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Published in final edited form as:
Radiat Res. 2017 April ; 187(4): 483–491. doi:10.1667/RR0010CC.1.

Germicidal Efficacy and Mammalian Skin Safety of 222-nm UVLightManuela Buonannoa, Brian Ponnaiyaa, David Welcha, Milda Stanislauskasb, Gerhard
Randers-Pehrsona, Lubomir Smilenova, Franklin D. Lowyc, David M. Owensb,d, and David J.
Brennera,1aCenter for Radiological Research, Columbia University Medical Center, New York, New York

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bDepartment of Dermatology, Columbia University Medical Center, New York, New YorkcDepartment of Medicine and Infectious Diseases, Columbia University Medical Center, New

York, New YorkdDepartment of Pathology and Cell Biology, Columbia University Medical Center, New York, NewYork

Abstract

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We have previously shown that 207-nm ultraviolet (UV) light has similar antimicrobial properties
as typical germicidal UV light (254 nm), but without inducing mammalian skin damage. The
biophysical rationale is based on the limited penetration distance of 207-nm light in biologicalsamples (e.g. stratum corneum) compared with that of 254-nm light. Here we extended ourprevious studies to 222-nm light and tested the hypothesis that there exists a narrow wavelength
window in the far-UVC region, from around 200–222 nm, which is significantly harmful tobacteria, but without damaging cells in tissues. We used a krypton-chlorine (Kr-Cl) excimer lampthat produces 222-nm UV light with a bandpass filter to remove the lower- and higher-wavelengthcomponents. Relative to respective controls, we measured: 1. in vitro killing of methicillinresistant Staphylococcus aureus (MRSA) as a function of UV fluence; 2. yields of the main UVassociated premutagenic DNA lesions (cyclobutane pyrimidine dimers and 6-4 photoproducts) in a
3D human skin tissue model in vitro; 3. eight cellular and molecular skin damage endpoints in
exposed hairless mice in vivo. Comparisons were made with results from a conventional 254-nm
UV germicidal lamp used as positive control. We found that 222-nm light kills MRSA efficiently
but, unlike conventional germicidal UV lamps (254 nm), it produces almost no premutagenic UVassociated DNA lesions in a 3D human skin model and it is not cytotoxic to exposed mammalianskin. As predicted by biophysical considerations and in agreement with our previous findings, farUVC light in the range of 200–222 nm kills bacteria efficiently regardless of their drug-resistantproficiency, but without the skin damaging effects associated with conventional germicidal UV
exposure.

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Address for correspondence: Center for Radiological Research, Columbia University Medical Center, 630 West 168th St., New York,NY 10032; djb3@cumc.columbia.edu.

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INTRODUCTION
The use of ultraviolet (UV) light for inactivating bacteria and viruses is well established (1,2). However, UV radiations emitted by typical germicidal lamps with a peak emission at 254
nm represent a human health hazard, causing skin cancer (3, 4) and cataracts (5, 6).

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We have developed an approach to kill bacteria without harming human cells in skin tissue
models (7) and mouse skin in vivo (8) that employs single-wavelength UVC light generated
by inexpensive filtered excilamps (9). The approach is based on the limited penetration
distance of UVC light in the wavelength range of 200–222 nm in biological samples.
Specifically, while far-UVC light has enough range to traverse microbes that are muchsmaller in size than human cells [less than 1 μm in diameter (10, 11), compared to thediameter of typical human cells ranging from about 10–25 μm (10)], it is strongly absorbedby the proteins in the cytoplasm of human cells (12, 13) and is drastically attenuated before
reaching the human cell nucleus. It follows that far-UVC light is not able to penetrate thestratum corneum of skin and reach the underlying critical basal cells or melanocytes (4).
Another organ especially sensitive to UV damage is the lens; however, the lens is positioned
distal to the cornea that is sufficiently thick [~500 μm (14))]. Therefore, penetration of farUVC ~200-nm light through the cornea to the lens is predicted to be essentially zero (15).

