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Abstract Worldwide, the rise of antibiotic resistance and the increase in the incidence of multidrug resistance (MDR) limits chemotherapeutic options and complicates the treatment. Alteration of drug target, changes in target accessibility, degradation by enzymes and efflux, can all be utilized by microorganisms to evade antibiotics. In this study we provide an emphasis on MDR mechanisms including efflux, permeability barrier and production of - lactamases. And it was hoped to find the proper treatment for MDR organisms using antimicrobial combinations. A total number of 135 clinical isolates were recovered from hospitalized patients in El-Demerdash Hospital, of these, 66 were recovered from swabs obtained from wound and burn infections, 27 from sputum specimens, 37 from urine specimens and 5 from blood cultures. The clinical isolates were screened for their suscebtibitlty to different antibiotics using the disk diffusion method. The prevalence of antibiotic resistance to the recovered Gram-positive isolates was as follows, tetracycline 50% > streptomycin 34.8% streptomycin 34.8% > ampicillin 34.7% > cefotaxime 30.4% > ceftazidime 26% = ciprofloxacin 26% = chloramphenicol 26% = erythromycin 26% > ofloxacin 17.4%. And For Gram-negative isolates the resistance prevalence was ampicillin 67.7% > tetracycline 46% > cefotaxime 43% > streptomycin 36.9% > erythromycin 30.7% > chloramphenicol 29.2% > ciprofloxacin 27.7% > ceftazidime 21.5% > ofloxacin 16.9%. All isolates were found to be resistant at least to one antibiotic, 84 isolates out of 135 were selected for further study as they were resistant to more than three antibiotics of different classes (according to their resistance profile) and charecterized microscopically to be 49 Gram-negative isolates and 35 Gram-positive isolates. The antibiotic resistance profiles of the 84 isolates against 11 antibiotics and ciprofloxacin were determined, the minimum inhibitory concentrations (MICs) values were determined and the range of MIC for -lactams, cephalosporins, macrolides and aminoglycosides was higher than that for chloramphenicols and tetracyclines and the lowest range was for quinolones and rifampicin. The prevalence of resistance to 11 antibiotics and ciprofloxacin for the 35 Gram-positive isolates resistant isolates was as follows: flucloxacillin 45.7% > cefotaxime 40% = cefdroxil 40% > ampicillin 37.1% = streptomycin 37.1% > clindamycin 31.4% > azithromycin 25.7% > erythromycin 22.9% > tetracycline 17.1% > ciprofloxacin 11.4% = chloramphenicol 11.4% > rifampicin 8.5% and for the 49 Gram-negative isolates, it was ampicillin 65.3% = flucloxacillin 65.3% > cefdroxil 57.1% > cefotaxime 53% > clindamycin 51% > erythromycin 42.9% > streptomycin 42.8% > chloramphenicol 26.5% > azithromycin 22.4% = rifampicin 22.4% > ciprofloxacin 16.3% > tetracycline 14.3%. The 11 most MDR (resistant to more than seven antimicrobial agents) were identified by culture charecteristics and biochemical tests to be E. coli (3 isolates), Staphylococcus aureus (1 isolate), Pseudomonas aeruginosa (4 isolates), and Proteus mirabilis (3 isolates). Of these isolates, only 4 were selected and the identity of the 3 Gram-negative isolates were confirmed by an API 20-E system (figure 8) to be E.coli isolate D102 , Pseudomonas aeruginosa isolate D105 and Proteus mirabilis isolate D128 and used for studying MDR mechanisms in addition to the fourth isolate Staph. aureus isolate D107. The four multidrug resistant isolates (E.coli isolate D102, Pseudomonas aeruginosa isolate D105, Staph.aureus isolate D107 and Proteus mirabilis isolate D128) were subjected for studying the efflux activity to EtBr. In all cases, the uptaken amount of EtBr remained constant with all refrence strains while the MDR isolates showed a progressive decrease in fluorescence. For verification the addition of EPIs maintained the fluorescence at relativily higher level compared to the control (EtBr loaded cells with no added inhibitor) indicating the acquisition of efflux phenomenon by the test MDR isolates. Another verification was carried out by determining the MIC for EtBr in absence and presence of some