الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
S. aureus is a dangerous pathogen capable of causing a variety of superficial or invasive diseases e.g. pneumonia, bloodstream infections, endocarditis, urinary tract infections, gastrointestinal tract infections, osteomyelitis, thrombophlebitis, and meningitis.
S. aureus colonizes the skin and mucosae of human beings and several animal species. Although multiple body sites can be colonized in human beings, the anterior nares of the nose are the most frequent carriage site for S aureus. MRSA nasal colonization is considered a major risk factor for subsequent nosocomial infection, and pre-surgical decolonization of MRSA has been associated with a 58% reduction in post-surgical infections.
Methicillin was put to clinical use in 1959 as the first semisynthetic β-lactamase resistant penicillin. Within a year of its introduction, MRSA emerged. MRSA contains a mecA gene, which encodes a modified PBP (PBP2a) that has low affinity for β-lactams. mecA confers resistance to the entire class of β-lactam drugs except for ceftaroline and ceftobiprole.
S. aureus causes a wide range of skin and soft tissue infections e.g. boils, furuncles, sties, impetigo, folliculitis, cellulitis, and surgical wounds. Many topical antimicrobial drugs have some effect against staphylococcal infections e.g. macrolide antibiotic gentamicin, fusidic acid and tetracyclines. However, it is difficult to completely eradicate the pathogenic S. aureus because the organism rapidly develops antimicrobial resistance and the drugs cannot act in the central necrotic part of a suppurative lesion.
In 1976 MUP was introduced as a promising drug to treat SSTIs. MUP is bactericidal at higher concentrations including those applied to the skin with the 2% formulation, and after just 1 day of exposure. 2% MUP has showed between 1 and 20% inhibition of the metabolic activity of MRSA biofilms after exposure times of up to 3.5 hours. MUP is also used to eliminate nasal MRSA carriage from patients and healthcare workers.
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Decolonization protocol include a 5-days topical MUP course with or without bathing using a topical antiseptic e.g. chlorhexidine.
MUP is an isoleucine analog that competitively bind IRS required for the transfer of this amino acid, consequently interfering with bacterial protein synthesis. According to antibiotic susceptibility testing, there are three groups of MUP susceptibility for S. aureus isolates. At a MIC of ≤4 μg/ml these isolates are susceptible to MUP, low level resistance at MICs of 8 μg/ml to lower than 256 μg/ml and the high-level resistance with MIC of ≥512 μg/ml. Low-level MUP resistance is caused by point mutations in the ileS gene, leading to a change in the MUP-binding site of the IRS. High level MUP resistance is mediated by acquisition of a conjugative plasmid encoding either mupA or mupB genes, both encode an alternate IRS for which MUP has no affinity.
The aim of this work was to detect MUP resistance among clinical isolates of MRSA by phenotypic and genotypic methods.
Isolates were collected from different types of clinical samples from inpatients and outpatients attending the Medical research institute hospital, Alexandria University and received at the Microbiology department over a period of one year. Clinical samples included surgical wound swabs, swabs from superficial and deep skin and subcutaneous tissue infections and nasal swabs from carriers. Identification of S. aureus was done by both conventional methods, including morphology, culture characteristics, Gram stain, catalase test and coagulase test. S. aureus identification was confirmed molecularly by femA gene using PCR.
Detection of MRSA was done phenotypically using cefoxitin antibiotic disc and genotypically by detection of mecA gene using PCR.
Susceptibility of MRSA isolates to different antibiotics including high level MUP resistance was determined by Kirby-Bauer disk diffusion method. Inducible clindamycin resistance was detected using the D test. MUP MIC was determined using E-test method using Ezy MIC™ strip test.
Genotypic determination of high level MUP resistance was verified by multiplex PCR detecting mupA and mupB genes. In low level MUP resistant isolates, amplification
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and sequencing of a 1081 base-pair segment of ileS gene which contains some common mutations implicated in development of low level resistance.
One hundred and fifty S. aureus isolates were identified as Gram-positive, catalase positive, and coagulase positive cocci.
Fifty methicillin resistant isolates out of the 150 S. aureus isolates were phenotypically detected by cefoxitin antibiotic discs. MRSA identification was confirmed genotypically by detection of both mecA and femA by multiplex PCR in all 50 isolates.
The majority of MRSA isolates were obtained from wound swabs 23 (46%) followed by nasal swabs 18 (36%) and pus swabs 9 (18%). The majority of samples included in this study were obtained from male patients (70 %), while female patients represented only 30%.
Susceptibility of the 50 MRSA isolates to different antibiotics was done using the Kirby-Bauer disk diffusion method according to CLSI guidelines 2019. All the isolates were susceptible to tigecycline and linezolid. (68%) of the isolates were susceptible to trimethoprim- sulfamethoxazole and rifampin. While 66% of the isolates were susceptible to chloramphenicol, 58% were susceptible to fusidic acid, and 50% were susceptible to levofloxacin. The isolates were most resistant to gentamicin (62%), doxycycline (58%), while 50% were resistant to erythromycin and ciprofloxacin.
Inducible clindamycin resistance was detected using the D test in 5 (15.15%) of 33 clindamycin susceptible/intermediate MRSA isolates.
MUP (200μg) disk was used to detect high level MUP resistance. 3 (6%) were positive for high level resistance. MUP MIC was determined by E-test using Ezy MIC™ strip test. 47 (94%) isolates had an MIC ≤ 2 μg/ml (from 0.5 -2 μg/ml). Interestingly, none of the 50 isolates showed low level MUP resistance. While the 3 isolates (6%) with high level MUP resistance had an MIC ˃ 1024 μg/ml.
Genotypic determination of high level MUP resistance by multiplex PCR detecting mupA and mupB genes revealed the presence of mupA gene in the three isolates that were also high level MUP resistant phenotypically. mupB was not detected in any of the isolates.