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The potential use of UVC light for microbe sterilization purposes in the presence of humans
paves the way to numerous clinical applications, including reduction of surgical site
infections (SSI) that are the second most common healthcare-associated infections resulting
in read-missions, prolonged hospital stays, increased morbidity and mortality, and an overall
higher medical cost (16, 17). A key factor contributing to the severity of SSI is the incidenceof drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) (18,19).
To address the issue of reducing SSI, we have developed an approach that involves the use of
inexpensive excimer lamps that, appropriately filtered, emit monoenergetic wavelengths in
the far-UVC range. A crucial property of UVC-mediated germicidal killing is that it isessentially independent of acquired drug resistance (20, 21).

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We have previously shown that 207-nm light emitted by a filtered krypton-bromine (Kr-Br)
excilamp has bactericidal efficacy while being minimally cytotoxic to human cells in a 3D
skin tissue model in vitro (7) and in a hairless mouse skin model in vivo (8). Thus,
continuous exposure of the wound to far-UVC light during surgery may inactivate themicrobes alighting directly onto the surgical wound from the air. If proven to be safe to eyes
as well as skin, continuous operation of far-UVC light would not require the use of
cumbersome protective clothing, hoods and eye shields for the surgical staff and the patient(22, 23).
Here we extended those studies to a filtered krypton-chlorine (Kr-Cl) excimer lamp that
produces essentially monoenergetic UV light at 222 nm and established that a wavelength
window exist in the far-UVC region (from 200–222 nm) that inactivates bacteria efficiently
but is not cytotoxic or mutagenic to mammalian cells.

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We describe measurements of MRSA survival and of typical UV-induced premutagenic
DNA lesions in a 3D human skin model immediately after exposure to different fluences of
222-nm light. We compared the results to those measured in samples exposed to similar
fluences from a typical germicidal lamp emitting at 254 nm.
We further tested eight cellular and molecular endpoints relevant to skin damage in vivo indorsal skin of hairless mice 48 h after exposure to 222-nm or 254-nm light used as positive
control (8), at a fluence at which the 254-nm light is predicted to produce a significant
decrease in SSI rates (22).
In agreement with our previous results using 207-nm light (7, 8), here we showed that 222nm light has similar antimicrobial properties as a conventional germicidal lamp but withoutcausing mammalian skin damage.

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The finding that a far-UVC wavelength window (200–225 nm) is differentially cytotoxic tobacteria relative to mammalian cells is novel and can be used for various applications thatwould require the presence of humans, including room sterilization and reduction of surgicalsite infections, without the need of additional personal protective equipment.

MATERIALS AND METHODSUV Lamps

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We used an excimer lamp based on a krypton-chlorine (Kr-Cl) gas mixture that emits
principally at 222 nm. The lamp (High Current Electronics Institute, Tomsk, Russia) was aircooled with a 6,000-mm2 exit window (24). A custom bandpass filter (Omega Optical,
Brattleboro, VT) was used to remove essentially all but the dominant 222-nm wavelength
emission.
A UV spectrometer (Photon Control, BC, Canada) sensitive in the wavelength range from
200–360 nm was used to characterize the wavelength spectra emitted by the excimer lamp,
and a deuterium lamp standard with a NIST-traceable spectral irradiance (Newport Corp,
Stratford, CT) was used to calibrate the UV spectrometer.

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Studies were also carried out with a conventional mercury germicidal lamp (Hygeaire,
Atlantic Ultraviolet Corporation, Hauppauge, NY) with peak emission at 254 nm and usedas positive control. An ILT1400 sensor equipped with a SEL220 detector (International
Light Technologies, Peabody, MA) was used to measure the fluence rate from both lamps.
For acute exposures (MRSA and 3D tissues), the 222- and 254-nm lamps were positioned 9
and 99 cm, respectively, from the sample at which corresponded a power density of 0.036
mJ/cm2. For chronic exposures of mice, delivery of 157 mJ/cm2 in a 7 h period was obtainedby locating the 222- and the 254-nm lamps 41 and 205 cm from the samples, respectively;
the corresponding power densities were 0.0062 mJ/cm2 for the 222-nm lamp and 0.0008
mJ/cm2 in the case of the conventional germicidal lamp.
Figure 1 shows the measured spectra emitted from the Kr-Cl excimer lamp that, together
with the main excimer emission at 222 nm, also includes lower fluences of higher
wavelength light (i.e. ~237 and ~258 nm). To remove these more penetrating higher

