الفهرس 
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Abstract
Pseudomonas aeruginosa is an opportunistic human pathogen and is the most common Gram negative bacterium causing nosocomial infections. Furthermore,P. aeruginosa is characterised by a high degree of clinical pathogenicity which is associated with a wide range of mechanisms and virulence factors. One of the reasons that P. aeruginosa is a successful opportunistic pathogen is that it produces this array of virulence factors.
QS is the mechanism whereby an individual bacterium produces small diffusible molecules that can be detected by surrounding organisms. In P. aeruginosa and most Gram negative bacteria, these signal molecules are acyl homoserine lactones (AHLs). Only when the concentration of AHLs in the environment increases, potentially because of increasing numbers of bacteria, the intracellular levels of AHLs are sufficient to maximally induce the activation of transcriptional regulators. This mechanism of communication enables bacteria to act as a community in the coordinated regulation of gene expression. This regulated expression of virulence genes is thought to give the bacteria a selective advantage over host defenses and thus is important for the pathogenesis of the organism. As a consequence, QS is an ideal target for anti-virulence strategies.
P. aeruginosa is characterized by high level of intrinsic resistance to a variety of antimicrobial agents; only a limited number of antimicrobial agents are active against it. In addition, P. aeruginosa has acquired multiple mechanisms of resistance against all available antipseudomonal agents. Resistance mechanisms are regulated and coregulated to make P. aeruginosa one of the greatest therapeutic challenges.
Efflux pumps are one of the different mechanisms through whichP. aeruginosa mediates antimicrobial resistance. These efflux pumps are Mex efflux pumps; members of the RND family. There is accumulating evidence that efflux pumps that confer clinically relevant antibiotic resistance are also important for the pathogenicity of the bacterium. These efflux pumps that export antimicrobial agents also export virulence determinants, such as adhesins, toxins or other proteins that are important for the colonization and infection of human and animal cells. In addition, AHLs that are involved in QS are substrates of the MexAB–OprM system. So far, evidence of a role for these pumps in pathogenicity has been obtained for members of the RND and MATE families.
Alternative mechanisms for targeting P. aeruginosa, other than traditional antibiotics, have been the focus of much research. Therapeutics that target and inhibit QS in P. aeruginosa would attenuate the virulence of the bacterium and thus potentially assist the host immune response in clearing the infection.
The therapeutic effect of AZM as an antimicrobial is mainly due to its ability to impair bacterial growth. However, in P. aeruginosa its effect cannot exclusively be attributed to growth impairment. Macrolides, like AZM, are neither bactericidal nor bacteriostatic for P. aeruginosa at clinically achievable concentrations and are therefore unlikely to select resistant clones. However, at these concentrations, AZM inhibits QS in vitro.
A large number of interesting molecules, acting on a series of efflux pumps in different bacteria, have been designed. The most advanced compounds in the series are broad-spectrum inhibitors of Mex pumps in P. aeruginosa. The most widely used compounds as Efflux Pump Inhibitors (EPIs) for Pseudomonas overexpressing Mex efflux pumps are the group of peptidomimetic molecules with phenylalanine arginyl β-naphthylamide (PAβN) as a leading compound.
The aim of the present work was to detect and study the effect of AZM onQS system and QS-dependent virulence factors in P. aeruginosa isolates of different clinical sources. Therefore, the production of C4HSL and four QS-dependent virulence factors (pyocyanin, rhamnolipid and protease production and biofilm formation) was investigated in absence and presence of AZM. In addition an extra point was examined; the effect of EPI on the production of C4HSL and the four QS-dependent virulence factors was studied and the results were compared to that of AZM.
In this study a total of 50 P. aeruginosa isolates that were isolated from different clinical specimens were obtained from Microbiology department of Medical Research Institute Alexandria University, Egypt.Isolates were recovered from various sources; sputum, BAL, ETT, swab from infected wounds and urine samples. These isolates were identified as P. aeruginosa using the standard biochemical tests and confirmed using MALDI-TOF Mass Spectrometer. Isolates were stored at -80°C in Luria broth (LB) supplemented with 10% glycerol until were needed for further investigations.
All the isolates were tested for:
I- Antibiotic senstivityusing Disk Diffusion Susceptibility Test (Kirby-Bauer Method)
The sensitivity to the following antibiotics was carried out: Gentamicin(CN), Amikacin (AK), Imepenem (IPM), Ceftazidime (CAZ), Levofloxacin(LEV) and Ciprofloxacin(CIP), in addition to Azithromycin (AZM)
II- Determination of sub-inhibitory concentration (sub-MIC) of azithromycin using microdilution test.
III- Phenotypic detection of QS system molecules by Micro-AHL Cross-feeding BioassayusingChromobacterium violaceum CV026strain as abiosensor for C4HSLproduction. The production of C4HSL by the test organism induced the pigment production by the biosensor strain.The production of C4HSL was detected in absence and presence of each of sub-MIC AZM and 20 µg/ml EPI PAβN.
IV- Phenotypic detection of QS dependant Virulence Factors:
1- Pyocyanin production.
