Indigo-Clean White Paper: #1

Bactericidal Performance Testing of Indigo-Clean Upon Bacterial Species

Healthcare

HC
Bactericidal Performance Testing of Indigo-Clean Upon Bacterial
Species
Clifford J. Yahnke, Director, Clinical Affairs, Kenall Manufacturing, 10200 W. 55th St. Kenosha, WI 53144

ABSTRACT
Susceptibility of a variety of medically-relevant Gram positive and Gram negative vegetative bacteria to 405 nm
visible light was investigated. Bacteria, deposited onto agar surface and stainless steel coupons, were exposed
to a high-intensity 405-nm visible light generated from a light-emitting diode array. The degree of bacterial
inactivation was calculated by determining the number of surviving CFUs after exposure to light at a pre-
determined distance and exposure times when compared to the controls that were not exposed to the
bactericidal light. The studies showed broad-spectrum activity of light against Gram positive bacteria such as
Staphylococcus aureus and Gram negative bacteria such as Enterobacter aerogenes, Klebsiella pneumoniae,
and Pseudomonas aeruginosa when seeded onto an agar surface. The bactericidal light was least effective
against Enterococcus faecalis (Gram positive organism) and Acinetobacter baumannii (Gram negative
organism). When tested on stainless steel surface, the significant light-dependent inactivation of population
was observed with E. aerogenes, S. aureus, and P. aeruginosa whereas least inactivation was observed with K.
pneumoniae, A. baumannii, and E. faecalis. The results of the study showing effective inactivation of medically
important bacteria offers the potential to provide continuous decontamination technology in a clinical setting
and other industries such as pharmaceutical and food manufacturing.

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INTRODUCTION
Healthcare Acquired Infections (HAI) are a substantial concern for healthcare providers with approximately
1.7M infections, 99,000 deaths and ~$96B-147B in excess costs each year1. In an effort to improve this
situation, the Affordable Care Act has created a series of reimbursements and penalties directly related to
provider’s performance in reducing the number of Healthcare Acquired Infections.
As part of this effort, healthcare providers are turning to a variety of solutions such as improved handwashing,
antimicrobial stewardship, isolation precautions, patient and staff education, and better policies and
procedures. The risk of pathogenic transmission via the environment is frequently overlooked or given far less
attention that any of the other modalities. Given the rise of antibiotic resistant bacteria, improving
environmental hygiene to prevent the bacteria from getting into the body will take on an increasing role.
Healthcare providers routinely clean the environment, typically on a daily basis (or episodically). The limitation
of this approach is that immediately after cleaning, bacteria begin to repopulate the space. This suggests a
need for an environmental disinfection system that operates continuously and which allows people to work in
the space while it’s in use.
The use of 405nm visible light as a potential method for reduction or inactivation of bacteria in the
environment has been the subject of academic interest since 20012 and has recently been introduced
commercially under the brand name Indigo-Clean. It is deployed clinically in an overhead light fixture operating
in one of two modes depending upon whether or not tasks are being performed in the room as shown below:

White Disinfection Mode = Indigo Disinfection Mode =

Ambient White Light + Disinfection Increased Disinfection Only

Visible light disinfection constantly emits a narrow spectrum of visible light at 405 nm to kill harmful bacteria in
the environment (air, hard and soft surfaces) safely, automatically and continuously. Specifically, the light is
first absorbed by porphyrin molecules inside the bacteria creating toxic and biocidal reactive oxygen species
(ROS) which inactivates the pathogen. Because it uses visible light, the room can be in use while the
disinfection is occurring. This technology has been recently commercialized and the capability of an Indigo-
Clean™ light unit was evaluated in the laboratory to assess the inactivation of variety of medically important
bacteria (ESKAPE organisms) on surfaces wherein the disinfection efficiency was calculated. The ESKAPE group
of organisms have become highly relevant in hospital settings due to the rise in antibiotic resistance and the

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fact that they are responsible for ~90% of nosocomial infections. This group includes Enterobacter aerogenes,
Staphylococus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterococcus faecalis.
Visible light disinfection constantly emits a narrow spectrum of visible light at 405 nm to kill harmful bacteria in
the environment (air, hard and soft surfaces) safely, automatically and continuously. Specifically, the light is
first absorbed by porphyrin molecules inside the bacteria creating toxic and biocidal reactive oxygen species
(ROS) which inactivates the pathogen. Because it uses visible light, the room can be in use while the
disinfection is occurring. Thus, the product can be used to continuously disinfect the environment.
Regardless of approach, all of these measures are ultimately graded by their ability to prevent infections. This
outcome data is highly sought after but can be very difficult and time consuming to collect due to the generally
low infection rates in US healthcare institutions (~1 in 25) and the number of variables to control throughout
the study. This becomes even more challenging for environmental disinfection technologies whose clinical
benefit is often obscured by larger effects such as handwashing compliance.
Therefore, the performance of environmental disinfection technologies is typically measured by its ability to
reduce bacteria on surfaces (or in the air) within a clinical setting. While clinical measurements such as this are
generally regarded as the most effective means of characterization, laboratory performance measurements
offer a faster alternative by which to make an initial assessment. Additionally, laboratory measurements offer
a more controlled environment in which the bactericidal performance can be measured against specific
organisms on various surfaces using known amounts of disinfectant.
With this in mind, the capability of an Indigo-Clean™ light unit was evaluated in the laboratory to assess the
inactivation of a variety of medically important bacteria (known as the ESKAPE organisms) on surfaces wherein
the disinfection efficiency was calculated.