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wavelengths and therefore potentially more harmful to human cells, we used a custom
bandpass filter (Omega Optical, Brattleboro, VT). As shown in the inset in Fig. 1, thefiltered lamp effectively emitted only the characteristic single wavelength of 222 nm.

IN VITRO STUDIESMRSA Cell Survival
We used methicillin-resistant S. aureus (MRSA USA300, multilocus sequence type 8, clonal
complex 8, staphylococcal cassette chromosome mec type IV), which is a major cause ofboth community and nosocomial infections (25–27). Survival of MRSA as a function of
UVC fluence was assessed immediately after exposure with the standard colony forming
unit (CFU) assay, as previously reported (7).

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UV-Associated Premutagenic DNA Lesions in a 3D Human Skin Model
We used a 3D human skin model EpiDerm-FT (MatTek Corp., Ashland, MA) consisting ofstratum corneum and 8–12 human cell layers to reproduce human epidermis and dermis(28). We measured induction of the two most abundant premutagenic DNA lesions in theepidermis, cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone 6-4
photoproducts (6-4PP) (4), as a function of UVC fluence in 3D human skin constructs
immediately after exposure using the immunohistochemical approach previously described(7).

IN VIVO STUDIES
Mouse Irradiations

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We used six- to eight-week-old male hairless mice (SKH1-Elite strain 477, Charles River
Laboratories, Stone Ridge, NY); the strain has UV action spectra for histological, physicaland visible changes similar to those of human skin (29, 30). Moreover, the typical thicknessof the stratum corneum of SKH1 mice (5–10 μm) (31, 32) is comparable to that of humanskin (5–20 μm) (33).
One group of three mice was exposed to fluence of 157 mJ/cm2 from 222 nm light delivered
in a 7 h period by a filtered Kr-Cl excimer lamp while another group of three mice was shamirradiated to zero UV fluence. Mice exposed to 157 mJ/cm2 delivered in 7 h period from 254
nm light represented the positive controls (8).
All animal procedures were carried out in accordance with federal guidelines and protocols
approved by the Columbia University Medical Center IACUC.

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Mouse Skin Safety-Specific Endpoints
Following the same protocols described in our previous mouse skin safety study (8), 48 h
after UV exposure we measured the following endpoints: 1. Epidermal thickness measured
in hematoxylin and eosin stained samples; 2. The percentage of proliferating keratinocytes
expressing the Ki-67 antigen (34, 35). 3. Yields of UV-induced cyclobutane pyrimidine
dimers (CPD) and 6-4 photoproducts (6-4PP); 4. The number of neutrophils and mast cells

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as markers of skin inflammation (36); 5. The expression of keratin (K) K6A as marker ofskin differentiation (37, 38).
Tissues were examined with an Olympus IX70 microscope equipped with a Photometrics®
PVCAM high-resolution, high-efficiency digital camera; Image-Pro Plus 6.0 software andFiji/Image J software were used to analyze the images. For each mouse, each endpoint wasmeasured in at least six randomly selected fields of view.
Statistical Analysis
Comparisons of mean values between treatment groups and controls were performed using
Student’s t test, and comparison of proportions were assessed with standard χ2 tests.

