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1-1-2012
Blue light (470 nm) effectively inhibits bacterial and fungal growth
A.J. De Lucca
USDA-ARS , anthony.delucca@v
C. Carter-Wientjes
USDA-ARS , Carol.Carter@v
K.A. Williams
USDA-ARS , karen.williams@v
D. Bhatnagar
USDA-ARS , Deepak.Bhatnagar@v
Follow this and additional works at:digitalcommons.unl.edu/usdaarsfacpub
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De Lucca, A.J.; Carter-Wientjes, C.; Williams, K.A.; and Bhatnagar, D., "Blue light (470 nm) effectively inhibits bacterial and fungal growth" (2012).Publications from USDA-ARS / UNL Faculty.Paper 1096.digitalcommons.unl.edu/usdaarsfacpub/1096
ORIGINAL ARTICLE
Blue light (470nm)effectively inhibits bacterial and fungal growth
A.J.De Lucca,C.Carter-Wientjes,K.A.Williams and D.Bhatnagar
Southern Regional Research Center,USDA,ARS,New Orleans,LA USA
Significance and Impact of Study:Light from two arrays
of different blue LEDs significantly reduced bacterial (Leuconostoc mesenteroides ,Bacillus atrophaeus and Pseudomonas aeruginosa )viabilities.Sig-nificant in vitro viability loss was observed for the filamentous fungi,Penicillium digitatum and Fusari-um graminearum when exposed to pure blue light only plus a aminearum viability was significantly reduced by blue light alone.Results suggest that (i)the amount of significant loss in bacterial viability observed for blue light that is pure or with traces of other wavelengths is genus dependent and (ii)depending on fungal genera,pure blue light is fungicidal with or without a photo-sensitizer.
Keywords
antibacterial,antifungal,visible blue light.Correspondence
Anthony J.De Lucca,Southern Regional
Research Center,USDA,ARS,1100Robert E.Lee Blvd.,New Orleans,LA 70124,USA.E-mail:anthony.delucca@v 2012/1233:received 9July 2012,revised 18September 2012and accepted 18September 2012
doi:10.1111/lam.12002
Abstract
Blue light (470nm)LED antimicrobial properties were studied alone against bacteria and with or without the food grade photosensitizer,erythrosine (ERY)against filamentous fungi.Leuconostoc mesenteroides (LM),Bacillus atrophaeus (BA)or Pseudomonas aeruginosa (PA)aliquots were exposed on nutrient agar plates to Array 1(AR1,0Á2mW cm À2)or Array 2(AR2,80mW cm À2),which emitted impure or pure blue light (0–300J cm À2),respectively.Inoculated control (room light only)plates were incubated (48h)and colonies enumerated.The antifungal properties of blue light combined with ERY (11Á4and 22Á8l mol l À1)on Penicillium digitatum (PD)and Fusarium graminearum (FG)conidia were determined.Conidial controls consisted of:no light,room light-treated conidia and ERY plus room light.Light-treated (ERY +blue light)conidial samples were exposed only to AR2(0–100J cm À2),aliquots spread on potato dextrose agar plates,incubated (48h,30°C)and colo
nies counted.Blue light alone significantly reduced bacterial and FG viability.Combined with ERY,it significantly reduced PD viability.Blue light is lethal to bacteria and filamentous fungi although effectiveness is dependent on light purity,energy levels and microbial genus.
Introduction
As early as 1887,visible blue light was known to be the most effective part of the light spectrum to stimulate pho-totrophism in plants (Sachs 1887).It is also important as a ‘cue’for fungal metabolism,growth,pigment formation,tropism and spore production (Siegel et al.1968;Casas-Flores et al.2006;Purschwitz et al.2006).Blue light sig-nal transduction pathways have been studied in Neuros-pora crassa,which has two major blue light photoreceptors,(i)white collar (WC)-1and (WC)-2,which control dark to light transition and (ii)VVD,a
protein important in the second light signalling system.Together,they control responses to daily changes in light intensity (Linden and Macino 1997;Schwerdtfeger and Linden 2001,2003).