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MATERIALS AND METHODS
Bacteria
The organisms used in this study were: Enterobacter aerogenes ATCC 13048, Staphylococus aureus ATCC 6538,
Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC
9027, and Enterococcus faecalis ATCC 19433 were used in the study. Dilutions of each microorganism
suspension were prepared in phosphate buffer and were titered concurrent to test inoculations.
LED Light Source
An Indigo-Clean™ fixture (Manufacturer Part No: M4SEDIC-24-150I/100L) from Kenall Manufacturing was used
to create a uniform distribution of disinfecting light at a task plane 1.5m beneath the fixture. This irradiance
was measured at various points across the plane to be 0.498 mW/cm2 to 0.558 mW/cm2 using a NIST-
calibrated, Ocean Optics portable spectrometer optimized for short wavelength measurements (Manufacturer
Part No: USB4000). All measurements were made over the spectral range 400-420nm consistent with other
published works on this subject. The average irradiance was calculated to be 0.525 mW/cm2 and was used for
all calculations during the experiment. This level of irradiance is typically used in a clinical setting. Agar plates
(100 mm diameter) and stainless steel coupons (1 in x 3 in) were exposed to an average irradiance of 0.525
mW/cm2.
Experimental Design
Light Exposure of Bacteria Seeded onto Agar Surfaces. Dilutions of each bacteria were prepared in phosphate
buffer (PB) such that 100 µL contained approximately 200-300 CFU. Tryptic Soy Agar plates (BBL, Franklin
Lakes, NJ, USA) were inoculated with 100 µL of bacteria and spread over the surface of the agar using a sterile
spreader and then lids placed on the plates. The test plates were exposed to the light at a distance of 1.5 m for
duration of 24 hours. Non-exposed control plates were prepared for each light-exposed sample. At 24 hours,
test plates were removed and test plates and non-exposed control plates were incubated at 30-35 °C for at
least 48 hours. Upon incubation, the plates were enumerated and results reported as CFU per plate. For
varying exposure time study, the seeded plates were exposed to the light for 2, 6, 12, and 24 hours whereas E.
faecalis was exposed for 24, 48, 72 and 96 hours. Figure 1 shows an example of plates being exposed to the
visible light generated from 405-nm light emitting diodes (LEDs).
Light Exposure of Bacteria Seeded onto Stainless Steel Coupons. Dilutions of each bacteria were prepared in
phosphate buffer (PB) such that 100 µL contained approximately 1E07 CFU. Stainless steel coupons were
inoculated with 100 µL of bacteria. The test coupons were exposed to the light at a distance of 1.5 m for
durations of 4 hours and/or 24 hours. Non-exposed control coupons were prepared for each light-exposed
sample. Following exposure at 4 hours and 24 hours, coupons were suspended in Fluid D and vortexed to
recover bacteria from the coupons. Serial dilutions were prepared and plated using Tryptic Soy Agar. The test
plates and non-exposed control plates were incubated at 30-35 °C for at least 48 hours. Upon incubation, the
plates were enumerated and results reported as CFU per coupon.

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 Figure 1. Indigo-Clean™ light fixture and experimental set up for light exposure
of bacteria seeded onto agar surfaces.

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RESULTS
Figure 2 shows the bactericidal effect of visible light onto an agar surface seeded with 6 different ESKAPE
pathogens after an exposure period of 24 hours.

Figure 2. Inactivation of various ESKAPE pathogens in a nutrious media after 24 hours of

exposure to Indigo-Clean. This test showed the potential level of disinfection that could be
achieved in a clinical setting and was used to determine the sampling interval for subsequent
measurements.
In order to understand the inactivation for shorter (or longer) time periods, a more detailed investigation
included the exposure of agar plates seeded with 5 of the 6 ESKAPE pathogens for 2-24 hours as shown in
Figure 3. A separate investigation of the enterococcus faecalis organism was performed due to the low level of
inactivation achieved during the 24-hour exposure.