IN VITRO RESULTS
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MRSA Survival
We measured MRSA survival immediately after exposure to different fluences of 222-nm
light generated by a filtered Kr-Cl excimer lamp (Fig. 2). We compared the results tosurvival of MRSA exposed to similar fluences from a conventional germicidal UV lamp
(254 nm) (7). As illustrated in Fig. 2, at relatively high fluences at which the 254-nm light ispredicted to produce a significant decrease in SSI rates (1), 222-nm light kills MRSA almost
as efficiently as a conventional germicidal UV lamp. However, 254-nm light is almost
equally efficient at killing human cells (7).
Induction of Premutagenic DNA Lesions in Human Skin

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Figure 3 shows the comparison of the induced yields of CPD (left panel) and 6-4PP (rightpanel) in 3D skin tissue models exposed to 222-nm or 254-nm light. Unlike conventional
germicidal light (254 nm), exposure to the 222-nm light at any of the studied fluences did
not induce yields of either lesions that was significantly higher than the sham-irradiatedsamples.

IN VIVO RESULTS
Epidermal Thickness and Keratinocyte Proliferation

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At 48 h after exposure, fixed dorsal skin sections were stained with hematoxylin and eosin(H&E) for analysis of epidermal thickness (Fig. 4A). As shown by the typical H&E-stained
cross-sections of dorsal skin of sham-exposed mice (top panel), of mice exposed to 157
mJ/cm2 from 254-nm light (middle panel) or 222-nm light (bottom panel), we found that,
unlike the 254-nm light, the epidermal thickness of skin of mice exposed to the 222-nm
excimer lamp was not statistically different from skin of unexposed mice (P = 0.18) (Fig. 4B
and Table 1).
Figure 4C shows representative cross-sectional images of skin samples comparing Ki-67
expression in sham-exposed mice (Fig. 4A, top panel), in skin of mice exposed to 157
mJ/cm2 from 254-nm light (Fig. 4A, middle panel) or 222-nm light (Fig. 4A, bottom panel).
We found that Ki-67 expression in keratinocytes of skin exposed to the 222-nm excimer

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lamp was not statistically different from control (P = 0.52) (Fig. 4D and Table 1). In
contrast, the same fluence from the 254-nm germicidal lamp triggered keratinocytes hyperproliferation (Ki-67 expression increased by 2.5-fold compared to controls, P < 0.0001)(Fig. 4D and Table 1); this was consistent with severe tissue hyperplasia induced by 254-nm
light as measured by a ~3-fold increase in epidermal thickness (P < 0.0001) (Fig. 4B and
Table 1).
Premutagenic UV-Associated DNA Lesions

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Figure 5A shows representative cross-sectional images of sham-irradiated mice or skin ofmice exposed to 157 mJ/cm2 from 222-nm light or 254-nm light, comparing premutagenic
skin lesions CPD (Fig. 5A, top panels) and 6-4PP (Fig. 5A, bottom panels). In agreementwith our previous findings (7, 8), we measured no increase in UV-induced DNA
photodamage in 222-nm exposed skin relative to controls (P = 0.18 for CPD and P = 0.41
for 6-4PP). On the contrary, exposure to the same fluence from a conventional germicidal
lamp (254 nm) caused a dramatic increase of both lesions relative to respective controls (P <0.0001) (Fig. 5B and C, respectively, and Table 1).
Skin Inflammation
To assess UV-induced cell inflammation, we measured the density of the markers mast cells
and neutrophyls [i.e., myeloperoxidase (MPO)-positive cells] (Fig. 6). Although 254-nm
light caused a statistically significant increase of both markers (P < 0.0005) (Fig. 6 and Table1), the density of mast cells and neutrophyls induced by the 222-nm light (Fig. 6A and B,
respectively) was not statistically distinguishable from controls (P = 0.68 and P = 0.59,
respectively).

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Skin Differentiation
We measured keratinocyte differentiation as keratin (K) 6A expression in skin of mice
exposed to 222-nm light and compared the results to K6A expression in sham-exposed miceand in mice exposed to 254-nm light used as positive control. Figure 7 illustrates typicalcross-sectional images of sham-exposed mice (Fig. 7, top panel), skin of mice exposed to
157 mJ/cm2 from 254-nm light (Fig. 7, middle panel) or from 222-nm light (Fig. 7, bottom
panel). The expression level of K6A in 222-nm exposed skin was not statistically different
from controls (P = 0.22) (Table 1). This is in contrast with a ~3-fold increase of newly
synthesized K6A in samples exposed to light from a conventional germicidal lamp (P <0.0001).

