الفهرس 
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Abstract
Diabetes mellitus (DM) is a chronic disease that characterized by high glucose concentrations as a result of the inability of the body to adequately produce or use insulin effectively. The World Health Organization predicts that diabetes will be the seventh leading cause of death worldwide in 2030. Moreover, it was expected that the range of diabetics increased from 382 million to 592 million from 2013-2035 according to Centers for Disease Control and Prevention. Egypt is among 10 top countries with the highest prevalence of diabetes and ranked ninth in the world, where there are 7 million and a half million Egyptians are living with diabetes and up to 15–25% of those with diabetes will develop a foot ulcer during their lifetime.
Diabetic foot ulcer (DFU) represented the most feared diabetic complication that caused the hospitalization of the diabetic patient. DFU usually characterized with delayed healing as, the diabetic neuropathy, angiopathy and become infected (40%–80%) that leads to the development of gangrene and which even necessi¬tates limb amputation. In recent years, the emergence of antibiotic resistant bacteria has made it increasingly difficult to select appropriate antibiotics for the treatment of Diabetic foot infection (DIF) resulting in a significant upturn of morbidity and mortality, emphasizes the importance of developing new treatment strategies to effectively eradicate these infections as phage therapy due to its specificity and stability under harsh environmental conditions and suitable for use in humans since phage do not infect eukaryotic cells and it replicates at the site of infection, ensuring their safety and absence of side effects.
In the present study, a total of 85 selected DFIs specimens were obtained from patients with foot ulcers hospitalized in the Al Demerdash Hospital, Nasr City, Health Insurance hospital, Sednawy Health Insurance hospital and diabetic foot clinics from December 2017 till May 2019. All collected specimens were transported to laboratory of microbiology at faculty of Education Ain shams University within 20 min for detection of bacteria and inoculated onto blood and MacConkey’s agar media. The isolated bacterial colonies were purified and identified based on morphological characteristics and gram staining.
The present study revealed that 85 specimens were obtained from diabetic foot patients. Sixty eight male (76.4%) and nineteen female (22.35%) of diabetic cases with the age ranged from 40 to 80 years. From85 collected wound swab specimens, only 78 (91.8 %) were positive for bacterial isolation with an average of 2.6 organisms per ulcer. from 78 positive cultures cases, 10 patients (12.8%) had mono microbial infection and 68 patients (87.2%) had poly microbial infections.
The results also indicated that the proportion of Gram negative bacilli was higher than Gram positive cocci and the commonest isolates in this study according their morphological characteristics on selective media were Staphylococcus sp. (24%), followed by Pseudomonas aeruginosa (18.2%), Klebsiella sp. (17.3%), Proteus sp. (15.6%), Escherichia coli (12.9%), whilst, the lower prevalence was recorded for Acinetobacter sp. (4.8%), Enterococcus sp. (4.8%) and Enterobacter sp. (2.4%).
The antibiotic resistance patterns of the isolated gram negative bacilli and the gram positive cocci were studied using different twenty five antibiotics. It was found that, the isolates were multi-drug resistant with percentage of 80%, 73.7%, 83.3%, 68.75, 92%, 80%, 90% and 100% for Staphylococcus sp., P. aeruginosa, Klebsiella sp., Proteus sp., E. coli, Acinetobacter sp., Enterococcus sp. and Enterobacter sp., respectively.
According to the different morphological characteristics on selective media and the multidrug resistance with high index, eight bacterial isolates were chosen and characterized biochemically using vitek 2 and identified as: S. aureus, P. aeruginosa, K. pneumonia, P. mirabilis, E. coli, A. baumannii, E. faecalis and E. cloacae.
The most resistant and prevalent five bacterial isolates S. aureus, P. aeruginosa, K. pneumonia, P. mirabilis and E. coli were confirmed genotypically using molecular analysis by 16S rRNA gene and used in phage isolation.