In vitro lethality of blue light for the bacteria Escherichia coli ,aerobic methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa (PA)has also been reported (Guffy and Wilborn 2006;Brovko et al.2009).Blue light causes photoexcitation of endogenous bacterial photosen-
sitive porphyrins and subsequent bactericidal reactive oxygen species production (Lavi et al.2004;Lipovsky et al.2008,2009,2010).Bacteria without internal light
460
Letters in Applied Microbiology 55,460--466©2012The Society for Applied Microbiology
Letters in Applied Microbiology ISSN 0266-8254
This article is a U.S. government work, and is not subject to copyright in the United States.
reactive compounds can be killed with a combination of blue light and nontoxic photoactivatable dyes,such as cationic phenothiazinium dyes(Alves et al.2009)that, together,generate singlet oxygen and reactive oxygen spe-cies(Wainwright1998;Hamblin and Hasan2004;Tegos et al.2005).
While blue light is an important‘cue’in the asexual development of fungal spores,the combination of it with certain photosensitive dyes is fungicidal.Such dyes include the phenothiazinium dyes,toluidine blue O and dimethylmethylene blue and,combined with blue light, reduce the viability of Candida(Jackson et al.1999;Phoe-nix and Harris2003).
In recent years,safe chemicals with greater photosensi-tizing efficacy due to strong absorbance of blue, red or white),lipophilicity with a delocalized positive charge and stable to photodegradation have been identi-fied.They include benzo[a]phenoxazinium chalcogen ana-logues(BCA)and cationic fullerenes(Tegos et al.2005; Foley et al.2006).
Other visible light wavelengths combined with photo-sensitizing agents can also reduce fungal viability.White light(polychromatic)combined with BAM-SiPc,an unsymmetrical bisamino phthalocyanine,reduces Candida albicans viability(So et al.2010).White light combined with cationic bis-and tris-cationic fullerenes reduces bac-terial and C.albicans viability after only10min of incu-bation(Tegos et al.2005).Red light is also active against C.albicans in the presence of methylene blue,BCA or BAM-SiPc(Foley et al.2006;de Souza et al.2006;So et al.2010).C.albicans growth and germ tube formation is inhibited by red light plus methylene blue(Munin et al.2007)and is due to increased permeability(Giroldo et al.2009).Among the compounds with photosensitizing properties is erythrosine(FD&C Red no.3),a common food dye(Yang and Min2009).
This study determined the effect of(i)different LEDs producing blue light(peak:470nm)and(ii)incubation temperature after exposure to blue light on bacterial via-bility.Three bacteria used in this study include Leuconos-toc mesenteroides(LM),a soil-borne bacteri
um which is the major factor in US sugarcane and sugarbeet deteriora-tion(De Bruijin2002;Eggleston and Monge2005);Bacil-lus atrophaeus(BA),a surrogate in experiments for Bacillus anthracis(Weber et al.2003),and P.aeruginosa which causes serious burn wound infections,colonization of medical atheters)as well as contact dermatitis(Yue et al.2007;Lundov et al.2009;Bak et al. 2010).
The anti-Candida properties of combined extracellular photosensitizers and light have been published(So et al. 2010).However,no data on the effect of monochromatic light and photosensitizers onfilamentous fungi have been reported.This study also investigated the effect of blue light with and without erythrosine(ERY)on the viability of nongerminated and germinatingfilamentous conidia. The fungi studied were Penicillium digitatum(PD)that causes rot in stored citrus and Fusarium graminearum (FG),which produces potent mycotoxins and renders harvested wheat unsafe when postharvest factors allow naturally occurring fungi to grow(Magan et al.2010). Results and discussion
Bacteria
Leuconostoc mesenteroides only grew only at25°C with no growth at the other postlight treatment te
mperatures. Array2light(pure blue)had no effect on LM.However, Array1light(impure blue)significantly reduced colony-forming units(CFU)beginning at150J cmÀ2,and a via-bility loss of about80%was observed at180J cmÀ2.
In contrast to the results with LM,both LED arrays reduced the CFU of BA although a difference was observed in the effect of the two arrays.Array1(Table1) achieved significant CFU reduction beginning at 40J cmÀ2,with approximate100%losses observed at 80J cmÀ2at incubation temperatures of25and30°C, which were much lower energy levels than needed to achieve similar results with Array2at any incubation temperature.
Significant CFU reduction of BA was not observed using Array2below100J cmÀ2,while approximately 100%viability reduction was achieved at300J cmÀ2. This energy level was much higher than that needed from Array1to achieve similar viability loss and indicates that blue light with trace levels of other wavelengths produced by Array1was more active against BA than pure blue light(Array2).
