الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
The aim of this work was to study the microbiological effect of linezolid, a relatively new antibiotic, against gram positive cocci in particular the staphylococci. This was achieved through studying the bacteriostatic action of linezolid, its antibiofilm activity, the mechanisms of linezolid resistance and the combination therapywith this antibioticto overcome resistance.
This study comprised 232 clinical staphylococcal isolates, collected in 2011-2012 and 2015. The identity of the isolates was checked by microscopical examination following gram stain which revealed violet grape like structures of spherical cells.
Linezolid resistance was checked using the disc diffusion technique which showed thatthe resistance increased from 0% in the 2011-2012 collection to 3% (n=3) among the 2015 collection. The three resistant isolates along with ten randomly selected susceptiblestaphylococcal isolates were identified using phenotypic characterization like catalase, MSA and DNase test agar and MALDI-TOF.MS.
These tests revealed that the three resistant and four susceptibleisolates were S. hemolyticusandthesix susceptibleisolates were S. aureus.
The MIC of linezolid against thethreeresistant and selected susceptible isolates was investigated using agar and broth microdilution techniques.
MIC ranged between 2-4 μg/ml among the ten susceptible isolates, and 128-256 μg/ml among the resistant ones.
Linezolid resistant mutants were selectedthroughthe serial passages of the susceptible isolates insub-inhibitory concentrations of linezolid. This resulted in three resistant S. aureusmutants as revealedby the increase in their linezolid MIC by 32-128 foldswhendetermined by broth microdilution method.
However, no single step mutants were generated following exposure to linezolid supra-inhibitory concentrations.The resistant S. hemolyticusisolates, resistant S. aureusmutants and the original susceptible isolates before mutation were investigated for their susceptibility towards 13 different antibiotics using the disc diffusion method.
The resistant isolates and mutants were multidrug resistant to almostall tested antibiotics except tetracycline, vancomycin, nitrofurantoin, andquinupristin-dalfopristin. On the other hand, by comparing the resistance patterns of the mutants withtheir parent susceptible isolates, it was found that tetracycline resistancewas lost during serial passage. Moreover, resistance towards chloramphenicol, rifampicin, gentamicin, ciprofloxacin, sulphamethoxazole-trimethoprim, clindamycinand erythromycin was gained.
To investigate the killing pattern of linezolid, the survival ofthe 6 resistant isolates and mutants together with 2 susceptible isolates was tested using time kill assays and growth curves at 1/2×MIC, 1×MIC and 2×MIC at time intervals 0, 6 and 24 hr.
These assays revealed that linezolid was generally a bacteriostaticagent at sub-inhibitory, inhibitory or supra-inhibitory concentrations. Linezolid combinations with different antibiotics: vancomycin, doxycycline and imipenemweretested at subinhibitory concentrations using time kill assaysagainst the resistant isolates and selected mutants.Linezolid-vancomycin combinations were indifferent.
However, the combination with doxycycline and imipenem showed synergistic activity. In addition,the combined effect of anti-inflammatory agents and linezolid, tested using modified checkerboard assay, revealed a synergistic effect to some extent
Summary, Conclusions and Recommendations75Eighteen linezolid susceptible S. hemolyticus(MIC range 2-4 μg/ml) isolates were selected and their biofilm formation potential was investigated by crystal violet staining technique.
Theisolates were found to be weak to moderate biofilm producers. Then, the anti-biofilm activity of linezolid was investigated against the same isolates by measuring the BPC, MBIC, and MBEC. They were 4-128 μg/ml, 16-128 μg/ml, and > 512 μg/ml, respectively.To study the mechanisms of resistance of linezolid, the three resistant isolates were screened for the presence of cfrgene using PCRtechnique.
Only one isolate: S16 was found to carry the cfrgene which is usually plasmid borne. Its role in resistance mediation was confirmed by performing a curing experiment by successive cultivation at 42°Cwhich led to the loss of linezolid and chloramphenicol resistance.
The absence of cfrgene was confirmed by PCR.Transformation using various methods have been performed to study the transferability of the plasmid.
Attemptsto transformDH5α chemically competent cellsusing heat shock or susceptible staphylococcal competent cellsusing electroporation with the cfrcarrying plasmidfailed to produce linezolid resistanttransformants.
To detect the effect of the othermechanismsof linezolid resistance, namely mutations in the V domain of the 23S rRNA gene, PCR was used to amplify the different alleles of 23S rRNAin the threeresistant isolatesusing published and designed primers.
S16 was found to carry four alleles, S60 carried 3 alleles and S42 carried one allele.
The V domain in these alleles was sequenced and aligned against the published sequences of the gene in NCBI database.
The analysis revealed G2603Tpoint mutation in the different alleles of S16 and S60which was suggested to have a role in linezolid resistance.