الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Escherichia coli (E. coli) is an important member of the intestinal microflora of
humans and other mammals, it is a common pathogen linked with community-associated
as well as nosocomial infections. By far it is the most common cause of urinary tract
infections (UTIs), and medically important bacterial agent of diarrhea and may spread to
the bloodstream causes Gram negative endotoxic shock, a dreaded and often fatal
complication.
In the last few years, the emergence and wide dissemination of E. coli strains
showing resistance to broad-spectrum of antimicrobial agents has been reported.
Emergence of resistance to multiple antimicrobial agents in pathogenic E. coli has become
a significant public health threat especially in developing countries as a result of overuse
and misuse of antibiotics.
It is known that bacteria showing a multi-drug resistance phenotype use several
mechanisms to overcome the action of antibiotics. Active efflux of antibiotics is one of the
major mechanisms of drug resistance in bacteria. Efflux pumps have been reported to play
vital roles in mediating multidrug resistance in clinical isolates from varied geographic
locations and varied populations.
Genomic analysis has revealed a number of genes encoding putative drug efflux
pumps on the chromosomes of most bacteria. The entire genome sequence of E. coli was
determined in 1997. Among nearly 4300 open reading frames (ORFs) identified on the 4.6
M bp chromosomes of E. coli, 354 ORFs (approx. 77 transporters per Mb of genome) are
predicted to be transporter genes. Of these genes, 37 are putative drug efflux genes.
Among these efflux pump genes, several are reported to actually contribute to drug
resistance. Among the 37 ORFs, 20 efflux genes were found to contribute to drug
resistance to E. coli. Among five families, drug efflux pumps belonging to the RND family
were shown to particularly contribute to higher resistance to a wide variety of compounds
The E. coli AcrAB–TolC complex, which consists of the polytopic inner membrane
protein AcrB, the periplasmic adaptor protein AcrA, and the outer membrane channel
TolC, has been studied extensively as a model for multidrug efflux pumps. The AcrAB–
TolC multidrug efflux pump confers resistance to a wide variety of antibiotics and other
compounds in E. coli.
The extremely wide substrate specificity of this pump was indicated by the fact that
inactivation of the acrAB genes made E. coli hyper-susceptible to not only to several dyes,
but also to detergents (such as SDS, Triton X-100, and bile salts) and to a wide range of
antibiotics, including macrolides, β-lactams, tetracycline, chloramphenicol, fusidic acid,
and novobiocin (but not aminoglycosides)
In order to address the problem of efflux pumps and their consequences on decreasing
the intracellular active concentration of antibiotics, it is necessary to search for and develop
new strategies to circumvent efflux activity. Inhibition of efflux pumps appears to be an
attractive approach to combat the problem of drug resistance. Efflux pump inhibitors can be
utilized for increasing the antibiotic concentration inside a pathogenic cell making these drugs
more effective. and English Diagnosis of efflux mediated resistance based only on phenotypic methods is often
ill-informed. Nucleic acid –based diagnostic techniques are used not only for detection and
identification of microbial pathogens, but also for genotyping as applied to the
determination of antibiotic resistance or to microbial fingerprinting.
Therefore, the aim of this work is to study the multi-drug resistance (MDR) among
E. coli clinical isolates and to screen for the efflux pump mediated resistance against
various antibiotics of different families among these isolates using phenotypic and
molecular methods.
In this study a total of 40 MDR E. coli clinical isolates were collected from different
clinical specimens (urine, wound swabs, blood and sputum) from patients admitted to
different medical facilities, medical facilities (Medical Research Institute, Alexandria
University and Alexandria Main Hospital, Alexandria University) over a period of three
months (November 2011 to January 2012). The collected isolates were stored at -80˚C in
Luria Bertani broth (LB) supplemented with 10% glycerol for further investigations.
Strains were tested for:
1. Antibiotic susceptibility: The selected isolates were challenged against the following
panel of antibiotics using Kirby-Bauer technique: Amikacin; Tobramycin; Gentamicin;
Ofloxacin; Ciprofloxacin; Norfloxacin; Levofloxacin; Tetracycline; Ceftriaxone;
Ceftazidime; Cefotaxime; Ampicillin-Sulbactam; Amoxicillin-Clavulinic acid; and
Trimethoprim-Sulphamethoxazol.
2. Determination of MIC for Levofloxacin, Ciprofloxacin, Gentamicin and Ceftriaxone;
alone and in the presence of Mefloquine hydrochloride as an efflux pump inhibitor
(EPI) to evaluate MDR reversal activity.
3. Detection of acrA and acrB genes using conventional PCR.
4. Quantifying the expression of AcrA-AcrB-TolC MDR tripartite efflux pump system in
E. coli with respect to a reference strain.
The results of this study revealed the following:
1- Out of the 40 collected isolates, 7 (17.5 %) were from blood, 21 (52.5 %) from urine, 4
(10 %) from sputum and 8 (20 %) from wound.
2- All the 40 clinical isolates were resistant to more than one class of antibiotics, all the
isolates show resistance to Cefotaxime, Ceftriaxone, Ceftazidime. Norfloxacin,
Ofloxacin, Levofloxacin, Ciprofloxacin, Tobramycin, and Gentamicin (100%).
Resistance to Amikacin, Ampicillin-Sulbactam, Amoxicillin-Clavulinic acid,
Tetracycline, and Sulphamethoxazol-Trimethprim were demonstrated by (55%),
(95%), (92.5%), (92.5%), and (95%) isolates, respectively.
