Lasers in Surgery and Medicine 48:311–317 (2016)
Violet 405 nm Light: A Novel Therapeutic Agent Against
b-Lactam-Resistant Escherichia coli
Nathaniel L.R. Rhodes, MS,1 Martin de la Presa, BA,2 Mitchell D. Barneck, BS,3 Ahrash Poursaid, BS,1
Matthew A. Firpo, PhD,2 and John T. Langell, MD, PhD, MBA1,2
1
Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112
2
Department of General Surgery, University of Utah, Salt Lake City, Utah 84132
3
School of Medicine, Oregon Health and Sciences University, Portland, Oregon 97239
Background and Objective: Approximately 1.7 million
patients are affected by hospital-acquired infections every
year in the United States. The increasing prevalence of
multidrug-resistant bacteria associated with these infections prompts the investigation of alternative sterilization
and antibacterial therapies. One method currently under
investigation is the antibacterial properties of visible light.
This study examines the effect of a visible light therapy
(VLT) on b-lactam-resistant Escherichia coli, a common
non-skin flora pathogen responsible for a large percentage
of indwelling medical device-associated clinical infection.
Materials and Methods: 405 nm light-emitting diodes
were used to treat varying concentrations of a common
laboratory E. coli K-12 strain transformed with the pCIG
mammalian expression vector. This conferred ampicillin
resistance via expression of the b-lactamase gene. Bacteria
were grown on sterile polystyrene Petri dishes plated with
Luria-Bertani broth. Images of bacterial growth colonies on
plates were processed and analyzed using ImageJ. Irradiance levels between 2.89 0.19 and 9.45 0.63 mW cm2
and radiant exposure levels between 5.60 0.39 and
136.91 4.06 J cm2 were tested.
Results: VLT with variable irradiance and constant
treatment time (120 minutes) demonstrated significant
reduction (P < 0.001) in E. coli between an irradiance of
2.89 mW cm2 (81.70%) and 9.37 mW cm2 (100.00%).
Similar results were found with variable treatment time
with constant irradiance. Log10 reduction analysis produced between 1.98 0.53 (60 minute treatment) and
6.27 0.54 (250 minute treatment) log10 reduction in
bacterial concentration (P < 0.001).
Conclusions: We have successfully demonstrated a
significant bacterial reduction using high intensity
405 nm light. Illustrating the efficacy of this technology
against a b-lactam-resistant E. coli is especially relevant to
the need for novel methods of sterilization in healthcare
settings. These results suggest that VLT using 405 nm
light could be a suitable clinical option for eradication of
b-lactam-resistant E. coli. Lasers Surg. Med. 48:311–317,
2016. ß 2015 Wiley Periodicals, Inc.
Visible light kills statistically significant concentrations
of E. coli.
Antibiotic-resistant Gram-negative bacteria exhibits
sensitivity to 405 nm light.
ß 2015 Wiley Periodicals, Inc.
Greater than 6 log10 reduction in b-lactam-resistant
E. coli when treated with visible light therapy.
Key words: antibiotic resistance; sterilization; ultraviolet (UV) light; visible light therapy; visible spectrum light
INTRODUCTION
Hospital-acquired infections (HAIs) occur in approximately 1 in 25 patients, resulting in over 1.7 million patients
affected annually in the United States [1,2]. It is estimated
that 25.6% of these infections are device-associated [1]. Over
150 million intravascular catheters are placed in the Unites
States each year with 2.65 billion dollars spent to treat the
resulting device-associated infections [3]. The current
standard of care for the treatment of device-associated
infections involves removal of the infected hardware and
treatment with first-line broad-spectrum antibiotics until
culture and sensitivity data allow for a more tailored
antibiotic regimen. The increasing prevalence of multidrugresistant bacteria often necessitates a use of second-line
antibiotics with a greater side-effect profile and may lead to
loss of efficacy of these critical therapeutics [4]. Drug
resistance is considered a natural evolutionary process and
over 90% of Staphylococcus aureus strains are resistant to
penicillin and other related antibiotics [5]. The National
Healthcare Safety Network reported as much as 83% of
blood stream infections and 20–40% of all HAIs were caused
by drug resistant species [4]. It is clear that standard drug
Conflict of Interest Disclosures: All authors have completed
and submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest and have disclosed the following: This
research was paid in part from a grant through the National
Science Foundation Innovation Corps proposal number 1347345.