After exposure to Array1,cells incubated post-treat-ment at37°C displayed less viability loss at60J cmÀ2 than did cells incubated at25and30°C,but this differ-ence was not significant.In contrast,no te
mperature effect was observed with cells exposed to Array2. Pseudomonas aeruginosa was more susceptible to blue light than the other bacteria and both arrays(Table1) significantly reduced viability.An8J cmÀ2energy dose from Array1saw the greatest CFU reduction(approxi-mately84%)with cells incubated at25°C,a suboptimum temperature for this bacterium.CFU reductions were approximately58–54%at10J cmÀ2with cells grown at 30and37°C(the optimum incubation temperature), respectively.
An8J cmÀ2energy dose from Array2caused the greatest viability loss,approximately96%,when PA was incubated at37°C after light treatment.In comparison,
Letters in Applied Microbiology55,460--466©2012The Society for Applied Microbiology461 A.J.De Lucca et al.Blue light(470nm)has antimicrobial properties
T a b l e 1E f f e c t o f b l u e l i g h t (470n m )o n B a c i l l u s a t r o p h a e u s a n d P s e u d o m o n a s a e r u g i n o s a v i a b i l i t y (P e r c e n t o f c o n t r o l v i a b i l i t y m e a n s )
J c m À2B .a t r o p h a e u s
P .a e r u g i n o s a
A r r a y 1†
A r r a y 2‡A r r a y 1
A r r a y 2
25°C 30°C 37°C
25°C
30°C
37°C 25°C
30°C
37°C 25°C
30°C
37°C
2§§
§
§
§
§
65Á9±16Á485Á1±8Á965Á8±14Á9§
§
§
4§§
§
§§
§40Á0*±11Á967Á1*±7Á560Á0*±17Á5§
§
§
5§§
§
§§
§§§§66Á2*±21Á7
55Á5*±10Á0
14Á6*±3Á5
6§§
§
§§
§47Á3*±12Á562Á3*±8Á565Á8*±15Á3§
§
§
首都圈8§§
§
§§
§16Á0*±7Á642Á1*±4Á146Á2*±13Á338Á4*±13Á043Á1*±12Á13Á5*±1Á01089Á5±8Á092Á7±4Á491Á1±5Á1§
§
§12Á1*±5Á140Á5*±6Á944Á2*±14Á9
28Á0*±10Á631Á1*±8Á40Á8*±0Á52086Á6±4Á7
89Á5±2Á9
92Á5±4Á7
§
§
§§§§
19Á1*±8Á422Á8*±6Á31Á9*±1Á030§§
§
§
§
§§§
§
16Á4*±7Á420Á9*±6Á81Á5*±0Á64071Á0*±7Á354Á0*±10Á6
79Á5*±4Á1
§
§
§§§
§
16Á1*±6Á822Á2*±8Á50Á6*±0Á450§§
§
§
§
§§§
§
15Á5*±
6Á420Á4*
±5Á93Á5*±
水电站与
电力网技术
1Á6
6016Á0*±7Á212Á9*±6Á653Á0*±11Á7§
§
§§§
§
§
§
§
800Á0*±0Á00Á7*±0Á112Á9*±0Á3§
§
§§§
§
§
§
§
1000Á0*±0Á0
0Á0*±0Á00Á0*±0Á0
74Á3±5Á463Á5±7Á775Á0±3Á5§
§
§
§
§
§
150§§
§
69Á9*±7Á544Á0*±7Á150Á1*±9Á2§
§
§
§
§
§
200§§
§
27Á6*±8Á149Á0*±5Á657Á1*±5Á1§
§
§
wpu
§
§
§
250§§
§
2Á1*±0Á86Á4*±2Á54Á4*±1Á4§
§
§
§
§
§
300
§§
§
0Á5*±0Á21Á1*±0Á40Á6*±0Á3
§
§
§
§
§
§
*S i g n i fic a n t l y (P <0Á001)l o w e r t h a n t h e c o n t r o l v i a b i l i t y m e a n s .†A r r a y 1:L E D s ,2Á2m W c m À2(L E D t r o n i c s ,T o r r a n c e ,C A ,U S A ).‡A r r a y 2:L E D s ,80m W c m À2(C R E E ,D u r h a m ,N C ,U S A ).
§N o t d o n e .
