PMID- 11722914
OWN - NLM
STAT- MEDLINE
DCOM- 20020304
LR  - 20191210
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 67
IP  - 12
DP  - 2001 Dec
TI  - Green fluorescent protein as a novel indicator of antimicrobial susceptibility in 
      Aureobasidium pullulans.
PG  - 5614-20
AB  - Presently there is no method available that allows noninvasive and real-time 
      monitoring of fungal susceptibility to antimicrobial compounds. The green 
      fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a 
      potential reporter molecule for this purpose. Aureobasidium pullulans was 
      transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden 
      Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, 
      BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright 
      fluorescence that was amenable to quantification using fluorescence 
      spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were 
      directly proportional to the number of viable cells determined by CFU plate 
      counts (r(2) > 0.99). The relationship between cell viability and GFP 
      fluorescence was investigated by adding a range of concentrations of each of the 
      biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions 
      of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 
      25-min) loss of fluorescence of greater than 90% when used at concentrations of 
      150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. 
      Further, loss of GFP fluorescence from A. pullulans cells was highly correlated 
      with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP 
      fluorescence and cell viability were highly dependent on external pH; maximum 
      losses of fluorescence and viability occurred at pH 4, while reduction of GFP 
      fluorescence was absent at pH 8.0 and was associated with a lower reduction in 
      viability. When A. pullulans was attached to the surface of plasticized 
      poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly 
      than in cell suspensions, with > 95% loss of fluorescence after 27 h. This 
      technique should have broad applications in testing the susceptibility of A. 
      pullulans and other fungal species to antimicrobial compounds.
FAU - Webb, J S
AU  - Webb JS
AD  - School of Biological Sciences, University of Manchester, Manchester, United 
      Kingdom.
FAU - Barratt, S R
AU  - Barratt SR
FAU - Sabev, H
AU  - Sabev H
FAU - Nixon, M
AU  - Nixon M
FAU - Eastwood, I M
AU  - Eastwood IM
FAU - Greenhalgh, M
AU  - Greenhalgh M
FAU - Handley, P S
AU  - Handley PS
FAU - Robson, G D
AU  - Robson GD
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Fungicides, Industrial)
RN  - 0 (Indicators and Reagents)
RN  - 0 (Luminescent Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
SB  - IM
MH  - Ascomycota/*drug effects/genetics/growth & development/*metabolism
MH  - Fungicides, Industrial/*pharmacology
MH  - Green Fluorescent Proteins
MH  - Indicators and Reagents/*metabolism
MH  - Luminescent Proteins/genetics/*metabolism
MH  - Microbial Sensitivity Tests/methods
MH  - Spectrometry, Fluorescence
MH  - Spores, Fungal/drug effects/genetics/growth & development/metabolism
MH  - Transformation, Genetic
PMC - PMC93351
EDAT- 2001/11/28 10:00
MHDA- 2002/03/05 10:01
PMCR- 2001/12/01
CRDT- 2001/11/28 10:00
PHST- 2001/11/28 10:00 [pubmed]
PHST- 2002/03/05 10:01 [medline]
PHST- 2001/11/28 10:00 [entrez]
PHST- 2001/12/01 00:00 [pmc-release]
AID - 0840 [pii]
AID - 10.1128/AEM.67.12.5614-5620.2001 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2001 Dec;67(12):5614-20. doi: 
      10.1128/AEM.67.12.5614-5620.2001.