One (or more) of the authors recognizes their equity interest
related to the commercialization of this research.
Contract grant sponsor: National Science Foundation Innovation Corps; Contract grant number: 1347345.
Correspondence to: Nathaniel L.R. Rhodes, MS, 138 W.
Fireclay Avenue #204 Murray, Utah 84107.
E-mail: [email protected]
Accepted 25 November 2015
Published online 29 December 2015 in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/lsm.22457
312
RHODES ET AL.
treatments are not always appropriate to stop HAIs and
more effective methods of sterilization should be implemented. Medical device manufacturers have attempted to
solve the issue of hardware-associated infections through
several novel approaches including coating at-risk surfaces
with bactericidal and bacteriostatic agents such as chlorhexidine or silver compounds. These coatings are intended
to inhibit bacterial and fungal colonization of the device.
Unfortunately, these solutions have had minimal efficacy
in the clinical setting in reducing device-associated
infections [6,7]. Given the magnitude of the problem of
indwelling medical device-associated infections, more efficacious solutions must be found. One novel approach under
investigation is harnessing the potential of visible light
therapy (VLT). VLT has shown substantial antimicrobial
efficacy against pathogenic bacteria and has received
clearance for clinical application for acne treatment by
the United States Food and Drug Administration (FDA) [8].
In addition, bacterial resistance to VLT has not been
observed. This is thought to be due to a different
antimicrobial mechanism, compared to traditional pharmaceutical antibiotics, which avoids the method in which
bacteria become drug-resistant [9,10]. The most commonly
used classes of antibacterial drugs target cell wall biosynthesis, protein synthesis, or DNA replication and repair.
However, once an antibiotic is introduced clinically, it will
only have a period of months to years until a statistically
significant efficacy reduction appears [11]. The most
common methods of antibiotic resistance seen in bacteria
are: chemical or enzymatic modification of the drug,
transmembrane pumps which reduce intracellular drug
concentrations below therapeutic levels, and bacterial
target structure modification resulting in a reduced affinity
for the drug [10].
Although others have demonstrated the bactericidal efficacy
of 405 nm light on the drug resistant Gram-positive bacterium
methicillin-resistant S. aureus (MRSA) and non-resistant
Gram-negative bacteria, few studies have investigated its
efficacy against common drug-esistant Gram-negative organisms [12–19]. In particular, Escherichia coli are Gramnegative bacilli commonly present in the gastrointestinal
tract of humans. According to the United States Center for
Disease Control, E.coli is one of the most common pathogens
that cause nosocomial infections in addition to S. aureus and
Pseudomonas aeruginosa [20]. Wavelength specific VLT
against the spectrum of pathogens responsible for indwelling
device-associate clinical infections may offer a novel, costeffective, and sustainable agent against these life-threatening
infections. In this study we explore the efficacy of 405 nm VLT
against E. coli, one of the most common isolates of HAIs and
indwelling medical device-associated infections [21,22].
MATERIALS AND METHODS
plasmid confers ampicillin resistance to the E. coli strain via
expression of the b-lactamase gene. The pCIG plasmid also
conferred expression of green fluorescent protein, which
was used to verify strain selection. Sterile polystyrene
100 15 mm2 Petri dishes were plated with Luria-Bertani
(LB) broth (Lennox L3022). LB selective media contained
0.1 mg/ml ampicillin in all experiments. For consistency in
counting colony forming units (CFUs) between experiments,
dilution series were made from cultures inoculated with 3–4
well-separated bacterial colonies and grown to saturation.
