الفهرس 
Literature ReviewOnly 14 pages are availabe for public view
Abstract
The present study aimed to emphasize the importance of performing genotypic studies beside phenotypic ones for characterization of carbapenemase producing GNB among pediatric cancer patients. Out of 187 GNB isolates recovered from different clinical specimens during the course of the study, 174 were enterobacterial isolates and 13 were NF GNB. The most common enterobacterial isolates were E. coli (88, 50.57%), followed by Klebsiella spp. (72, 41.37%), while A. baumannii (7, 53.84%) was the most common NF GNB. The majority of the recovered GNB isolates (79.67%) were from blood specimens.
The overall antibiogram analysis of GNB isolates revealed a remarkable high resistance pattern towards the tested antimicrobial agents, especially β-lactam group including amoxicillin/clavulanic acid, 3rd and 4th generation cephalosporin and aztreonam. Additionally, 173 (93.51%) GNB showed resistance to at least one agent in three or more antimicrobial classes and were considered MDR. On the other hand, a remarkable reduced resistance profile was recorded for polymyxins group, tigecycline, and amikacin.
from the antibiogram results, 125 (67.56%) GNB showed resistance to one or more carbapenem (doripenem, ertapenem and imipenem) by Kirby Bauer method and were considered carbapenem resistant isolates. Additionally, the results of MIC by broth microdilution method reveals that out of 73 isolates of E. coli 50 (68.49%) and 51 (69.86%) were ertapenem and imipenem resistant, respectively. Out of 46 isolates of Klebsiella species 32 (69.56%) and 42 (91.30%) were ertapenem and imipenem resistant, respectively. Subsequently, isolates that showed reduced sensitivity by disk diffusion and MIC were selected for performing a series of phenotypic tests.
The ability of E. coli and Klebsiella spp. to produce carbapenemase enzyme was determined by using MHT, mCIM, BCT and CDT. Preliminary screening of carbapenemase producers was done using MHT and of the 90 enterobacterial isolates tested 42 (46.66%) with ertapenem disk and 34 (37.77%) with meropenem disk showed cloverleaf like indentation. To discriminate between the classes of carbapenemases, boronic acid and EDTA were used in addition to suitable substrate to inhibit class A and Class B, respectively in the CDT. Out of 21 isolates 4 (19.04%) and 15 (71.42%) were positive for class A and class B carbapenemase, respectively. Out of 15 phenotypically confirmed carbapenemase producer by previous methods, 7 (46.6%) and 12 (80%) of enterobacterial isolates were mCIM and BCT test positive, respectively.
In addition to the previously mentioned tests, we further screened the phenotypically confirmed carbapenemase producer isolates for plasmid mediated carbapenem resistance genes. Almost 90.6% of the tested isolates (29 out of 32) showed bands in their electrophoresed plasmid extracts. In a trial to confirm the association of these plasmid bands with carbapenem resistance, transformation experiment was performed using E. coli DH5α competent cells and four plasmid extracts were successfully transformed. Subsequently, all the phenotypically confirmed plasmid mediated carbapenemase gene (29 isolates) were subjected to multiplex PCR using blaKPC, blaIMP, blaVIM, blaNDM and blaOXA-48 specific primers. The results revealed that blaOXA-48 was the most prevalent 17 (58.62%), followed by blaNDM 8 (27.58%), then blaVIM 3 (10.3%) and finally blaKPC 2 (6.89%).
With regard to limited therapeutic options for treating severe infections caused by multidrug resistant GNB, in vitro and in vivo evaluation of different antibiotics was tested. Checkerboard broth microdilution technique was used for in vitro evaluation of different antibiotic combinations. Colistin combination with meropenem or ertapenem and double carbapenem regimen with ertapenem as suicidal agent showed fraction inhibitory concentration index less than or equals 0.5 (synergy) in most of the tested isolates. However, in vivo efficacy of colistin with meropenem was not superior to monotherapy in thigh infection model.