الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
DM is a one of the major public health problem worldwide. People with DM have about a 25% chance of developing a foot ulcer in their lifetime, about half of which are infected at the time of clinical diagnosis. Bacterial strains resistant to widely prescribed antibiotic agents, especially highly resistant gram negative aerobes, are becoming more common in DFIs. Moreover, Bacteria isolated from DFIs frequently form biofilms. PRP is an autologous derivative of whole blood that contains platelet concentrations above the average level, also contains the full complement of clotting factors, chemokines, cytokines, a range of GFs, and other plasma proteins. Current evidence suggests PRP as a good antibacterial agent that could help to prevent postoperative infections and treat chronic wound or bone infections. The advantages of PRP are apparent since it is easy to prepare, cheap, autologous in nature, and less likely to induce antibiotic resistance. Therefore, more research is needed to assess the in vitro antibacterial and antibiofilm activity of PRP against MDR bacteria to lead up to clinical trials of localized PRP implementations in DFIs.
The present study aimed to:
1- Isolate and identify certain MDR bacterial strains from infected DFUs.
2- Assess the antibacterial effect of PRP against the isolated MDR bacterial strains.
3- Examine the isolated MDR bacterial strains for their ability to form biofilm.
4- Assess the effect of PRP against biofilm formed by the isolated MDR bacterial strains.
The study was conducted over the period from April 2021 to May 2022 in the Microbiology Laboratory at HIPH and the Clinical Pathology Laboratory at AMUH. A total of 78 swab samples were randomly collected, using Levine’s technique, from clinically suspected infected diabetic foot lesions of patients admitted to the Vascular Surgery and Diabetic Foot Unit at the Surgery Department in AMUH. All samples were subjected to the conventional microbiological methods to isolate and identify bacterial pathogens. Bacterial isolates other than MRSA, MDR K. pneumoniae, and/or MDR P. aeruginosa were excluded from the PRP experimental study. All MDR K. pneumoniae isolates were tested for ESBL production and all MDR P. aeruginosa isolates were tested for carbapenemase production by mCIM, as recommended by the CLSI.
For PRP preparation, venous blood samples were obtained from 16 adult volunteers. The volunteers were all females and the mean age was 31.17 ± 2.79. The mean platelets count was 233.17 ± 79.81 ×109/L in whole blood, and increased to 823.67 ± 282.05 × 109/L in PRP with an average increase of 3.53 fold of platelet concentration after processing.
Antibacterial activity of PRP was determined using Kirby-Bauer disk diffusion method on MHA plates, broth microdilution assay, chekerboard synergy testing, and time-kill assay. Biofilm forming ability of the identified isolates was tested by tissue culture plate method. Biofilm inhibition and eradication assays were performed to evaluate the effect of PRP on biofilm formation and established biofilms, respectively.
The results of the present study revealed that:
1. Mono-microbial growth was observed in 63 (80.77%) samples, while 8 (10.26%) showed poly-microbial growth leading to 79 bacterial isolates.
2. The most common bacterial isolate was K. pneumoniae (29.11%) followed by S. aureus (25.32%), and then P. aeruginosa (20.25%).
3. The majority of S. aureus isolates were MRSA (80.00%) and all P. aeruginosa and the majority of K. pneumoniae (91.30%) isolates were MDR.
4. Among the 21 MDR K. pneumoniae isolates, 11 (52.40%) were ESBL-producers and among the 16 MDR P. aeruginosa isolates, 6 (37.50%) were carbapenemase producers.
5. PRP could not inhibit the growth of MRSA, MDR K. pneumoniae, or P. aerugionsa isolates by neither Kirby-Bauer disk diffusion method nor broth microdilution assay.
6. By using the time-kill assay, there was a highly statistically significant antimicrobial effect of PG (activated PRP) against the planktonic cells of the three tested MDR strains when compared to the controls after 1, 2, and 5 hours of incubation (p-value < 0.0001).
7. The peak point of effectiveness for PRP inhibited nearly 99.50%, 91.35%, and 97.23% of growth rates of MRSA, MDR K. pneumoniae, and MDR P. aeruginosa isolates, respectively.
8. There was a decrease in the effectiveness of PPR after the second hour of incubation for MRSA and MDR P. aeruginosa and the first hour of incubation for MDR K. pneumoniae but the effect was maintained up to 5 hours with no effect of PRP on the tested strains at 24 hour of incubation.
9. By using the checkerboard synergy testing of PRP-antibiotic combinations against the 53 studied bacterial isolates, the synergistic activity of PRP was more prominent against P. aeruginosa isolates, where PRP had a synergetic effect for 37.50% of P. aeruginosa isolates when combined with ceftazidime. In contrast, the antagonistic activity of PRP was more prominent against MRSA isolates when combined with cefixitin (37.50%).
10. The effect of PRP-antibiotic combinations was indifferent against half of MRSA (50.00%) and the majority of K. pneumoniae (80.95%) isolates.
11. Most of the studied bacterial isolates (69.81%) were biofilm producers.
12. The majority of ESBL-producing K. pneumoniae isolates (81.82%) were biofilm producers. However, those results were not statistically significant.
13. PRP had not shown any biofilm inhibition or eradication activity.
It can be concluded from the present study that:
1. Antibacterial activity of PRP is transient, depending on the bacterial strain.
2. Only activated PRP is able to inhibit microbial growth.
3. The inhibitory effect of PRP reaches its peak point at the second hour of incubation for MRSA and MDR P. aeruginosa isolates and at the first hour of incubation for MDR K. pneumoniae.
4. There is a decrease in the effectiveness of PPR after the peak point and by the 24th hour of incubation, PRP display no antibacterial activity.
5. PRP can neither inhibit biofilm formation nor eradicate firmly established biofilms.
from the results of the current study, the following recommendations are suggested:
1. Treatment of DFIs should rely on the results of culture and antibiotic susceptibility tests instead of the usual trend of empirical treatment to avoid overuse of unnecessary antibiotics that eventually lead to the emergence of MDR bacteria.
2. Further research is required to meticulously assess the antibacterial properties of PRP, especially in vivo, before definitive conclusions regarding its effectiveness can be made.
3. Prospective, randomized, controlled clinical studies on a large scale are essential for establishing standardized guidelines regarding the indications, contraindications, and specific procedures for PRP utilization in clinical settings.