Serial dilutions were prepared by taking 0.5 ml of the
saturated bacterial solution and adding it to 4.5 ml of the
LB broth stock solution. Time and power trial plates were
diluted in this fashion by a factor of 105 for each control and
treated plates. Log10 reduction plates were diluted by a factor
of 105 for control and between 101 and 103 for treated. Each
plate was seeded with 200 ml aliquots and the liquid was
spread until uniformly distributed. Optimal plating densities
were determined in pilot experiments in order to yield 10–
15 CFUs cm2.
Experimental Setup
The 405 nm light-emitting diodes (LEDs) were selected
(Bivar #: UV5TZ-405-15) and wavelength was verified using
a visible light spectrometer (Ocean Optics USB2000). LEDs
had a peak wavelength at 404.63 nm 1.74 and had a
spectrum of 16.57 nm 0.89 at 50% relative peak intensity.
Nine LEDs were wired in a parallel circuit and arranged in a
3 3 grid pattern. This setup was placed onto a tray
customized for an incubator. Custom partitions were
created to ensure no light transferred between treatment
areas (Supplementary Fig. S1).
Light intensity from the LEDs were measured to
determine overall output power using a digital power
meter (PM100USB, Thorlabs, Newton, NJ) and sensor
(S140C, Thorlabs, Newton, NJ). Diode intensity was
measured pre- and post-treatment to determine irradiance. Untreated and treated (exposure to 405 nm light)
areas within the same plate were measured for CFUs in
order to control for potential plate-to-plate differences in
bacterial plating densities. A flexible opaque polyethylene
sleeve was placed to cover half of the plate. The other half of
the plate was left unobstructed and light sources were
placed approximately 3.75 cm above the surface of the
plate. Circular areas on both the control and treated sides
with a radius of 1.02 cm were marked on the plate and used
to focus the light in a treated area. All measurements of
CFUs were done with respect to bacteria inside this
1.02 cm radius circle. Radiant exposure levels are defined
as the intensity of light in an area for a certain amount of
time giving it units of mWsec
or cmJ 2 . Varying power levels of
cm2
light intensity as well as irradiation time led to several
different radiant exposure levels tested.
Bacteria Preparation
The common laboratory E. coli K-12 strain (fhuA2 lac(del)
U169 phoA glnV44 F80’ lacZ(del)M15 gyrA96 recA1 relA1
endA1 thi-1 hsdR17) transformed with the pCIG mammalian
expression vector was used for all experiments. The pCIG
Bacterial Growth Analysis
After 24 hours of growth, high-resolution images were
taken of each plate using a digital 18-megapixel camera
(Canon T2i DLSR). The camera was mounted on a stand
VISIBLE LIGHT TREATMENT ON A b-LACTAM-RESISTANT E. COLI
313
with the lens 61 cm from the plate surface. Images were
processed and analyzed using ImageJ (National Institute of Health). Supplementary Figure S2 shows various
stages of image processing including a whole Petri dish
photograph, a binary image, and individual colony count
outlines. Individual colonies were selected based on a
size and circularity protocol. Size was determined by
pixel count. A range of 200–4000 pixel was determined
based on the average isolated colony size [2]. A
circularity value range of 0.2–1.0 was selected. A value
of 1.0 indicates a perfect circle, whereas a value
approaching 0.0 indicates an increasingly elongated
polygon.
The resulting CFUs counted at different radiant
exposures were plotted as survival fraction versus
radiant exposure. The survival fraction values were
generated based on a log10 normalization of the resulting
CFUs.
Statistical Analysis
Continuous data of individual survival fractions were
defined as the quantity of colony forming units normalized by taking log10 of said value in treated and untreated
control groups. Differences in continuous data were
assessed using a Wilcoxon signed-rank test using JMP
software (SAS, Cary, NC). Data was binned based on
variable component in each experimental setup. For
example, in time variable testing, data groups were
binned by time, in log10 reduction testing, groups were
binned based on concentration and time. Differences in
binned data were assessed using a paired t-test using
Microsoft Excel 2013. A P-value of less than 0.05 was set
as the a priori level of significance.
