APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2009, p. 2987–2990
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0099-2240/09/$08.00⫹0 doi:10.1128/AEM.02180-08
UV Light Inactivation of Bacterial Biothreat Agents䌤
L. J. Rose* and H. O’Connell
Centers for Disease Control and Prevention, Atlanta, Georgia
Received 19 September 2008/Accepted 25 February 2009
Seven species of bacterial biothreat agents were tested for susceptibility to UV light (254 nm). All gram-
negative organisms tested required <12 mJ/cm2 for a 4-log10 reduction in viability (inactivation). Tailing off
of the B. anthracis spore inactivation curves began close to the 2-log10 inactivation point, with a fluence of
approximately 40 mJ/cm2, and 3-log10 inactivation still was not achieved with a fluence of 120 mJ/cm2.
The security of our nation’s water supply is a concern for
water providers and public health officials. Questions have
been asked regarding the possibility of our drinking water
becoming contaminated with biothreat agents and the efficacy
of current disinfection practices for the reduction in viability
(inactivation) of biothreat agents (5, 8, 14). The use of UV
irradiation as a supplemental water disinfection practice is
increasing for several reasons, among them improving control
of protozoa, such as Cryptosporidium spp., and decreasing dis-
infection by-products created by chemical disinfectants (21).
This study employed a bench-scale collimated beam test to
determine the UV fluence (dose) required to inactivate seven
representative bacterial biothreat agents.
Seven species, two isolates each, from the Health and Human
Services and U.S Dept. of Agriculture lists of select agents (http://
www.selectagents.gov/resources/List%20of%20Select%20Agents
%20and%20Toxins_111708.pdf) were used in this study: Bacillus
anthracis Ames spores, B. anthracis 34F2 (Sterne) spores, Bru-
cella melitensis ATCC 23456, B. melitensis IL195, Brucella suis
KS528, B. suis MO562, Burkholderia mallei M9, B. mallei M13,
Burkholderia pseudomallei ATCC 11688, B. pseudomallei
CA650, Francisella tularensis LVS, F. tularensis NY98, Yersinia
pestis A1122, and Y. pestis Harbin. B. anthracis was grown on
soil extract-peptone-beef extract agar (SEA) (1) or in Schaef-
fer’s sporulation medium (SSM) (10) for 7 days, resulting in
⬎99% spores as determined by phase-contrast microscopy.
The cells and spores were then washed by centrifugation (8,000 ⫻
g), resuspended in ultrapure water, transferred to centrifuge
tubes, treated with 50% ethanol for 1 h at room temperature,
and washed five times with sterile ultrapure water before being
stored in reverse-osmosis water at ⫺70°C. F. tularensis isolates
were grown on cysteine heart agar (1) and all other isolates on
Trypticase soy agar with 5% sheep blood (TSA II; Becton
Dickinson Microbiology Systems, Sparks, MD) for 24 h before
testing. B. anthracis spores were adjusted to 107 CFU/ml and
other bacterial suspensions to 108 CFU/ml in Butterfield buffer
(3 mM KH2PO4, pH 7.2; Becton Dickinson Microbiology Sys-
tems), and then isolates were sonicated (40-Hz ultrasonic
cleaner; VWR, Suwanee, GA) for 1 min to disperse aggre-
* Corresponding author. Mailing address: Centers for Disease Con-
trol and Prevention, NCID/DHQP/ILB, 1600 Clifton Rd. NE, MS
C-16, Atlanta, GA 30329. Phone: (404) 639-2161. Fax: (404) 639-3822.
E-mail: lrose@cdc.gov.
䌤
Published ahead of print on 6 March 2009.
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Vol. 75, No.
gates. The suspensions were diluted 1:100 in Butterfield buffer
for final test concentrations. Five milliliters of each suspension
were placed into a small petri dish (50-mm diameter) along
with a small sterile stir bar, and the petri dish was placed on a
stir plate.
UV irradiation was performed by using a collimated beam
apparatus (Calgon Carbon, Pittsburgh, PA) equipped with a
low-pressure lamp (254 nm) according to the standard method
developed by Bolton and Linden (2). The surface of the sus-
pension was placed 5 cm from the end of the collimating tube.