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 Figure 3. Inactivation of various ESKAPE pathogens in a nutrious media as a function of time.
Note that 5 of the 6 organisms show a greater than 90% reduction after 6 hours of exposure to
an irradiance typically found in a clinical setting.
While these measurements are instructive, they do not represent the full range of clinical conditions than an
organism could experience. While is impossible to re-create all of these conditions in a laboratory setting, one
can get a better indication of how Indigo-Clean would perform in a clinical environment by seeding bacteria
onto surface types found in a clinical environment and exposing them to the disinfecting light. One example of
this is stainless steel. Figure 4 below shows the results of this effort, similar to the measurements shown in
Figure 2.

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 Figure 4. Inactivation of various ESKAPE pathogens on a stainless steel surface after 24 hours
of exposure to Indigo-Clean. This test showed the potential level of disinfection that could be
achieved in a clinical setting.

To better illustrate the effect that the surface has upon the disinfection, Figure 5 compares the inactivation of
the bacteria studied in agar and on a stainless steel surface.

Figure 5. Comparison of inactivation achieved for various organisms seeded onto agar and
stainless steel after 24 hours of exposure to Indigo-Clean

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DISCUSSION
This study demonstrated the bactericidal effect of 405 nm visible light on the variety of Gram positive and
Gram negative organisms when seeded onto different surfaces. E. aerogenes, S. aureus, and K. pneumoniae
showed a greater than 99% reduction on the agar surface while A. baumannii and P. aeruginosa showed an
approximately 90% reduction. E. faecalis was the most resistant of the group showing negligible or least
reduction on the agar surface. Comparison of these results with those for a normal disinfectant are challenging
because narrow-spectrum light (Indigo-Clean) operates continuously, 24/7. As shown in Figure 6 below, a
normal disinfectant operates episodically, killing a high proportion of bacteria, but only during the time it is
applied. After cleaning, bacteria begin to quickly repopulate the space. Thus, a more objective frame of
reference in which to compare the two solutions is the total amount of bacteria killed between routine
cleanings (approx. 24 hours).

Figure 6. Comparison of continuous and episodic disinfection. Note that the best method of
comparison between these two types of disinfectants is the total number of bacteria killed
over a normal, daily cycle.
High levels of inactivation (> 95%) were obtained on stainless steel for E. aerogenes, S. aureus, P. aeruginosa. A.
baumannii and E. faecalis showed substantial inactivation (> 70%) on this surface as well. K. pneumoniae
showed the lowest level of inactivation on this surface (~40%).
In general, neither the Gram-positive bacteria nor the Gram-negative bacteria showed any preferential level of
inactivation on either agar or stainless steel. Any comparison is complicated by the natural die-off of organisms
on the stainless steel surface itself, forcing a much higher inoculum to be used. This could, in turn, create a
self-shielding effect from the narrow-spectrum light (or any disinfectant for that matter) leading to differences
in observed inactivation. The differences in activation for the various organisms on each surface type could be
explained by this hypothesis and additionally, the differences in how each organism reacts to the difference
between a nutrious media and a clinical surface.
In addition to bactericidal effect against vegetative cells, the effect of light-inducing inactivation of Bacillus and
Clostridium spores has been investigated3,4 and showed that although bacterial spores were sensitive to light
inactivation, such reduction in spore population required a higher dose of light compared to that required for
inactivation of vegetative cells. In other studies, inactivation of yeasts and molds due to lethal effects of light
at 405-nm has also been investigated5 with similarly positive results.

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CONCLUSIONS
Indigo-Clean can be used to achieve significant, continuous inactivation of pathogenic organisms on both
nutrious media and clinically relevant surfaces. These results are consistent with those previously obtained and
suggest that the clinical performance of the product would be consistent with that found in previous clinical
studies6,7.

ACKNOWLEDGEMENTS
The author wishes to thank Dr. Manish Parekh, Trisha Pankratz and Anna Chowaniec of SGS Chicago for
designing and performing the inactivation studies and useful discussions during analysis and interpretation of
the data.

REFERENCES
1. Marchetti, A., and R. Rossiter, “Economic burden of health-care associated infection in US acute care
hospitals: societal perspective”, J. Med. Econ. 2013;16:1399-1404.
2. Maclean, M., S. J. MacGregor, J. G. Anderson, and G. Woolsey, “Inactivation of Bacterial Pathogens
following Exposure to Light from a 405-Nanometer Light-Emitting Diode Array”, Appl. Environ.
Microbiol 2012; 75:1932-1937.
3. Maclean, M., L. E. Murdoch, S. J. MacGregor, and J. G. Anderson, “Sporicidal Effects of High-Intensity
405 nm Visible Light on Endospore-Forming Bacteria”, Photochem. and Photobio. 2012; 88:1280-
1286.
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405 nm Light Exposure Demonstrated by Inactivation of Escherichia, Salmonella, Shigella, Listeria, and
Mycobacterium Species in Liquid Suspensions and on Exposed Surfaces”, Sci. World J. 2012; 2012:1-8.
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Watson, B. Thakker, G. Gettinby, “Environmental decontamination of a hospital isolation room using
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Hosp. Inf. 2014; 88:1-11.

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