الفهرس 
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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.