RESULTS
Variable Irradiance With Constant Exposure Time
Analysis
The bactericidal effect of the 405 nm light on ampicillinresistant bacteria was first determined using light of
varying irradiance. Irradiance was modulated by controlling the voltage and current applied to each diode
determined as a percentage based on the manufacturer’s
suggested forward voltage and current (3.4 volts, 30 mA).
The values were 25%, 50%, 75%, and 100% of this voltage,
which produced 9.43 0.72, 16.05 0.79, 26.15 2.30, and
30.30 1.25 volts across each diode, respectively. Figure 1
shows the results for the exposure of ampicillin-resistant
E. coli to 405 nm light delivered at increasing light
irradiance and with time being held constant at 120
minutes. There was a significant reduction in E. coli
starting at an irradiance of 2.89 mW cm2 resulting in an
81.70% reduction in bacterial growth (P < 0.001). Each
subsequent increase in irradiance yielded a statistically
significant bacterial reduction (P < 0.001). Maximum bacterial eradication (100.00%) was observed after an irradiance of 9.37 mW cm2 for 120 minutes (65.90 J cm2) was
applied (P < 0.001). Table 1 lists the bacterial counts
observed at each treatment intensity.
Fig. 1. E. coli reduction at variable power density: (Top) A scatter
plot showing the survival fraction of each plate in control and
treated areas. Logarithmic trend lines are added to show the
general relationship between control and treated plates as a
function of radiant exposure. (Bottom) A bar graph showing
binned survival fraction of grouped by relative power (25%, 50%,
75%, and 100%) and tested for statistical significance in treated
versus control. The relative power 25%, 50%, 75%, and 100%
corresponds approximately to 20.8, 35.4, 58.1, and 67.5 J cm2,
respectively. Statistical significance of P < 0.001 is noted with a
double asterisk ( ).
Variable Exposure Time With Constant Irradiance
Analysis
The antimicrobial effect of 405 nm light was also
determined by varying irradiation time. Voltage and
current levels of each diode were held at the manufacturer’s suggested values (3.4 volts and 30 mA, respectively). The average irradiation during the time protocols
was 9.24 0.44 mW cm2. Figure 2 depicts the results for
the exposure of E. coli to 405 nm light delivered at
increasing duration of time with irradiance being held
constant. There was a significant reduction (P ¼ 0.030,
P < 0.001) in bacterial growth seen at each time point. It
is interesting to note that statistically significant changes
were shown in a duration of treatment as short as 10
minutes (5.59 J cm2), in which a mean of 26% bacterial
reduction was seen. Table 2 lists the bacterial counts
observed for each treatment duration.
Log10 Reduction Analysis
Using the ASTM E2315-03 protocol (Standard Guide for
Assessment of Antimicrobial Activity), E.coli was plated in
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RHODES ET AL.
TABLE 1. Variable Irradiance With Constant Treatment Time
Irradiance
(mW cm2)
Time
(minutes)
Radiant
exposure
(J cm2)
Treated
(CFU)
Control
(CFU)
Mean %
reduction
P-values
2.89 0.19
4.91 0.23
8.07 0.06
9.37 0.03
120
120
120
120
20.80 1.39
35.37 1.71
58.09 4.78
67.49 2.21
3.67 2.92
2.11 1.83
0.44 0.72
0.00 0.00
19.67 2.33
20.00 3.80
17.30 4.13
19.68 5.35
81.7
89.74
96.46
100.00
<0.001
<0.001
<0.001
<0.001
Mean percent E. coli CFU reduction following exposure to 405 nm light with treatment time held constant.
high confluent concentrations and irradiated with different exposure times (60, 120, and 250 minutes). Untreated
control bacterial colony forming units averaged 2.16 0.17
103 CFUs cm2,
2.12 0.16 104 CFUs cm2,
and
5
1.56 0.13 10 CFUs cm2 for the 60, 120, and 250
minute treatments, respectively. The resulting reductions
in bacterial concentration produced between 1.98 0.53
(60 minute treatment) and 6.27 0.54 (250 minute
treatment) log10 reduction (Fig. 3). Statistically significant
results (P < 0.001) were found at all exposure times, which
are listed in Table 3.