To examine the therapeutic effect of bacteriophages in resolving diabetic foot infection in animal model, phages specific for S. aureus, P. aeruginosa, K. variicola, E. coli and P. mirabilis were isolated. Sixteen phage isolates were obtained from three sewage samples and formed distinct plaques which differ in size and transparency, then propagated and purified with syringe filter 0.2μm. Five lytic phages with high titers were chosen for further studies and designed as S2 Staphylococcus phage, Ps1 Pseudomonas phage, K4 Klebsiella phage, Pr2 Proteus phage and C3 coliphage (E. coli). The morphological, biological and physical characteristics of five selected phages were studied.
The results of morphological identification with Transmission Electron Microscope revealed that isolated phages belong to order Caudovirales and two families Podoviridae and Myoviridae.
.Biological properties of phages as host range pattern and rate of adsorption were done. It was found that all phages had broad spectrum. The adsorption rate of S2 phage on S. aureus was found to be 3 min, while 10min for PS1 phage on P. aeruginosa, 15 min for K4 phage on K. variicola and C3 phage on E. coli and 25 min for Pr2 phage on P. mirabilis.
Results of the thermal end point revealed that all phage isolates remain infective after the exposure to 40oC, 50oC and 60oC, variable in their stability between 60 to 80oC, but lost their abilities to lyse specific host at 90oC. The obtained results also revealed that the viral infectivity the maximum infectivity of all phages was in neutral medium at (pH=7) and lost their ability to lyse host at pH12. In the other hand, Ps1 phage was in alkaline medium (pH= 8), in contrast Pr2 which were in acidic medium (pH=5). Also, it was found that, all tested phages remain stable at refrigerator 4oC for eight months than at frozen temperature -20oC than at room temperatures.
Forty-five adult male Sprague-Dawely rats were assigned in 3 groups (15 rats each), namely, the diabetic infected wound group, diabetic infected wound ceftriaxone treated group, and the diabetic infected wound phage cocktail treated group. The results revealed that phage cocktail had a superior effect over the ceftriaxone in wound healing parameters; wound size, wound index, wound bacterial load, mRNA expression of; wound healing markers (Cola1a, Fn1, MMP9, PCNA, and TGF-β), inflammatory markers; (TNF-α, NF-κβ, IL-1β, IL-8, and MCP-1), anti-inflammatory markers (IL-10 & IL4), and diabetic wound collagen deposition, also, the histomorphic picture of the diabetic infected wound. Based on the current findings it could be speculated that, phage therapy could be considered a novel antibiotic substitute in the DFU with MDR-poly microbial infection therapeutic strategies.
Conclusion
Diabetic foot is one of the most feared complications of dia¬betes and characterized by several pathological complications such as foot ulceration which frequently become infected with bacteria. Healing of these ulcers is largely delayed by the concomitant infection. The looming threat of antibiotic resistance calls for immediate action. Phage therapy is well suited to be part of the multidimensional strategies to fight against it.
The present study revealed that 85 samples were obtained from diabetic foot patients. Only 78 samples (91.8 %) were positive for bacterial isolation. A total of 208 bacterial isolates were obtained with an average of 2.6 organisms per ulcer. The present study revealed that the proportion of Gram negative bacilli was higher than Gram positive cocci. The antibiotic resistance patterns of the isolated gram negative bacilli and the gram positive cocci were studied using different twenty five antibiotics and it was found that, the isolates were multi-drug resistant with percentage ranged between 68 to 100%.
The present study focuses on the use of five lytic phages cocktail specific for five selected multidrug resistant isolates in comparison with ceftriaxone as an effective treatment strategy against the polymicrobial infection in diabetic foot rats. Phage cocktail therapy in the current study increased the animal survival rates, decreased the wound index, bacterial loads and decreased the levels of inflammatory markers and oxidative stress, increasing the expression of regenerative markers.
Finally, we can conclude that phage therapy could be considered a novel antibiotic substitute in the DFU with MDR-poly microbial infection therapeutic strategies.
*Recommendation
The results of this study suggested the topical phage therapy was a promising method for the treatment of non-healing diabetic wounds and doesn’t exhibit any side effects that ensuring their safety. So, we recommend increasing the application scale of phage therapy to include the diabetic foot patients.
The proper manipulation of these highly active phages, full genome sequencing and protein analysis can be an ultimate key for better application.