2- Protease activity.
3- Rhamnolipid Production.
4- Determination of Bioflm forming Capacity.
The 50 P. aeruginosa isolates were tested for the production of these QS-dependant virulence factors in absence and presence of each of sub-MIC AZM and 20 µg/ml EPI PAβN.
V- Detection of QS genes (lasR, lasI, rhlR and rhlI) by conventional PCR in the 50 P. aeruginosa isolates.
VI- The lasR, lasI, rhlR and rhlI genes for P. aeruginosa isolates (that were genotypicaly positive and phenotypically defective) were sequenced to determine if QS genes have mutations.
The result of this study revealed the following:
I- The results of studying QS system and QS-dependant virulence factors production:
1- The isolates were collected from different clinical sources; urine samples (42%), swabs (46%) and respiratory specimens (12%).
2- Out of the 50 P.aeuginosa isolates 14 (28%) were defective in the production of the autoinducers C4HSL.
3- On screening for the production of AHLs dependent virulence factors, 40 (80%), 32 (64%) and 41(82%) P. aeruginosa isolates were positive for the production of pyocyanin, rhamnolipid and protease respectively. Regarding the biofilm formation, 90% of P. aeruginosa isolates produced biofilm whereas 10% of the isolates formed almost no biofilm.
4- There was a statistical association between the production of QS signaling molecules C4HSL and the production of QS dependant virulence factors; the36 P. aeruginosa isolates with QS positive signaling molecules C4HSL were positive for pyocyanin, rhamnolipid and protease production,; 88.9%, 83.3% and 100% respectively. For biofilm production, there was no statistical significant association with the production of QS signaling molecules C4HSL.
5- There was statistical significant difference between the proportion of positive QS autoinducer C4HSLand the presence of the four QS genes. (FEp <0.001) (All 36 (72%) isolates producing autoinducer C4HSL had the 4 QS genes present).
6- Six isolates had the four QS genes present and were defective in the production of the most of the virulence factors. Sequence analysis of their PCR products revealed that these six isolates appeared to carry various point mutations in one or more of the QS genes.
7- The susceptibility pattern of the 50 P. aeroginosa isolates for anti-pseudomonal antibiotics showed that the highest resistance was to the class fluroquinolones; 74% for levofloxacin and ciprofloxacin followed by imepenem (70%). Meanwhile, 32 (64%) isolates were Multidrug-resistant (MDR)
8- There was no statistical significant association between P. aeroginosaresistance to antibiotics and QS signalsC4HSL production. (p > 0.05).
II- The results of the effect of each of AZM and EPI PAβN onQS system and QS-dependant virulence factors production:
9- The AZM MIC values ranged from 16-250 µg/ml with a median 62 µg/ml and a mean value99.26 µg/ml. The chosen sub MIC of AZM was 8µg/ml and was used for all further phenotypic experiments.
10- All the 36 P.aeruginosa, producing QS signals C4HSL, failed to produce the signals in the presence of sub MIC of AZM (8µg/ml).
11- There was statistical significance difference in proportion of positive QS dependant virulence factors (biofilm, rhamnolipid, protease and pyocyanin) between isolates in absence and presence of sub MIC of AZM. (each of p<0.001)
12- In the presence of EPI PAβN (20 µg/ml) 14 out of the 36 isolates producing QS signals were affected; 6 isolates failed to produce the QS signals molecules C4HSL and 8 isolates showed reduction in the production of the QS signals.
13- Since EPI affected the production of QS signals C4HSL in only 14 P.aeruginosa isolates producing signals, therefore, its effect on the production of virulence factors among these 14 isolates was investigated and also compared to the effect of sub MIC of AZM on these 14 isolates.
14- There was statistical significance difference in proportion of positive QS dependant virulence factors (biofilm, rhamnolipid and pyocyanin) between isolates in absence and presence of either 20µg/ml EPI or sub MIC of AZM.
15- By comparing the results of the effect of 20µg/ml EPI PAβN on the QS dependant virulence factors in the tested isolates and sub MIC of AZM, the inhibitory effect of AZM was more observed but this was not statistically significant.
from this study we can conclude that:
1- QS-dependant virulence factors production among the 50 P. aeruginosa isolates included in the study is associated with the production of the QS signals C4HSL and the presence of the 4 QS genes.
2- Defects in QS-dependant virulence factors production is associated with either absence of or mutation in one or more of the 4 QS genes.
3- High level of P. aeruginosa resistance to fluroquinolones (74%) and imepenem (70%) was reported in this study and there was no statistical significant association between resistance to antibiotics and QS signals C4HSL production.
4- Sub MIC of AZM resulted in complete inhibition in the production of QS molecules C4HSL in all (100%) the 36 producing C4HSL P. aeruginosa isolates compared to 38.9 % in case of EPI (PAβN) where 14/36 showed either inhibition or reduction in the production of QS signals C4HSL.
5- The inhibitory effect of sub MIC AZM on QS-dependant virulence factors production was more observed than the effect of EPI (PAβN) but this difference was not statistically significant.