الفهرس 
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Abstract
Global human health is seriously threatened by the emergence and spread of XDR-CR GNB that have been dramatically increased in recent years causing a worldwide concern. The consequences of the high morbidity and mortality rates have made it essential to quickly identify CPs as soon as possible and to urgently find a solution to stop this hazardous public health disaster. Thus, the goal of this study is to search for new treatment options in an attempt to combat carbapenem resistant MDR-GNB infections that has emerged as a result of treatment failures along with the global rise in antibiotic resistance.
In this study, 59 non-duplicate CR GNB isolates from unidentified patients were recovered from the microbiology lab of the El Demerdash Tertiary Care Hospitals in Cairo, Egypt. Results revealed that a total of 21 (35.6%) and 38 (64.4%) CR GNB isolates were identified as enterobacterial isolates (including, 16 (27.1%) K. pneumoniae and 5 (8.5%) E. coli) and non-fermentative bacilli (including, 23 (39%), A. baumannii, and 15 (25.4%) P. aeruginosa).
The antimicrobial susceptibility tests were carried out using Standard procedures. The antibiogram analysis showed that all the 59 isolates (100%) had high resistance levels to different antimicrobials including carbapenems, in addition to the fact that the MIC of all isolates indicated that they are all CR and as a consequence, they are all categorized as CR-XDR isolates.
The isolates were investigated for carbapenemase production using major phenotypic techniques as mCIM, BCT and CDT. A combined disc test was initially used to screen CPs for MBLs
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identification. In our present study, 45.8% (27 out of 59) of the tested isolates tested positive for class B carbapenemase. The mCIM and blue- carba tests were also performed on the 59 CP isolates to guarantee compliance with the most recent carbapenemase production criteria. The mCIM test recognized 52 (88.1%) of the CP isolates, whereas the blue- carba test detected 57 (96.6%).
Polymerase chain reactions (PCR) were performed for molecular identification of the five main carbapenemase-coding genes. The multiplex PCR results showed that blaKPC was detected in 40 (67.8%), followed by blaOXA-48 that was amplified in 30 (50.8%) isolates, followed by blaVIM that was found in 16 (27.1%) isolates. Nevertheless, neither blaIMP nor blaNDM were found in any of the tested XDR isolates. Phenotypic and genotypic relatedness was carried out using the heatmap and ERIC PCR analysis. The heatmap and ERIC PCR analysis resulted in non-clonal 28 XDR CR isolates which were next chosen for further investigation.
Combinations of MEM with PR at a concentration of 0.5 -1 mg/mL against the resulting 28 non-clonal XDR-CR GNB pathogens were assessed by calculating the MIC decrease factor (MDF), Results revealed that PR, at a concentration of 0.5 mg /mL, decreased MICs values of the tested XDR CR isolates (28; 100%) and restored susceptibility of only 4 (14.3%) isolates. However, PR (1 mg/mL) when combined with MEM has completely (28; 100%) restored the susceptibility of the tested XDR CR GNB to MEM.
The effects of various combinations of MEM with AK or TGC on 28 chosen CR-GNB pathogens were studied in order to decrease or
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eradicate CR in some CR-GNB isolates using the checkerboard assay. Results showed that combinations of MEM/AK and MEM/TGC had additive effects of 92.8% and 71.4% and indifferent effects of 7.2% and 28.6%, respectively.
The inadequate therapeutic opportunities associated with CRPA clinical isolates impose a search for innovative strategies. This has prompted a reconsideration of bacteriophage therapy as a potentially beneficial therapeutic approach. As a result, we used an appropriate skin animal model to assess the lytic activity of two new isolated bacteriophages formulated as hydrogels to cure CRPA-infected burns.
This was done by isolating four clinical P. aeruginosa isolates from a third-degree burn patient who is suffering from significant complications and resistance to numerous antimicrobial drugs employed in treatment. The antibiogram analysis revealed that the isolates exhibited a high level of resistance to numerous antimicrobial drugs, including carbapenems, as well as the MIC of all isolates, indicating that they are all CR and so categorized as CR-XDR isolates.
For isolation of bacteriophages,15 environmental samples were taken from a hospital’s sewage source for this purpose. However, only two lysates continued to yield positive outcomes and named vB_Pae_SMP1 and vB_Pae_SMP5. They were active against two of the four tested XDR P. aeruginosa clinical isolates.
vB_Pae_SMP1 and vB_Pae_SMP5 phage suspensions were presented for Transmission electron microscopy (TEM) examination. Their morphological characteristics indicated that they belong to the
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class Caudoviricetes. Moreover, Phage vB_Pae_SMP5 was genotypically determined to be a member of the class Caudoviricetes and genus Septimatrevirus and therefore, we suggest that it can be categorized as the Septimatreviridae family.
For evaluating the in vitro activity of the isolated phages formulated as hydrogels on a CRPA-infected burn, The CRPA propagated phage lysate was combined with CMC to form the hydrogel, and the anti-CRPA activity of the tested hydrogel was evaluated using the cup-plate technique. The results demonstrated the formation of inhibition zones around both the positive control cups containing either vB_Pae_SMP1 or vB_Pae_SMP5 phage lysates and around the cups containing either the tested phage vB_Pae_SMP1 or vB_Pae_SMP5 hydrogel.
A thermal injury model in rats infected with the CRPA isolate (50S) was designed to assess the ability of bacteriophages formulated as hydrogels to alleviate such pathogenicity. The results of our study revealed that the tested hydrogel containing the bacteriophages increased survival rate, as all of the animals given this tested hydrogel survived.
The histological examination of different wounded groups revealed that groups treated with either Phage vB_Pae_SMP1, vB_Pae_SMP5, or a two-phage cocktail hydrogel showed cellular subepidermal granulation tissues with abundant records of fibroblastic activity and mixed inflammatory cell infiltrates and presented 17.2%, 25.8%, and 22.2% records of dermal mature collagen fibers, respectively.