DISCUSSION
This study has demonstrated that 405 nm light has a
significant bactericidal effect on b-lactam-resistant E. coli.
This study set out with three aims: [1] to test the relative
susceptibility and potential resistance of a medically common
b-lactam-resistant microbe to 405 nm light [2] to determine
the potential difference in efficacy between a brief highintensity and a long low-intensity treatment of equivalent
irradiant dose, and [3] to assess the potential log10 reduction
capability of 405 nm light on b-lactam-resistant E. coli.
Bactericidal Effect of 405 nm Light
Fig. 2. E. coli reduction at variable time points: Inactivation of E.
coli upon exposure to 405 nm light as a function radiant exposure.
Light was delivered at a constant power intensity (9.2 mW cm2)
with varying durations of time: 10, 50, 120, and 250 minutes (5.6,
27.7, 67.5, and 136.9 J cm2, respectively). (Top) A scatter plot of
all experimental data points. (Bottom) A clustered column graph
based on grouped by treatment time (10, 50, 120, and 250 minutes
respectively). Statistical significance of P 0.05 is noted with a
single asterisk ( ) and P-values of P < 0.001 is noted with a double
asterisk ( ).
The bactericidal effects of visible light, specifically violet
spectrum 405 nm light, have been investigated by several
groups. The most clinically established use of the
bactericidal properties of this light is for the treatment
of acne vulgaris. Ashkenzai et al., was able to demonstrate
a 5 log10 reduction of Propionibacterium acnes after
illumination with 407–420 nm light at a radiant exposure
of 225 J cm2 in an in vitro study [23]. The effectiveness of
this light therapy for acne vulgaris has also been shown in
multiple studies with clinical improvements reported from
64.7% to over 90% [24–28].
VLT has also been recently shown to be effective in the
treatment of gastritis. Elimination of Helicobacter pylori
has been demonstrated in vitro to have a 99.9% reduction
in bacterial proliferation after exposure to 20.0 J cm2 of
405 nm light [19]. Ganz et al. demonstrated between 91%
and 99% reduction of H. pylori colonies after a single
treatment of 40.5 J cm2 with a 405 nm diode laser [15].
These results were replicated by Lembo et al., showing an
86% to over 97% reduction in H. pylori after 15–60 minutes
of 408 nm VLT [29].
The efficacy of the broad-spectrum action of violet light
against both Gram-positive and Gram-negative bacterial
species has been well-documented [12,16–18,30,31]. A
study by Maclean et al. compared the relative efficacy of
violet light on several medically relevant bacterial species
with an LED array delivering an irradiance of 10 mW cm2
to a bacterial suspension [13]. It was found that Grampositive species were generally more susceptible (2.6–5.0
log10 reduction) than Gram-negative species (3.1–4.7 log10
reduction).
There has also been an interest in demonstrating the
effectiveness of violet light against drug-resistant species.
VISIBLE LIGHT TREATMENT ON A b-LACTAM-RESISTANT E. COLI
315
TABLE 2. Variable Treatment Time With Constant Irradiance
Irradiance
(mW cm2)
Time
(minutes)
Radiant
exposure
(J cm2)
Treated
(CFU)
Control
(CFU)
Mean %
reduction
P-values
9.32 0.51
9.22 0.55
9.37 0.03
9.13 0.27
10
50
120
250
05.59 0.31
27.67 1.66
67.49 2.21
136.91 4.06
18.94 7.71
4.11 3.59
0.00 0.00
0.00 0.00
27.42 13.42
27.54 12.69
19.68 5.35
35.57 23.38
26.40
82.11
100.00
100.00
0.030
<0.001
<0.001
<0.001
Mean percent E. coli CFU reduction following exposure to 405 nm light with variable treatment time and constant irradiance.
