رسائل دكتوراة
English
2021
Immunogenic Properties of Capsular Polysaccharides (CP5) against Methicillin Resistant Staphylococcus aureus in Rats
أمل محمد سعيد محمد جواد الموسوي, علاء هاني الجراخ
كلية الطب-جامعة بابل · العراق
The present study aimed to investigate active immunization strategies using capsular polysaccharides (CP5) in protection against Staphylococcus aureus clinical isolates in animal models of infection in order to provide protection against various Methicillin Resistant Staphylococcus aureus in the acute fatal bacteremia model. A total of 180 different clinical samples were collected from patients who were admitted to the main hospitals in Babylon province; Hilla Teaching Hospital and Babylon Teaching Hospital for Maternity and Children in Al-Hilla city during the period from January till November 2020. Samples obtained included blood, urine, cerebrospinal fluid, burns, wounds, and others (bronchial fluid, abscess).
Among 180 clinical samples, 46 (25.5%) gave positive growth on ordinary culture media while 134 (74.5%) gave no growth, yielding 46 bacterial isolates identified using biochemical tests, Vitek2-system ID cards, and Analytical Profile Index (API) system. Results revealed that out of 46 culture-positive samples, a high rate of isolation 16 (34.7%) was found in urine samples, followed by 15 wound (burn) samples (32.6%), blood 12 (26.1%), abscess 2 (4.3%), while only one sample (2.1%) from bronchial fluid showed positive growth.
Among 46 positive cultures, 22 (47.82%) were Gram-positive and 24 (52.17%) were Gram-negative bacteria. Among 22 Gram-positive bacterial isolates, 17 belonged to Coagulase-negative Staphylococci (CoNS) and 5 were identified as S. aureus. All 5 S. aureus isolates were tested for methicillin resistance using cefoxitin as an indicator of MRSA according to CLSI guidelines. Molecular detection of Staphylococcal cassette chromosome; mecA and capsular polysaccharides (CP5) genes revealed that 5 S. aureus isolates carried the mecA gene and one isolate (A.MS isolate) had capsular polysaccharides (CP5) gene.
Isolation, extraction, and partial purification of capsular polysaccharide 5 (CP5) from local methicillin-resistant S. aureus isolates (A.MS isolate) was carried out using previous methods with modifications. This is the first study in Iraq. Chemical composition of the partially purified extract of S. aureus capsular polysaccharide 5 (CP5) from A.MS isolate was measured by UV-visible spectrophotometer. The results found that the absorption of CP5 at 206 nm was 0.279; Conc. 6 μg/mL, the absorption of protein (0.017); Conc. 3.4 μg/mL at 280 nm, and the absorption of nucleic acid content (0.018); Conc. 3.6 μg/mL at 260 nm. Concentration of the partial extract of S. aureus CP5 was 6 mg/L.
The present study aimed to investigate active immunization strategies using CP5 emulsified with polysorbate 80 (Tween-80) in animal models. In the experimental group, 8 female rats were treated intraperitoneally with CP5-Tween80 in two doses (50 μg/10 μL per animal, 7 days between doses), and the control group (8 female rats) received phosphate buffer saline in two doses (10 μL per animal). After 3 weeks, blood was collected for hematological and serological analysis before challenge dose. At 30 days after the first immunization, rats were challenged intraperitoneally with 0.5 mL inoculum containing 10^7 CFU S. aureus.
The degree of protection was assessed by the number of rats surviving 7 days after infection. Blood samples were collected to measure total and differential leukocytes, bactericidal power, interleukin-4 (IL-4), and interleukin-12p40 (IL-12p40) by ELISA before and after the challenge dose. Hematological analysis before challenge showed non-significant increase in total leukocyte count (TLC) in group I (11093 cells/mm³) compared with group II (9650 cells/mm³), p < 0.05. Differential leukocyte counts were not significantly different. IL-4 levels increased significantly in group I (4.775 pg/mL) compared with group II (0.709 pg/mL), p < 0.05. IL-12p40 levels showed no significant change (group I: 7.496 pg/mL, group II: 12.115 pg/mL, p < 0.05). Bactericidal power of blood showed higher clearance in group I (71.5%) compared with group II (33.4%).
After challenge dose, TLC significantly increased in group I (9407 cells/mm³) compared with group II (4682 cells/mm³), p < 0.05. IL-4 levels were significantly higher in group I (8.6676 pg/mL) than group II (2.8531 pg/mL), p < 0.05. IL-12p40 showed non-significant change (group I: 10.483 pg/mL; group II: 5.792 pg/mL, p < 0.05). Histopathological examination of spleen slices showed white pulp enlargement with lymphoid follicular changes in group I, while control group slices showed shrinkage and infiltration.
The study concluded that CP5 extracted from A.MS S. aureus isolate improved immune responses against MRSA, enhancing humoral immunity and inducing histopathological changes in spleen that stimulate immune defense against antigens.