الفهرس 
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Abstract
The present study aimed to study macrolides resistance among S. aureus isolates that has been collected from Tanta University hospital to study possible resistance mechanism to macrolides at molecular level and evaluated for certain drug combination to macrolides resistant S. aureus. A total of 921 clinical samples from different sources including urine (88), blood (39), sputum (264), nasal swabs (168), ICU (45) and wound pus (181) were obtained from different departments of Tanta university hospital during the period from March 2019 to August 2020. Microscopical and biochemical identification of the recovered isolates revealed that 399 isolates were Staphylococci, 300 (75.19%) of them were S. aureus and the remaining 99 (24.81%) were CoNS. All the recovered (300) S. aureus isolates were subjected to antibiotic susceptibility testing by disk agar diffusion method for preliminary screening of macrolides resistant S. aureus. About 176 isolates were macrolides resistant & about 124 isolates were sensitive. These macrolides resistant isolates subjected to susceptibility testing against oxacillin and cefoxitin discs for identification of methicillin resistant S. aureus (MRSA). The highest percentage of macrolides resistance was isolated from wound pus (35.23%) followed by blood (21.6%) while the least percentage was from nasal swabs (5.11%) and bone (1.7%) The susceptibility of macrolides resistant isolates to 18 different antimicrobial agents was performed using disk diffusion method. Macrolides resistant isolates showed high resistance to most of the tested antimicrobials. All isolates were resistant to ampicillin and cefotaxime. The highest susceptibility percentage to pristinamycin, azithromycin, oxacillin, cefoxitin, ampicillin, clindamycin, penicillin and erythromycin was ranging from (69 – 97%), moderate percentage of resistance ranging from (30 – 66%) was detected in nitrofurantoin, cotrimoxazole, clarithromycin, tetracycline, ciprofloxacin, moxifloxacin and lowest percentage of resistance was ranging from (16 – 29%) was detected in chloramphenicol, gentamycin and rifampicin. On the other hand, only 2 (2.53%) isolates were resistant to vancomycin. Based on the resistance patterns of macrolides resistant isolates, the multiple antibiotic resistance (MAR) index and the multidrug resistance (MDR) character were identified. The isolate that showed resistance to at least one agent in ≥ 3 antimicrobial categories was considered MDR accordingly, all macrolides resistant S. aureus isolates in this study exhibited MDR character. Analysis of MAR index of isolates revealed that all the isolates had a high MAR index value (> 0.2). This suggested that all isolates would have originated from a high-risk source of contamination where several antimicrobials were used. Analysis of MAR index of antibiotics revealed that both cotrimoxazole and Nitrofurantoin were the most effective antimicrobials against macrolides resistant S. aureus isolates. Disk approximation test (D-test) was performed on all 77 erythromycin resistant isolates. we found that 40.26% of erythromycin resistant isolates exhibited inducible resistance, whereas 50.65% exhibited constitutive resistance. S. aureus isolates with macrolides and methicillin resistance were selected for PCR studies. The PCR amplified products were electrophoresed, and the amplicons were visualized with approximate size of 139, 142, 190, 163, 722 and 815 bp for erm(A), erm(B), erm(C), msr(A) and mph(C) genes, respectively. It was found that About 25 isolates were tested for the presence of MLSB resistant genes. erm(A) was detected in 10 isolates MRSA, and 9 isolates MSSA, while erm(B) was detected in 11 MRSA in addition to 8 isolates MSSA. erm(C) was detected in 15 MRSA isolates and in 10 of MSSA isolates. Both erm(A) and erm(B) were in 10 MRSA and 9 MSSA. Also, both erm(B) and erm(C) were detected in 11 MRSA and 8 MSSA. All three genes erm(A), erm(B) and erm(C) were detected in 10 MRSA and 9 MSSA. The erm(A) gene was detected in 19 isolates (76%) (10 isolates MRSA and 9 isolates MSSA), the erm(C) gene was detected in 25 isolates (100%) (15 isolates MRSA and 10 isolates MSSA) which was statistically significant (p < 0.001) and the erm(B) was detected in 19 isolates (76%) (11 isolates MRSA and 8 isolates MSSA). Combination of erm genes was