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DISCUSSION
Here we showed that 222 nm is essentially equi-effective at killing antibiotic-resistantbacteria as conventional germicidal UV lamps (254 nm). However, compared to the latter,
222-nm light does not induce typical UV-associated premutagenic DNA lesions in humankeratinocytes in a 3D human skin model and appears to be safe for skin of exposed hairless
mice, as assessed by eight cellular and molecular endpoints associated with damaged skin.

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The results are consistent with previous in vitro and in vivo findings using a filtered kryptonbromine lamp that emits light at 207 nm (7, 8); this suggests that there is a range of
wavelengths, specifically between 200 and 222 nm, which are equitoxic to bacteria as
typical germicidal lamps emitting at 254 nm, but without associated skin damage risks.
The lack of induced damage is due to the limited penetration of far-UVC light in biologicalsamples (39): while light in the 200-222 nm region can traverse microbes that are muchsmaller in size (<1 μm) (10, 11, 40) than a typical mammalian cell (~10–25 μm in diameter)(10), it cannot penetrate mammalian cells cytoplasm (7) as well as all tissues with a stratum
corneum (8).

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A central application of our approach is reduction of SSI, which still represent a majorcomplication of surgical procedures (41). Current evidence suggests that the majority of SSI
result from bacteria alighting directly onto the surgical wound from the air (22, 42–44).
Based on our previous studies (7, 8) and on the preclinical results reported here, lamps
emitting far-UVC light in the 200–222 nm range could potentially be used for continuous
low-fluence/low-rate exposures during the course of a surgical procedure to inactivate
bacteria before they penetrate into the interior of the wound. At bactericidal fluences, farUVC light cannot traverse tissues with a stratum corneum such as skin and lens of the eyes.
The use of this light would therefore not require additional protective clothing for patients ormedical staff and could become a standard in hospital environments to reduce SSI rates,
particularly those due to drug-resistant pathogens. In addition, targeting bacteria as theyalight onto the wound would prevent the formation of bacterial clusters or biofilms (45),which are difficult to eradicate and impede wound healing (46).

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Other potential applications of far-UVC light is sterilization of any environment with a highlikelihood of airborne-based pathogen transmission, including tuberculosis, small pox,
severe acute respiratory syndrome (SARS) and pandemic influenza, which collectively
affects one billion people annually (47). Although upper-room UV-irradiation systems basedon conventional broad-spectrum UV lamps (48) have long been considered for roomsterilization (49–51), they cannot be widely used due to safety concerns relating to skincancer and cataract risks (49, 52, 53).
Collectively, our studies suggest that far-UVC light (200–225 nm), unlike conventional UV
germicidal lamps, has considerable promise to be a safe and inexpensive modality for SSI
reduction, while being cytotoxic to both drug-resistant and drug-sensitive microbes (22).

Acknowledgments
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We are very grateful to Lynn Shostack for her support and advice. We thank Ms. Elizabeth Flores of the Departmentof Medicine and Infectious Disease at Columbia University for her expertise and for the use of the laboratory
equipment. We thank Drs. Alan W. Bigelow and Yanping Xu for their insight in the UV dosimetry. GR-P receives
royalty payments from Ushio Inc. (Tokyo, Japan) pursuant to exclusive license and research agreements with
Columbia University.

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FIG. 1.

Nonfiltered and filtered measured emission spectra from a Kr-Cl excimer lamp (main peak
222 nm).

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FIG. 2.

Bacterial cell killing induced by 222- and 254-nm UV light. Killing of MRSA (strain
USA300) is shown relative to zero-fluence controls, expressed as surviving fraction or as
logs of cell kill (–log10[surviving fraction]), produced by different fluences of UV lightfrom a filtered 222-nm Kr-Cl excimer lamp (▲) or a germicidal 254-nm lamp (●).

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FIG. 3.