The UV intensity was measured with a radiometer at 0.5-cm
intervals across the test area and variability compensated for
according to the UVCalc software directions (International
UV association [http://www.iuva.org]). The fluences (UV
doses) were determined using the UVCalc software, and the
petri dishes were placed under the beam for at least five time
periods to deliver a range of appropriate fluences to the or-
ganisms. Each irradiation test was conducted at room temper-
ature (23 ⫾ 2°C) in triplicate and in a random order of flu-
ences. After exposure, 10-fold serial dilutions were performed,
and the dilutions were spread plated in triplicate. Plates were
placed in a dark incubator within 10 min of plating and incu-
bated at the temperature appropriate for the organism, and the
colonies were counted at 24 h and 48 h for B. anthracis and at
3 and 5 days for the remaining organisms. Colonies were
counted, and the log10 inactivation at each fluence was deter-
mined for each organism. A linear regression of the fluence
response data determined the fluence required for 2-, 3-, and
4-log10 inactivation.
The UV fluences required for inactivation of each organism
are reported in Table 1. Little difference in UV susceptibility
was seen between the gram-negative organisms. B. suis KS528
and B. melitensis ATCC 23456 required the greatest UV flu-
ence of the gram-negative organisms for 4-log10 inactivation
(10.5 and 10.2 mJ/cm2, respectively), while the two Y. pestis
isolates required the lowest UV fluence (4.1 and 4.9 mJ/cm2)
for the same 4-log10 inactivation (Table 1). Generally, the two
isolates of each species differed no more than 3 mJ/cm2 in the
UV fluence required for 4-log10 inactivation.
The spores of the two B. anthracis isolates were more resis-
tant to UV than the gram-negative organisms tested but were
similar to each other in UV susceptibility (Fig. 1). B. anthracis
Sterne and B. anthracis Ames spores were inactivated by 90%
(1 log10), with fluences of 23.0 and 25.3 mJ/cm2, respectively. B.
anthracis spores produced on SEA and plated after UV ex-
2988 ROSE AND O’CONNELL APPL. ENVIRON. MICROBIOL.

TABLE 1. UV fluence required for given log10 inactivation of each organism

Fluence (mJ/cm2) for log10 inactivation of:
Organism
1 2 3 4

B. anthracis Ames 25.3 (5.1) ⬃40 ⬎120 (tailing off) ⬎120 (tailing off)
B. anthracis Sterne 23.0 (0.7) ⬃40 ⬎120 (tailing off) ⬎120 (tailing off)
B. suis MO562 1.7 (0.0) 3.6 (0.1) 5.6 (0.2) 7.5 (0.3)
B. suis KS528 2.7 (0.2) 5.3 (0.3) 7.9 (0.4) 10.5 (0.5)
B. melitensis ATCC 23456 2.8 (0.2) 5.3 (0.2) 7.8 (0.3) 10.3 (0.5)
B. melitensis IL195 3.7 (0.2) 5.8 (0.2) 7.8 (0.2) 9.9 (0.3)
B. pseudomallei ATCC 11688 1.7 (0.2) 3.5 (0.1) 5.5 (0.2) 7.4 (0.3)
B. pseudomallei CA650 1.4 (0.2) 2.8 (0.1) 4.3 (0.3) 5.7 (0.6)
B. mallei M-9 1.0 (0.3) 2.4 (0.2) 3.8 (0.2) 5.2 (0.3)
B. mallei M-13 1.2 (0.5) 2.7 (0.2) 4.1 (0.1) 5.5 (0.4)
F. tularensis LVS 1.3 (0.0) 3.1 (0.0) 4.8 (0.0) 6.6 (0.1)
F. tularensis NY98 1.4 (0.1) 3.8 (0.0) 6.3 (0.1) 8.7 (0.2)
Y. pestis A1122 1.4 (0.5) 2.6 (0.5) 3.7 (0.6) 4.9 (0.6)
Y. pestis Harbin 1.3 (0.1) 2.2 (0.0) 3.2 (0.1) 4.1 (0.1)
Bacillus anthracis Sternea 27.5 ⬃36 ⬃53 —d
Bacillus subtilisb 28 39 50 62
E. colic 3.0 4.8 6.7 8.4
Cryptosporidiumc 2.5 5.8 12 22
Giardiac 2.1 5.2 11 22
Virusc 58 100 143 186
a
Data from reference 12 (estimated from graph).
b
Data from reference 4.
c
Data from reference 21.
d
—, 4-log10 inactivation not achieved with a fluence of 60 mJ/cm2.