detected in 19 isolates (76%) (10 MRSA and 9 MSSA). All S. aureus isolates with MS resistance phenotype (4 isolates) were MSSA and carried the three genes (erm(A), erm(B) and erm(C)). Moreover, most of the isolates with iMLSB resistance phenotype carried both erm(A) and erm(B) genes (12 isolates, 8 MRSA and 4 MSSA), 8 isolates carried erm(B) and erm(C) genes and 4 isolates carry erm(B) gene only. We found that most of the isolates with cMLSB resistance phenotype were carrying both erm(B) and erm(C) (13 isolates, 9 MRSA and 4 MSSA) and 12 isolates were carrying erm(B) gene. S. aureus isolates with macrolides MICs ranging from 512 to 1024 μg/ml were used to study virulence factors. Out of 176 isolated strains of S. aureus, 108 isolates (61%) identified as biofilm producing isolates. The Result obtained showed that ascorbic acid treatment presented a concentration-dependent inhibition in terms of biofilm forming ability by more than 73%, 44% & 29% of the tested isolates showed a potent anti-biofilm effect (50 – 87%, 40 – 70% & 30 – 66% reduction) was excreted by 0.5 – 0.25 – 0.125 MIC of ascorbic acid respectively. We are selected about 15 tested S. aureus that have a strong biofilm producer. Vit K3 (menadione) is considered an (efflux Pump inhibitor), Vit K3 decreased the MICs by 7-12-fold changing it to the sensitive range and the presence of efflux mechanism of resistance was investigated in macrolides resistant isolates. All the tested isolates were efflux positive where they showed accumulation of fluorescent dye ethidium bromide inside the cells that remained relatively higher when the cells were treated with ⅛ mic of the efflux inhibitor menadione (Vit K3). In this work, vitamin K significantly inhibited the efflux pump by reducing the minimum inhibitory concentration (MIC) of erythromycin, Azithromycin and EtBr by a factor of at least seven-fold. An enhanced antimicrobial effect was recorded after combination of Vit K3 & Vit C with macrolides resistant S. aureus synergistic effect of Erythromycin in combination with vitamins K and C. The synergistic impact of Vit K3 plus erythromycin was 78.18%, 63.64%, and 56.36%. On the other hand, Vit K3 plus azithromycin showed 70.37, 57.41 and 53.7% synergistic effect at (0.5 – 0.25 – 0.125 MIC), respectively. The synergistic impact of Vit C plus erythromycin was 76%, 64%, and 52%. On the other hand, Vit C plus azithromycin showed 72.92, 47.91 and 39.58% synergistic effect at (0.5 – 0.25 – 0.125 MIC), respectively. In conclusion, this study has demonstrated that emergence of macrolides resistance among S. aureus isolates has increased in Tanta area. Resistance genes were mostly plasmid mediated which is alarming and may lead to rapid dissemination of these strains among health care workers and patients. This underscores the need for programs to prevent the spread of antimicrobial resistant strains and control the use of antimicrobial drugs in health care settings. Knowledge about the emerge of resistance of macrolides among S. aureus is the aim goal of the present study in order to develop the hypothesis of prevention and control measures of infection caused by this bacterial pathogen. Our study indicated many different mechanisms of macrolide resistant S. aureus such as biofilm formation, efflux mechanism and different erm genes. Combination of some vitamins such as vitamin K3 and ascorbic acid combined with antibiotics had significant synergistic effect as antibacterial agents for resistant S. aureus isolates. Data of our work and that of others in the same field strongly recommend the following: • Scientists, clinicians, and other healthcare professionals should identify and report macrolides resistant S. aureus promptly. • Macrolides should not be used (prescribed) except if it is highly indicated and only after susceptibility tests. • Ensure proper use of antimicrobials to preserve their efficacy and minimize the development of antimicrobial resistance. • Vancomycin is the first drug of choice and Sulphamethoxazole/ trimethoprim might be the second choice for the treatment of infections caused by macrolides resistant S. aureus • Discovery of new compounds with antimicrobial activity against multidrug resistant macrolides resistant S. aureus must be considered. • Further large-scale research is needed to explore new molecular techniques for early detection and control of macrolides resistant S.