reported EPIs. The results showed a decrease in MIC values for all the test isolates due to the incorporation of the EPIs along with EtBr except for Proteus mirabilis isolate D128 that exhibited no difference in MIC values against EtBr in presence or absence of the EPI omeprazole. Then the four multidrug resistant isolates were subjected for studying the efflux activity to ciprofloxacin fluorometrically. Time dependant increase in fluorescence indicates diffusion and accumulation of this quinolone. In all cases, the MDR isolates showed an initial accumulation of ciprofloxacin then a progressive decrease in the accumulated amount of ciprofloxacin by time which verify the presence ofefflux mechanism while the refrence strains showed a gradual increase of the amount of ciprofloxacin accumulated by time without any decline afterthat. In all cases, the addition of EPIs maintained the accumulated amount of ciprofloxacin and showed no further decrease compared to control (no EPI was included). Another verification approach for the acquisition of efflux pump by the MDR isolates was carried out by determining the MIC for ciprofloxacin in presence and absence of some reported EPIs, the results showed a decrease in MIC values for all the test isolates due to the incorporation of EPIs along with ciprofloxacin. Reserpine showed the highest effect for both ciprofloxacin accumulation (65% increase) and MIC value (16 fold reduction) for both P. aeruginosa isolate D105 and S. aureus isolate D107. Also verapamil behaved alike but in case of E. coli isolate D102 and Proteus mirabilis isolate D128. For each of E.coli isolate D102, Pseudomonas aeruginosa isolate D105, and Proteus mirabilis isolate D128, a time kill study was performed on the intact cells and their prepared spheroplasts against erythromycin, streptomycin, ciprofloxacin, tetracycline and rifampicin. There is marked differences between the susceptibility of the intact cells and their spheroplasts to different antimicrobials. The intact cells count was reduced only by 2 - 3 fold after 6 hrs by all tested antimicrobials while the same concentrations affect the spheroplasts differently. For E. coli isolate D102, erythromycin and rifampicin led to complete killing of spheroplasts after 6 hrs, streptomycin decreased the count of spheroplasts by 6 fold after 6 hrs but ciprofloxacin and tetracycline showed a little difference in reduction of spheroplasts count compared to the intact cells. While for Pseudomonas aeruginosa D105, erythromycin and rifampicin led to complete killing of spheroplasts after 6hrs, ciprofloxacin decreased the spheroplasts count by 6 fold after 6hrs but streptomycin and tetracycline showed almost no difference in reduction of spheroplasts count compared to the intact cells. As for Proteus mirabilis D128, No antibiotic led to complete killing after 6 hrs at the tested concentration. Erythromycin, streptomycin and ciprofloxacin decrease the count of spheroplasts by 4 fold after 6hrs but rifampicin and tetracycline showed almost no difference in reduction of spheroplasts count compared to the intact cells. The OMPs of the Gram-negative MDR isolates E. coli D102, P. aeruginosa D105, Proteus mirabilis D128 and their corresponding reference strains were prepared and analysed by polyacrylamide gel electrophoresis. The separation profiles of protein contents of the different preparations against the standard moleculer size protein marker showed differences in the amounts and types of proteins among the tested resistant isolates and their corresponding refrence strains. For E. coli isolate D102 but not with the reference strain, bands of molecular sizes 50, 45, 20 and 14 Kda OMPs were detected. On the other hand , the 37 and 39 Kda OMPs which were detected in the E. coli reference strain were absent in the resistant isolate. However, both the resistant isolate and the reference strain showed common protein bands of molecular sizes 60 and 41 Kda. For Pseudomonas aeruginosa D105 but not for the reference strain, bands of molecular sizes 50 , 54, 43,42 and 25 Kda OMP were detected. The band with molecular mass 46 Kda was lost in the resistant isolate D105 and it appeared in the reference