An in vitro study by Maclean et al. demonstrated a 5 log10
reduction of MRSA in a liquid suspension after irradiating
with 630–720 J cm2 of >400 nm light [32]. A following
study further showed that a 5 log10 reduction of MRSA can
be achieved with a significantly lower radiant exposure of
45 J cm2 when light is delivered specifically at the 405 nm
wavelength [13]. Another research study produced similar
results when irradiating both MRSA USA 300 and
MRSA IS-853 at 55 J cm2 with 405 nm light, achieving
a reduction of 92.1% and 93.5%, respectively [16]. In
Gram-negative bacteria, Hamblin et al. tested VLT on
a strain of H. pylori that was resistant to multiple
antibiotics [19].
Though studies have demonstrated the use of violet light
to target MRSA and H. pylori, data is lacking on the efficacy
of 405 nm light in the inactivation of b-lactam-resistant
E. coli. b-lactam antibiotics act through inhibition of
bacterial cell wall biosynthesis. MRSA and other b-lactam
-resistant bacteria produce b-lactamases that provide
resistance to commonly used b-lactam antibiotics like
penicillin, cephamycin, and carbapenem. Hydrolysis of
the b-lactam ring of the antibiotic will render it
ineffective [33].
This study has established the efficacy of using 405 nm
light to inactivate a medically common drug-resistant
bacterium. We have constructed dose-response curves
illustrating the irradiation necessary for the in vitro
eradication of E. coli on plated growth media. This
information may be useful for establishing the potential
for clinical use. Due to the increasing numbers of HAIs and
medically relevant drug-resistant microbes, this technology could provide another option for the clinical treatment
of antibiotic-resistant bacterial infections.
Mechanism of Action
Fig. 3. E. coli Log10 reduction at variable radiant exposure:
Inactivation of confluent concentrations of E. coli upon exposure to
405 nm light as a function time duration. Light was delivered at a
constant power intensity (9.1 mW cm2) with varying durations
of time: 60, 120, and 250 minutes. (Top) A scatter plot showing the
survival fraction of each plate in control and treated areas.
Logarithmic trend lines are added to show the general relationship
between control and treated plates as a function of radiant
exposure. (Bottom) A bar graph showing binned survival fraction
of grouped by concentration and treatment time of E. coli (101 at
250 minutes, 102 at 120 minutes, 103 at 60 minutes) Statistical
significance in treated versus control. Statistical significance of
P < 0.001 is noted with a double asterisk ( ).
Although the antimicrobial mechanism of action of violet
light has not been completely elucidated, research over the
past decade has identified several critical areas of activity.
It has been demonstrated that this process is oxygen
dependent. Studies have shown that bacteria exposed to
violet light under anaerobic conditions were able to resist
the phototoxic effects and that decreasing the amount of
reactive oxygen species (ROS) in an aerobic environment
also reduced the phototoxic effect [34]. Conversely, other
groups have shown that increasing the amount of oxygen
significantly increased the rate of bacterial inactivation [32]. It has also been demonstrated that violet light
illumination of endogenous porphyrins within bacteria
results in their decreased proliferation [23]. Further,
Hamblin et al. found that there is a strong positive
correlation between amount of porphyrins contained
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RHODES ET AL.
TABLE 3. Log10 Reduction
Irradiance
(mW cm2)
Time
(minutes)
Radiant exposure
(J cm2)
Treated
(CFU)
Control
(CFU)
Mean log
reduction
P-values
8.80 0.79
9.02 0.34
9.45 0.63
60
120
250
34.03 2.28
64.95 2.45
132.09 11.89
105.67 71.11
24.57 21.51
0.67 1.14
70.60 7.75
70.82 8.35
51.11 14.4
1.98 0.53
3.60 0.47
6.27 0.54
<0.001
<0.001
<0.001
Mean Log10 E. coli CFU reduction following exposure to 405 nm light.
within bacteria and the cytotoxic effects of violet light [19].
