الفهرس 
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Abstract
The emergence and increasing prevalence of MRSA strains that are virulent and resistant to available antibiotics had been reported by local health care settings in Egypt. This demands the discovery of alternative therapeutic approaches. Therefore, the aim of the present study was to search for agents that affect MRSA virulence determinants, as an alternative approach for MRSA infection control.
In order to achieve this goal, a total number of 164 bacterial isolates were recovered from different collected clinical specimens obtained from Ain Shams University hospital (El Demerdash). Of these, 99 isolates (60.4 %) were recovered from pus, 35 isolates (21.3 %) from sputum, 18 (11 %) isolates from nasal swabs, 11 (6.7 %) isolates from blood and only one isolate (0.6 %) was recovered from prostatic exudates. Gram staining and microscopical examination of the clinical isolates revealed that 61 isolates (37 %) were Gram positive cocci arranged in clusters. Of the Gram positive clusters, 59 isolates showed growth on mannitol salt agar with yellow zone as well as yellow pigment production and consequently were identified as Staphylococcus aureus. This could be confirmed by positive catalase and coagulase tests.
The results showed that out of the total 59 Staphylococcus aureus isolates, 46 isolates (77.97 %) were resistant (considered to be methicillin resistant Staphylococcus aureus isolates ”MRSA”), 2 isolates (3.39 %) were of borderline resistance (considered to be borderline resistant Staphylococcus aureus isolates ”BORSA”, while 11 isolates (18.64 %) were sensitive (considered to be methicillin sensitive Staphylococcus aureus isolates ”MSSA”) Of these 46 resistant isolates, 16 isolates (34.78 %) were highly resistant while the other 30 isolates were resistant.
Afterwards, all MRSA isolates were subjected to extracellular virulence factors assessment including protease, lipase and hemolysin assays. Quantitative protease assay revealed that MRSA isolates could be relatively categorized according to protease production into three groups; strong protease producers-(>2-units/ml; n=10;-21%), intermediate protease producers-(0.5-2-units/ml;-n=33;-69%) and weak protease producers (<0.5 unit/ml;-n=5; 10%) On the other hand, most of the isolates did not show any lipase activity. Regarding hemolysin, it was found that only 3 isolates (6.25%) showed high hemolytic activities, 9 isolates (18.75%) showed moderate hemolytic activities and 13 isolates (27%) showed low hemolytic activities. However, most of the isolates (48%) were non-hemolytic and didn’t show any hemolytic activity at all even after prolonged time of incubation. Moreover, no cell bounded hemolysin was detected in all MRSA isolates under the tested conditions.
In addition, biofilm production was assessed using microtiter plate assay. The results showed that 27% (n=13, OD590 > 0.6) of the isolates had strong ability to form biofilm, 42% (n=20, OD590=0.2- 0.6) had moderate ability while 31% (n=15, OD590 < 0.2) had weak biofilm forming capability. The statistical analysis showed that there was higher mean biofilm formation among highly resistant isolates compared to those of moderate and low resistant ones and the difference was highly significant statistically (P<0.01).
Since microbial adherence to host cells is a prerequisite step for development of infection, the adherence of some isolates to Vero cell line was investigated.
The results revealed that that the adherence of MRSA species were in the range of 0.082 - 0.5 %. On the other hand, invasion values ranged from 0.01 – 0.4 %. Although most of the isolates could adhere to the mentioned monolayer, most of them were weak invadors.
In addition, MRSA resistance profile against different antibiotics was studied. The results revealed that all tested MRSA isolates were resistant to amoxicillin clavulunic acid combination, 93.75% were resistant to ceftriaxone, and 87.5% were resistant to cefepime while only 68.5% of them were resistant to ampicillin sulbactam combination. On the other hand 75% of the tested isolates were sensitive to meropenem and didn’t show marked resistance to it. As a general observation, there was no correlation between virulence factors and level of resistance in MRSA isolates under study except in biofilm formation where the highly resistant isolates had higher biofilm production profile.
These multiresistant variants need more effective antimicrobial therapy. Therefore, targeting bacterial virulence represents a new approach to prevent and treat infectious diseases. Accordingly, attemps were made to find new antibiofilm and antiadherent agents against MRSA.
Since MRSA biofilms is an important virulence mechanism that complicates MRSA infections, fourty four agents belonged to different chemical classes were screened for their abilities to prevent biofilm formation in some selected isolates. Regarding mucolytics, the results showed that ambroxol HCl and ammonium acetate 1% had reduced biofilm formation by about 50% (median reduction) followed by ammonium acetate 0.1% that caused reduction by 34.5% and finally, bromohexine HCl that had the lowest ability to reduce biofilm formation in the tested isolates (only 24 % median reduction). Concerning heparin, it was found that the tested concentrations of heparin had an inhibitory effect on biofilm formation in which the higher the concentration of heparin used, the more reduction in biofilm was observed.