strain. However, the reference strain showed other bands at 60, 58, 40 and 25 Kda. For Proteus mirabilis D128 but not with reference strain, bands of molecular sizes 49 , 42, 25 and 21 Kda OMP were detected. In addition to the loss of 39 Kda OMP in this resistant isolate and its presence in the the reference strain. However, the reference strain showed other bands at 60, 58, 42 an 41 Kda The production of -lactamases by the four MDR isolates and the effect of inducers and inhibitors on -lactamases production as well as the effect of inhibitors on the enzyme activity were studied. Obviously, the data showed that all tested agents either alone or in combinations (benzyl penicillin/clavulanic and benzyl penicillin/cloxacillin) increased -lactamase production by all the test isolates. In addition, the use of test agents in combinations caused more increase in enzyme production relative to each agent alone. The results revealed that although both clavulanic acid and cloxacillin were reported as -lactamases inhibitors, they induced the enzyme production by the test isolates when they investigated either separately or in combination with benzyl penicillin. Benzyl penicillin/clavulanic acid combination caused the greatest induction for enzyme production by E. coli isolate D102 and S. aureus isolate D107. While for the other two isolates P. Aeruginosa isolate D105 and Proteus mirabilis isolate D128, this combination gave inducing effect equivalent to benzyl penicillin alone. P. aeruginosa isolate D105 recorded the highest production level of -lactamases compared to other tested isolates. The effect of clavulinic acid and cloxacillin when each is combined with benzyl penicillin, ampicillin or cefotaxime was tested by determining the MIC of these antibiotics against the four MDR isolates in the presence of each agent at one fourth its MIC. The results showed a decrease in MIC of the tested antibiotics against the tested isolates by their combinations with clavulinic acid (all cases) and with cloxacillin ( borderline effect). The effect of the combination of some agents belong to cephalosporins /aminoglycosides and cephalosporins/fluoroquinolones against the four isolates was studied using checkboard protocol . For all isolates no antagonistic effects were observed, all antibiotic combinations showed either additive or synergistic activity. For E. coli isolate D102, cephalosporins/ quinolones interaction showed additive effect while cephalosporins/ aminoglycosides showed synergistic effect. Cefotaxime (128 μg/ml)/ gentamicin (32 μg/ml) combination gave FIC equal to 0.187 and cefotaxime (128 μg/ml)/ streptomycin (32 μg/ml) gave FIC equal to 0.25. For P. aeruginosa isolate D105, ceftazidime showed additive effect when combined with both aminoglycosides and quinolones but cefotaxime showed synergistic effect with both classes. The two combination cefotaxime (32 μg/ml)/ofloxacin (16 μg/ml) and cefotaxime (32 μg/ml)/streptomycin (32 μg/ml) gave the same FIC of 0.09. For S. aureus isolate D107, all cephalosporins/aminoglycosides showed additive effect, and also all cephalosporins/quinolones showed additive effect except for ceftazidime (64 μg/ml)/ciprofloxacin (32 μg/ml) where it interacted synergistically giving FIC equal to 0.5. For Proteus mirabilis isolate D128, all cephalosporins/ aminoglycosides combinations showed additive effect while cephalosporins/ quinolones showed synergy. The two combinations cefotaxime (128 μg/ml/ ciprofloxacin (16 μg/ml) and ceftazidime (32 μg/ml/ofloxacin (8 μg/ml) gave the same FIC of 0.375. For each isolate, the combinations that showed the best synergistic interaction (which gave the lowest FIC) were selected to perform the killing kinetics. All the selected combinations were confirmed to be synergistic as they all reduced the viable count by > 2 log cycles after 6 hrs and showed nearly complete killing after 24 hrs. In conclusion, the prevalence of MDR bacteria is high among pathogenic microrganisms in Egypt. All the MDR isolates were proved to resist several antimicrobials by interplay between different mechanisms of resistance and the use of antimicrobials combinations showed a synergistic effect in killing these MDR isolates. |