3- The isolates showed decrease in MIC for the tested antibiotics in presence of the EPI.
In case of levofloxacin, its MIC values in absence of Mefloquine hydrochloride
were ranged from (8-128) µg/ml. On the other hand, in presence of Mefloquine hydrochloride, a reduction of the MIC values was observed (Range: 0.25-8)µg/ml.
The fold decrease in MIC values ranges were (8-128) folds.
For Ciprofloxacin, its MIC values in absence of Mefloquine hydrochloride were
ranged from (64-512) µg/ml. On the other hand, in presence of Mefloquine
hydrochloride, a reduction of the MIC values was observed (Range:1- 256)µg/ml.
The fold decrease in MIC values ranges were (2- 512) folds.
Concerning Gentamicin, its MIC values in absence of Mefloquine hydrochloride
were ranged from (16-512) µg/ml. On the other hand, in presence of Mefloquine
hydrochloride, a reduction of the MIC values was observed (Range: 2- 512)µg/ml.
The fold decrease in MIC values ranges were (0- 8) folds.
While for Ceftriaxone, its MIC values in absence of Mefloquine hydrochloride
were ranged from (16-512) µg/ml. On the other hand, in presence of Mefloquine
hydrochloride, a reduction of the MIC values was observed (Range: 0.25-128)µg/ml. The fold decrease in MIC values ranges were (4 -1024) folds.
4- Out of the Levofloxacin resistant isolates (95 %) recovered susceptibility in presence
of Mefloquine hydrochloride (MIC ≤ 2µg/ml), according to the levofloxacin
breakpoint mentioned in CLSI (S:≤ 2, I: 4, R: ≥ 8)µg/ml.
For Ciprofloxacin, resistant isolates (5 %) recovered susceptibility in presence of
Mefloquine hydrochloride (MIC ≤ 1µg/ml), according to the ciprofloxacin breakpoint
mentioned in CLSI (S:≤ 1, I: 2, R: ≥ 4)µg/ml.
For Gentamicin, resistant isolates (5 %) recovered susceptibility in presence of
Mefloquine hydrochloride (MIC ≤ 4µg/ml), according to the gentamicin breakpoint
mentioned in CLSI (S:≤ 4, I: 8, R: ≥ 16)µg/ml.
And in Ceftriaxone, resistant isolates (62.5 %) recovered susceptibility in presence of
Mefloquine hydrochloride (MIC ≤ 1µg/ml), according to the ceftriaxone breakpoint
mentioned in CLSI (S:≤ 1, I: 2, R: ≥ 4)µg/ml.
5- As acrA and acrB are intrinsic genes, so as expected, all the clinical isolates
demonstrated such genes upon testing by conventional PCR (100%)
6- Quantification of both acrA and acrB genes expression for all the studied isolates
showed overexpression of both genes. The fold increase in expression of both acrA
and acrB was of average 3.98 and 2.554 folds respectively. Relative to the
housekeeping gene rpsL, the mean increase in the levels of expression of acrA and
acrB genes in the MDR E. coli clinical isolates were (3.9 ± 1.58) and (2.6 ± 1.09) folds
respectively compared to those of ATCC standard strain. For both genes, the
differences between the levels of expressions of both genes in MDR strains and ATCC
strain were statistically significant (p ˂ 0.001). There was a strong linear correlation
between acrA and acrB genes expression levels. (r = 0593, p< 0.001).
7- Quantification of both acrA and acrB genes expression for 5 selected isolates in
absence and in presence of antibiotic (levofloxacin 3 µg/ml), Relative to the
housekeeping gene rpsL, a significant increase in the levels of expression of acrA and
acrB in the selected isolates was observed, being of averages 5.86 and 3.44 folds respectively, compared to that of the ATCC standard E. coli strain. However, the
difference in expression in absence and in presence of antibiotic did not reach the
statistical significance
from the present study we conclude that:
As the MDR bacteria are the principal cause of failure in the treatment of infectious
diseases, resulting in increases in the term and magnitude of morbidity, higher rates
of mortality, and a greater health cost burden. It is important to encourage careful
and limited use of antibiotics in the health care system to control the spread of these
MDR strains.
In the present study, the phenotypic and genotypic diagnostic methods used for
detection of acrAB tolC – mediated MDR in E. coli as well as the study of the
effect of exposure to antibiotics as a stress factors on the expression of acrAB genes
need to be considered with caution due to the small number of strains included.
Therefore further studies with larger sample size are recommended for
investigation of the effect of different factors on the gene expression putting into
consideration the effects of type and concentration of the tested antibiotic, presence
or absence of another mechanism of resistance and the role of interplay of such
mechanism if present with the efflux pump expression and finally the effect of time
of contact between the tested antibiotic with the sample and the time of harvesting
of the bacteria.
Options for treating infections caused by multidrug-resistant bacteria are limited.
However, as one of the main results of blocking the efflux pumps is the obvious
decrease in appearance of clinical resistance. Therefore, The EPIs are expected to
be beneficial in combating drug resistance. EPIs are expected to: (1) decrease
intrinsic resistance and consequently expand the spectrum of activity of some
antibiotics to previously non-susceptible species, (2) reverse acquired resistance,
and very importantly (3) decrease the frequency of emergence or resistance strains.
Consequently, the effect of efflux pumps needs to be considered in the design of
future antibiotics
Inhibition of acrAB tolC efflux pump in MDR E. coli by Mefloquine
hydrochloride appears to be an attractive approach to combat the problem of drug
resistance.
Further studies exploring novel strategies to interfere with efflux pump expression
and function are warranted.