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Antibiotic resistance mechanisms in ...

Gillis, Richard John.

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Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin./
Author:	Gillis, Richard John.
Description:	181 p.
Notes:	Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4072.
Contained By:	Dissertation Abstracts International66-08B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3185030
ISBN:	9780542263446

Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin.
Gillis, Richard John.

Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin. - 181 p.

Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4072.

Thesis (Ph.D.)--University of Rochester, 2005.

Pseudomonas aeruginosa biofilms found in the lungs of patients afflicted with cystic fibrosis (CF) are extremely resistant to antibiotic treatment. RND efflux pumps were investigated due to their implications in planktonic P. aeruginosa antibiotic resistance. Experiments done exposing biofilms of various single null efflux pump mutants to several different classes of antibiotics failed to establish a correlation between efflux pump expression and antibiotic resistance in biofilms. Bacteria containing mexA-gfp and mexC-gfp transcriptional fusions were cultivated in biofilms in the presence of carbenicillin. Even though beta-lactam antibiotics are known substrates for both the MexAB-OprM and MexCD-OprJ efflux pumps, expression of mexA and mexC was relatively low in four day biofilms; decreasing to near zero levels by day eight. Recent clinical trials have reported improvement in CF patients treated with azithromycin (AZM). The mechanism(s) of action of AZM against P. aeruginosa are unknown. A sub-MIC level of AZM delayed but did not inhibit P. aeruginosa (PAO1) biofilm formation and resulted in the development of a stable AZM-resistant phenotype (PAO1-BV). A new device, the 'stack reactor', was developed to collect RNA from biofilms for DNA microarrays. DNA microarrays were used to identify differentially-expressed transcripts of PAO1 biofilms developed in 2 mug/ml AZM. Significant transcript increases of RND efflux pump genes and type III secretion genes were observed. RND efflux pump and TTSS mutants were further investigated to substantiate microarray data. Only a DeltamexAB-oprM, DeltamexCD-oprJ mutant was unable to form a biofilm in AZM. To further demonstrate MexAB-OprM and MexCD-OprJ as the critical pumps, P. aeruginosa containing either a mexA-gfp or mexC-gfp transcriptional fusion, was cultivated as a biofilm during subMIC exposure to AZM. Biofilms exhibited expression of mexC from the outset of their formation, but only in the presence of AZM. mexA, constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. Using three independent methods, either MexAB-OprM or MexCD-OprJ efflux pumps were necessary to form a biofilm in the presence of AZM. These data indicate that expression of RND efflux pumps by P. aeruginosa is antibiotic specific and, moreover, can be biofilm specific.

ISBN: 9780542263446Subjects--Topical Terms:

1017734
Biology, Microbiology.

Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin.
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100 1 $a Gillis, Richard John. $3 1914231
245 1 0 $a Antibiotic resistance mechanisms in Pseudomonas aeruginosa biofilms: The contributions of RND efflux pumps in response to carbenicillin and azithromycin.
300 $a 181 p.
500 $a Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4072.
500 $a Supervisor: Barbara H. Iglewski.
502 $a Thesis (Ph.D.)--University of Rochester, 2005.
520 $a Pseudomonas aeruginosa biofilms found in the lungs of patients afflicted with cystic fibrosis (CF) are extremely resistant to antibiotic treatment. RND efflux pumps were investigated due to their implications in planktonic P. aeruginosa antibiotic resistance. Experiments done exposing biofilms of various single null efflux pump mutants to several different classes of antibiotics failed to establish a correlation between efflux pump expression and antibiotic resistance in biofilms. Bacteria containing mexA-gfp and mexC-gfp transcriptional fusions were cultivated in biofilms in the presence of carbenicillin. Even though beta-lactam antibiotics are known substrates for both the MexAB-OprM and MexCD-OprJ efflux pumps, expression of mexA and mexC was relatively low in four day biofilms; decreasing to near zero levels by day eight. Recent clinical trials have reported improvement in CF patients treated with azithromycin (AZM). The mechanism(s) of action of AZM against P. aeruginosa are unknown. A sub-MIC level of AZM delayed but did not inhibit P. aeruginosa (PAO1) biofilm formation and resulted in the development of a stable AZM-resistant phenotype (PAO1-BV). A new device, the 'stack reactor', was developed to collect RNA from biofilms for DNA microarrays. DNA microarrays were used to identify differentially-expressed transcripts of PAO1 biofilms developed in 2 mug/ml AZM. Significant transcript increases of RND efflux pump genes and type III secretion genes were observed. RND efflux pump and TTSS mutants were further investigated to substantiate microarray data. Only a DeltamexAB-oprM, DeltamexCD-oprJ mutant was unable to form a biofilm in AZM. To further demonstrate MexAB-OprM and MexCD-OprJ as the critical pumps, P. aeruginosa containing either a mexA-gfp or mexC-gfp transcriptional fusion, was cultivated as a biofilm during subMIC exposure to AZM. Biofilms exhibited expression of mexC from the outset of their formation, but only in the presence of AZM. mexA, constitutively expressed in planktonic cells, was uniformly expressed in biofilms regardless of the presence of AZM. Using three independent methods, either MexAB-OprM or MexCD-OprJ efflux pumps were necessary to form a biofilm in the presence of AZM. These data indicate that expression of RND efflux pumps by P. aeruginosa is antibiotic specific and, moreover, can be biofilm specific.
590 $a School code: 0188.
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710 2 0 $a University of Rochester. $3 515736
773 0 $t Dissertation Abstracts International $g 66-08B.
790 1 0 $a Iglewski, Barbara H., $e advisor
790 $a 0188
791 $a Ph.D.
792 $a 2005
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3185030