Letters in Applied Microbiology 55,460--466©2012The Society for Applied Microbiology
Blue light (470nm)has antimicrobial properties
A.J.De Lucca et al.
cells incubated at 25and 30°C showed approximately 62and 57%CFU loss,respectively.
With both Arrays,lethality increased with the amount of energy to which PA was exposed and suggests that for PA,Array 2(pure blue light)was slightly better than Array 1(blue light with traces of other wavelengths)as a bactericide.Fungi
Penicillium digitatum.Nongerminated conidia :Blue light (Array 2)or ERY alone did not reduce the viability of PD nongerminated conidia (Fig.1)when compared with the viability control means (no light or ERY treatments).In contrast,nongerminated conidia treated with blue light and 11Á4l mol l À1ERY significantly reduced CFU counts by about 40and 70%with blue light of 80and 100J cm À2,respectively,when compared with the conid-ial control.
Blue light plus 22Á8l mol l À1ERY showed significant CFU reduction of approximately 25%at 40J cm À2when compared to the conidial (no light or ERY)control.CFU losses increased to approximately 80and 95%when the blue light energy exposure was increased to 80and 100J cm À2,respectively.C
FU counts at these two energy levels in the presence of ERY (22Á8l mol l À1)were significantly lower than with blue light or ERY alone controls.
Germinating conidia :The blue light and ERY con-trol CFU for the germinating conidia (Fig.2)were not significantly lower than those for the conidial (no light or ERY)control means.Germinating conidia were much more susceptible than nongerminated conidia to a treat-ment of blue light plus ERY.The viability losses (approximately 80–98%)with blue light energy levels of
40–100J cm À2
in combination with ERY at 11Á4l mol l À1
were significantly lower than the CFU of the conidial (no light or ERY),blue light and ERY con-trols.Viability losses of approximately 95–98%were observed when blue light (40–100J cm À2)was combined with 22Á8l mol l À1ERY.The combination of light and ERY also significantly reduced CFUs in comparison to the blue light and ERY alone controls.
Fusarium graminearum.Nongerminated conidia :Figure 3shows that no significant viability loss was observed for the FG nongerminated conidia treated with only ERY at 11Á4and 22Á8l mol l À1as well
as conidia treated with blue light (20–100J cm À2)alone.However,significant viability losses of 80,95and 100%,were observed for conidia exposed to blue light energy levels of 40,80and 100J cm À2,respectively,in the presence of 11Á4l mol l À1ERY when compared with the conidial control.When the ERY concentration was increased to 22Á8l mol l À1,the viability loss was 100%when combined with a blue light energy value of 40J cm À2.
The germinating conidia were resistant to ERY alone at 11Á4and 22Á8l mol l À1when compared with the conidial control (Fig.4).In contrast,blue light alone at energy levels of 40,80and 100J cm À2significantly reduced conidial viability by approximately 36,42and 47%,respectively.Combining blue light with ERY (11Á4l mol l À1)produced a significant viability loss of about 90and 100%at 40and 80J cm À2,respectively.Blue light combined with ERY (22Á8l mol l À1)displayed
020
40
60*P < 0·001n = 16J cm –2
80*
白象百科100
20406080120140100**
*
*
Effect of blue light from Array 2with and without erythro-and 22Á8l mol l À1)on the viability of nongerminated Penicilli-digitatum conidia.Nongerminated P.digitatum +erythrosine
l À1
)only;Nongerminated P.digitatum +blue light only;Nongerminated P.digitatum +blue light +erythrosine (11Á4l mol l À1Nongerminated P.digitatum +erythrosine (22Á8l mol l À1)only;Nongerminated P.digitatum +blue light +erythrosine (22Á8l mol l À1020
40
60*P < 0·001n = 16
J cm –2
80
*100
20406080120140100*
*
*Effect of blue light from Array 2with and without erythro-and 22Á8l mol l À1)on the viability of germinating Penicil-digitatum conidia.Germinating P.digitatum +erythrosine
mol l À1
)only;Germinating P.digitatum +blue light only;Germinating P.digitatum +blue light +erythrosine (11Á4l mol l À1Germinating P.digitatum +erythrosine (22Á8l mol l À1)only;Germinating P.digitatum +blue light +erythrosine (22Á8l mol l À1).
Letters in Applied Microbiology 55,460--466©2012The Society for Applied Microbiology
463
A.J.De Lucca et al.Blue light (470nm)has antimicrobial properties
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