Premutagenic skin DNA lesion yields induced by 222- and 254-nm UV light. Yields ofcyclobutane pyrimidine dimers (CPD) (left panel) and pyrimidine-pyrimidone 6-4
photoproducts (6-4PP) (right panel) in keratinocytes in a 3D human skin tissue model,
induced by conventional germicidal UV radiation (●) and by 222-nm UV light (▲). In bothgraphs zero-fluence control measurements (<1%) have been subtracted from the data.

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FIG. 4.

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Epidermal thickness and keratinocyte proliferation in mouse skin exposed to UVC light.
Panel A: Representative cross-sectional images of H&E-stained mouse dorsal skincomparing the epidermal thickness in sham-exposed mice (top panel), in mice exposed to
157 mJ/cm2 from 254-nm light (middle panel) or from 222-nm light (bottom panel). Panel
B: Quantification of epidermal thickness. Panel C: Ki-67-positive keratinocytes (darkbrown-stained cells) in typical cross-sections of skin of sham-exposed mice (top panel), of
mice exposed to 254-nm light (middle panel) or to 222-nm light (bottom panel). Panel D:
Quantification of the percentage of keratinocytes expressing Ki-67 antigen. Values represent
the average ± SD of epidermal thickness measured in nine randomly selected fields of view
per mouse (n = 3). *Values from elsewhere (8).

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FIG. 5.

UVC-induced premutagenic DNA lesions in mouse skin. Panel A: Representative crosssectional images of dorsal skin samples comparing premutagenic skin lesions CPD (top row,dark-stained cells) and 6-4PP (bottom row, dark-stained cells) in the epidermis of shamexposed mice (left column), of mice exposed to 157 mJ/cm2 from 254-nm light (middle
column) or 222-nm light (right column). Quantification of the percentage of keratinocytes
showing (panel B) CPD and (panel C) 6-4PP dimers. Values represent the average ±SD ofcells exhibiting dimers measured in nine randomly selected fields of view per mouse (n = 3).
*Value from elsewhere (8).

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FIG. 6.

UVC-induced inflammation in mouse skin. Density of (panel A) mast cells and (panel B)
cells expressing the myeloperoxidase (MPO) enzyme (i.e., neutrophils) in the epidermis ofsham-exposed mice, of mice exposed to 157 mJ/cm2 from 254-nm light or 222-nm light.
Values represent the average ±SD of the number of cells/m2 measured in six randomly
selected fields of view per mouse (n = 3). *Value from elsewhere (8).

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FIG. 7.

Tissue differentiation in UVC-exposed mouse skin. Representative cross-sectional images ofmouse dorsal epidermis expressing K6A (brown stained area) with relative quantification.
Mice were sham exposed (top panel), exposed to 157 mJ/cm2 from 254-nm light (middle
panel) or 222-nm light (bottom panel). Values represent the average ± SD of the percentage
of keratin optical density measured in nine randomly selected fields of view per mouse (n =3). *Value from elsewhere (8).

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Values from elsewhere (8).

*15.9 ± 5.7

K6A optical density (%)

0.50 ± 0.06

6-4PP positive keratinocytes (%)

24.2 ± 9.1

0.43 ± 0.01

CPD positive keratinocytes (%)

Density of MPO positive cells (number/cm2)

14.2 ± 2.6

Ki-67 positive keratinocytes (%)

28.5 ± 11.9

21.0 ± 0.8

Epidermal thickness (μm)

Density of mast cells (number/cm2)

Controls

Endpoint

18.9 ± 6.7

27.2 ± 5.7

30.6 ± 14.2

0.45 ± 0.03

0.47 ± 0.03

15.8 ± 2.7

20.1 ± 0.4

222 nm

0.22

0.59

0.68

0.41

0.18

0.52

0.18

222 nm P value

45.8 ± 6.9

202.4 ± 11.3

86.6 ± 16.8

31.1 ± 1.4

52.3 ± 5.6

36.1 ± 7.7

60.9 ± 7.4

254 nm*<0.0001<0.0005<0.0005<0.0001<0.0001<0.0001<0.0001

254 nm P value

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Summary of the Combined Results for Each Endpoint

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TABLE 1
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