In addition, two sugars (glucose and mannose) were investigated at two different concentration levels (0.1, 1 %) but none of them had antibiofilm activity. Chitosan also (high molecular weight and low molecular weight) were tested at a concentration of 0.5%. Only, low molecular weight one exhibited 34.9% median reduction in the biofilm formation.
Besides, four non-ionic surfactants (tween 20, tween 80, poloxamer 188 and poloxamer 407) were tested at two different concentration levels (0.1, 0.5%). Both tween 20 and 80 had no effect on biofilm formation, poloxamer 188 and 407 showed very weak antibiofilm activity. Two anionic surfactants (sodium lauryl sulphate and sodium cholate) were tested. Both had some effect on prevention of biofilm formation; sodium lauryl sulphate at the two concentration levels (0.1, 0.5%) reduced the biofilm formation by 35.6% and 45.2% respectively. On the other hand, sodium cholate had lower antibiofilm activity at the same concentration levels (12.9 and 21.6%).
Moreover, the chelating agent, EDTA was tested at different concentration levels. EDTA at concentration of 0.01 M caused median reduction in biofilm by 37.9%. Moreover, higher concentrations of 0.02 M and 0.05 M of EDTA caused more prevention of biofilm formation (about 41% median reduction in both) but there was no significant difference between them. Afterthat, four metal ions (zinc sulphate, calcium chloride, silver nitrate and ferric chloride) were also tested; all of them had effect on biofilm formation except ferric ion. Silver ion reduced the biofilm formation by 58.7% (median reduction), zinc ion by 41% and calcium ion by 37.3%. Concerning anti-COX agent, such as acetyl salicylic acid and meloxicam both of them failed to inhibit biofilm formation of the isolates under test. In addition to the previously mentioned agents, some natural products were tested for their antibiofilm activities. Garlic extract at a concentration of 100% had shown significant reduction in biofilm formation in almost all tested isolates (about 60% median reduction). Moreover, many types of honey (honey citrus, clover honey, honey thyme, honey blackseed and honey propolis) were tested for their effects on biofilm formation. Clover honey and honey blackseed showed slight antibiofilm effect (about 30% median reduction for both) while honey thyme had more potent antibiofilm activity (about 52.1% median reduction). On the other hand, honey propolis had no capability to prevent biofilm formation of the tested isolates. Moreover, royal jelly was tested and it was found that it reduced biofilm formation significantly in the tested isolates (70% median reduction). Therefore, royal jelly could be considered as promising agent for the control of MRSA.
As it became widely accepted that bacterial adhesion to tissues was a prerequisite step in the infectious process. Therefore, targeting bacterial adherence using different agents was aimed. Two different mucolytics were investigated for their effects on adherence of the tested isolates; ambroxol HCl and bromohexine HCl at a concentration of 0.005%. Both of them showed antiadherent activity. Ambroxol HCl reduced adherence (median reduction) by about 36.5% while bromohexine had 20 % median reduction. Regarding heparin, the results revealed that the higher concentrations of heparin used (100 and 1000 IU/ml) showed higher antiadherent activity (both had median reduction of about 42%) than the lower one (10 IU/ml; median reduction of about 28%).
In addition, two sugars (glucose and mannose) were investigated at a concentration of 5 %. Both of them did not show any antiadherant activity, but moreover the glucose and mannose had increased the adherence by 15.9% and 27.5% respectively. Concerning surfactants, both tween 20 and 80 had nearly no effect on MRSA adherence. Poloxamer 188 (0.01%, 0.1%) had reduced the adherence of the tested isolates by 23.7% and 29.5% respectively, while poloxamer 407 (0.01%, 0.1%) had reduced it by 40, and 42% respectively in the tested isolates. At last some natural products were tested; the garlic extract had no significant effect on the adherence of the tested isolates. Honey blackseed and honey thyme showed marked antiadherant effect on the tested isolates (about 61% and 56% median reduction). On the other hand, clover honey had nearly no effect on the adherence of tested isolates. Besides, royal jelly was tested at a concentration of 50%. It had significantly reduced the adherence of isolates under test (about 81.7% median reduction in adherence). Therefore, royal jelly could be considered as protent antiadherent agent against MRSA isolates.
Finally, two promising agents, proved to have anti-virulence effect; royal jelly and garlic extract were selected for testing in rat model with MRSA skin infection in two different concentrations (50%, 100%). The results revealed that royal jelly could eradicate MRSA completely and in the same time promote healing even in the uninfected groups. On the other hand, garlic extract did not show healing promoting ability in the superficially infected rats. However, the wound in the garlic treated group was dry and no exudates or pus was observed when compared to the infected untreated group ,which showed oozing wound. This implies that pure garlic extract may had antiseptic effect but however, it failed to eradicate MRSA from the superficially infected rats.
Because royal jelly seemed to have effect on MRSA skin infected rats. Therefore, royal jelly was selected from the tested agents to test its effect on wound healing without establishing MRSA infection. The results implied the significant role of royal jelly not only in eradication of MRSA infection, but also in promoting the healing process in the wounds.