseases, 188(3), 469-479(2003).‏

17. Cuesta, A. I., Jewtuchowicz, V., Brusca, M. I., Nastri, M. L., & Rosa, A. C. Prevalence of Staphylococcus spp and Candida spp in the oral cavity and periodontal pockets of periodontal disease patients. Acta Odontológica Latino Americana, 23(1), 20-26(2010).‏

18. Abbas, Maysoon. K., Hussain, S. S., Noor, A. H. A., and Khadhom, I. Immunological and Molecular Study of Toll-Like Receptor-4 in Patients with Urinary Tract Infections. Annals of Tropical Medicine and Health, 23, 23-934(2020).‏

19. Hamdy, A. N., Abbas, Maysoon. K., Khudhair, M. A., and Tektook, N. K. Interactions between Interleukin-6 and MDA in Women with Preeclampsia. Systematic Reviews in Pharmacy, 11(3) (2020).‏

20. Abbas, M. K. " Changes in Immune Markers for Prostate Cancer Patients Pre and Post-operation". College of Science, University of Al-Mustansiriyah, Iraqi J. of Science, 55,3B:1218-1231.2014.

Received on 14.09.2021 Modified on 27.11.2021

Research J. Pharm. and Tech. 2022; 15(7):3119-3124.

DOI: 10.52711/0974-360X.2022.00522