These studies support an oxygen dependent mechanism
which allows violet light to excite endogenous porphyrins
to a high energy state. This energy is transferred to form
ROS that cause intracellular damage. Bacterial and fungal
proteins, lipids, and nucleic acids are particularly susceptible to these ROS [35].
The mechanism of action of 405 nm VLT is relevant when
assessing the potential for microbe resistance. Although
resistance to 405 nm light specifically has not been
investigated, the potential development of microbial resistance to photodynamic inactivation has been studied. After
repeated cycles of partial inactivation followed by regrowth,
different bacterial species failed to develop resistance to the
photodynamic process after 10 and 20 cycles [36,37].
It is possible that due to the multifactorial mechanism of
action viable bacteria are less likely or unable to develop a
resistance to VLT. Further studies are necessary to determine
the extent to which bacteria are able to develop resistance.
However, this study was able to illustrate that drug-resistant
bacteria are susceptible to 405 nm VLT. The significant
bacterial toxicity demonstrated makes VLT a potential
alternative treatment for associated clinical infections.
Equivalent Radiant Exposure Dose
An in vitro study by Murdoch et al. showed significant
differences in the bactericidal effect on Listeria
monocytogenes at equivalent radiant exposure levels but
varying irradiance and exposure time [18]. We performed
similar preliminary testing on E. coli by reducing power
levels and increased treatment time. The results supported
the findings of Murdoch et al. in that we demonstrated a
significant reduction of bacteria in treated plates even at 25%
power and fourfold longer treatment times. However, due to
the radiant exposure tested, both treatments in this study
resulted in a 100% reduction in bacterial CFUs. Because of
this, we cannot ascertain if one method is more efficacious
than the other given equivalent radiant exposure. Additional
testing will be performed in subsequent studies to determine
efficacy trends at equivalent radiant exposure and varying
irradiance and time levels.
Log10 Reduction
This study has also established the potential log10
reduction of 405 nm light on drug-resistant E. coli. The
FDA requires that a minimum of a four-log10 reduction be
achieved in order to be considered an antibacterial
solution [38]. In order to be considered for approval, the
FDA recommends following ASTM E2315-03 Standard
Guide for Assessment of Antimicrobial Activity Using a
Time-Kill Procedure or an equivalent method.
Using this ASTM protocol, we have demonstrated a
greater than 6 log10 reduction. This is 100 times the
reduction necessary to be defined as bactericidal by the
FDA. The results from this study also illustrate the time
and dose dependence of light technology. These results
suggest that VLT using 405 nm light could be a suitable
clinical option for sterilization in drug-resistant species of
bacteria. The log10 reduction information provided may
help direct future applications of VLT in a clinical setting.
CONCLUSIONS
We, along with other researcher groups, have successfully
demonstrated a high efficacy at bacterial reduction using
high intensity 405 nm light. Illustrating the efficacy of this
technology against a b-lactam-resistant E. coli is especially
relevant given the great need for novel methods of sterilization in healthcare settings. This information is also valuable
in the context of the increasing occurrence of drug-resistant
bacteria in HAIs and device-associated infections. The
increasing incidence of these infections illustrate that
current antimicrobial technologies are inadequate. Further
research directives will focus on other common nosocomial
infection causing agents, such as P. aeruginosa, S. aureus,
and other drug resistant bacteria.
ACKNOWLEDGMENTS
The authors would like to thank the University of Utah
and Center for Medical Innovation who assisted in funding
this research. The authors would like to thank the
University of Utah Department Of Surgery staff Elaine
Hillas, Patti Larrabee, and Dr. Jill Shay, as well as Sami
Barneck and the microbiology laboratories at Weber State
University. Also, they would like to thank the National
Institute of Health for allowing open access to ImageJ.
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