الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
S. aureus is a human and animal pathogen that is a global cause of morbidity and mortality. While S. aureus was previously recognized as a common cause of nosocomial infections, some strains have a propensity to disseminate between otherwise healthy individuals giving rise to community-acquired infections. Further amplifying the gravity of S. aureus infections is the emergence of multi-drug resistant strains such as MRSA that can demonstrate enhanced infectivity and virulence. These bacteria can colonize virtually every tissue in the body causing pathologies varying from minor SSTIs to fatal invasive diseases such as necrotizing pneumonia, osteomyelitis and sepsis.
Methicillin resistance in staphylococci is based on excessive production of PBP2a, an altered penicillin-binding protein that exhibits a low affinity for β -lactams antibiotics, encoded by the mecA gene. Because of its low affinity for all β-lactam antibiotics, resistance to methicillin confers resistance to all β-lactam agents, including cephalosporins.
Aminoglycosides still play an important role in antistaphylococcal therapies, although emerging resistance amongst staphylococci is widespread. They are often used in combination with either a β-lactam or a glycopeptide, especially in the treatment of staphylococcal endocarditis, as these drugs act synergically.
The main mechanism of aminoglycoside resistance in staphylococci is drug inactivation by AMEs. These enzymes are categorized into three classes AACs, APHs, and ANTs. Among them, three enzymes, named aminoglycoside - 6′ - N – acetyltransferase / 2′′ - O – phosphoryltransferase ([AAC(6′)Ie/APH(2′′)Ia], aminoglycoside - 4′ - O – nucleotidyltransferase – I [ANT(4′)-Ia], and aminoglycoside - 3′ - O phosphotransferase III [APH(3′)-IIIa], encoded by aac(6’)-Ie/aph(2’’)Ia , ant(4’)-Ia, and aph(3’)-IIIa genes, respectively, are of particular significance because they are the most common AMEs in staphylococci.
The aim of this work was to detect the genes encoding AMEs among clinical isolates of MRSA.
Clinical samples were collected from outpatients attending the microbiology department of the Medical Research Institute, Alexandria University; over a period of 5 months.
Identification of S. aureus was done by both conventional methods, including morphology, culture characteristics, Gram stain, catalase test and coagulase test, and molecular methods by the detection of amplified femA gene using PCR.
Phenotypic detection of MRSA was done using cefoxitin antibiotic discs and chrOMagar MRSA.
Detection of MRSA was confirmed genotypically by amplification of mecA gene using PCR.
Susceptibility of MRSA isolates to different antibiotics including aminoglycosides was determined by Kirby-Bauer disk diffusion method.
Inducible clindamycin resistance was detected using the D test.
Summary
90
Susceptibility to vancomycin was determined using broth microdilution method.
Genotypic detection of aminoglycoside resistance was determined by the amplification of genes encoding AMEs using different sets of primers by PCR.
One hundred S. aureus isolates were identified as Gram-positive, catalase positive, and coagulase positive cocci.
Fifty methicillin resistant isolates out of the 100 S. aureus isolates were phenotypically detected by cefoxitin antibiotic discs and by the growth on chrOMagar MRSA with the production of rose to mauve colonies. Methicillin phenotypical resistance was confirmed in all cases by PCR detection of mecA gene.
All our 50 MRSA isolates were also femA positive.
The majority of MRSA isolates were detected in aspirated pus 30 (60%) followed by wound swabs 13 (26%). The rate of isolation was higher in males 31 (62%) than in females.
Using the Kirby-Bauer disk diffusion method, all the isolates were susceptible to teicoplanin, tigecycline and linezolid. Most of the isolates were resistant to tetracycline 38 (76%), fusidic acid 37 (74%), ciprofloxacin 37 (74%), ofloxacin 36 (72%), and levofloxacin 35 (70%).
Inducible clindamycin resistance was detected using the D test in 6 (13.04%) of our clindamycin susceptible MRSA isolates.
All isolates were sensitive to vancomycin, using broth microdilution method, with the majority 35 (70%) having MIC of 1 μg/ml. No vancomycin intermediate isolates were found.
Ninety four percent 47 (94%) of our MRSA isolates were resistant to at least one of the tested aminoglycosides, 41 (82%) isolates were resistant to neomycin, 38 (76%) to tobramycin, 33 (66%) to gentamicin and kanamycin, 21 (42%) to sisomicin, 13 (26%) to amikacin, and only one isolate (2%) to netilmicin.
Genotypic detection of AGAs resistance was carried out by amplification of genes encoding AMEs using PCR. The aph(3’)IIIa gene was the most frequently encountered gene as it was detected in 36 (72%) of our isolates followed by ant(4’)Ia in 29 (58%) and aac(6’)Ie/aph(2’’)Ia in 20 (40%) of our 50 isolates.
The distribution of the genes encoding aminoglycoside resistance among our strains resulted in 8 genotypic resistance patterns, the most common pattern was the presence of both ant(4’)Ia and aph(3’)IIIa in 13 (26%) of our isolates.
Twenty percent 10 (20%) of our isolates possessed the 3 AME genes but showed 7 different phenotypic resistance patterns.
Seventeen phenotypic resistance patterns were described, none of them could be traced to a single genotypic pattern.
Summary
91
The most common phenotypic resistance pattern detected among 11 (22%) of our isolates, showed resistance to gentamicin, amikacin, tobramycin, neomycin, kanamycin, and sisomicin and was associated with 5 genotypic resistance patterns.
Absence of the three genes was observed in 6 isolates. Among these, 2 isolates were sensitive to all the tested AGAs, 2 were resistant to neomycin, 1 isolate was resistant to gentamicin, tobramycin, and amikacin, and 1 isolate was resistant to gentamicin and tobramycin.
Three isolates were susceptible to all the tested aminoglycosides, 2 of which showed complete absence of the genes encoding AMEs and one isolate possessed the ant(4′)-Ia gene.
Even when phenotypic resistance patterns were restricted to the 3 most commonly used AGAs in Egypt, gentamicin, tobramycin, and amikacin, no correlation was found between phenotypic and genotypic resistance patterns.