posure on TSA II required more than 40 mJ/cm2 for a
2-log10 inactivation, and further exposure to UV light did
not inactivate the sample further (Fig. 1), as seen in the
tailing off of the inactivation curve. In order to investigate
this tailing off further, B. anthracis spores produced in SSM
were also challenged with the same UV fluences and found
to require 40 mJ/cm2 for a 2-log10 inactivation as well, but
they continued to be inactivated to a slightly greater degree
than the spores produced on SEA (Fig. 1). An additional
experiment was conducted in which spores produced on
SEA were grown on two media (SSM with 1.7% agar and
TSA II) after UV exposure, and no difference in recovery
was observed (data not shown).
FIG. 1. UV inactivation curves of B. anthracis spores. B. anthracis
Sterne was grown and sporulated on SEA and SSM, and B. anthracis
Ames was grown and sporulated on SEA.
The inactivation results for Y. pestis, F. tularensis, Brucella
spp., and Burkholderia spp. reflect findings similar to those of
other waterborne pathogenic organisms, such as Escherichia
coli, Shigella sonnei, Yersinia enterocolitica, and Campylobacter
jejuni (3, 4). These reported values ranged from 1.8 to 6 mJ/
cm2 for a 3-log10 inactivation (Table 1).
Previous work established that bacterial spores are 10 to 50
times more resistant to UV at 254 nm than vegetative cells (11,
12). The DNA in spores is saturated with ␣/␤-type small acid-
soluble proteins during the sporulation process. This bound
small acid-soluble protein suppresses the formation of pyrim-
idine dimers (as seen in vegetative cells) when irradiated with
UV and instead promotes formation of a unique spore photo-
product, 5-thyminyl-5,6-dihydrothymine. During germination,
light-independent repair occurs by lyase activation of the spore
photoproduct and nucleotide excision repair, restoring the two
thymines (6, 18, 19). Variations in resistance to UV may be
attributed to differences in sporulation conditions, such as the
availability of metal ions present during sporulation, or germi-
nation conditions (10, 11, 13, 18).
The susceptibility of B. anthracis spores grown on SEA in
this study can be compared to the results found by Knudson
(6), in which a fluence of 120 mJ/cm2 was not sufficient to
achieve a 2-log10 reduction. However, Nicholson and Galeano
(12) did not observe tailing off of the disinfection curve occur-
ring after a 2-log10 reduction. We therefore produced spores in
the same manner as Nicholson and Galeano to determine if
the difference in spore preparation could account for the dif-
ferences in UV susceptibility. Though this study noted a
greater susceptibility of SSM-produced spores than SEA-pro-
duced spores, we did not see as great a reduction as did Ni-
cholson and Galeano (12) (Fig. 1). Rice and Ewell (15) also
reported tailing off of the inactivation curve in a similar study
using Bacillus subtilis spores and were unable to determine if
VOL. 75, 2009 UV INACTIVATION OF BACTERIAL BIOTHREAT AGENTS
the tailing off indicated the presence of a resistant subpopula-
tion of organisms or was an artifact of the testing protocol.
Subsequent work by Mamane-Gravetz and Linden (7) demon-
strated that the tailing off of UV inactivation curves is a result
of the presence of spore aggregates in the suspension, and the
degree of aggregation is directly related to the hydrophobicity
of the spores. The hydrophobicity of the spores used in this
study was tested in the same manner as in the study by
Mamane-Gravetz and Linden (7) and found to correlate with the
inactivation curves in Fig. 1. The SSM-produced spores were
less hydrophobic (P ⫽ 0.25) at 64.1% (standard deviation [SD],
5.6%) than the SEA-produced spores at 76.2% (SD, 2.8%),
whereas the SEA-produced Ames spores were closer (P ⫽
0.03) in hydrophobicity to the SEA-produced Sterne spores at
79.6% (SD, 3.4%). These observations agree with the previous
publication (7) in that the more hydrophobic spores tend to
aggregate together to a greater extent, shielding a greater
number of spores from exposure to UV radiation, thereby
creating a more pronounced tailing off of the inactivation
curve.
Since the finding that UV irradiation can control protozoa
much more effectively than chlorine, installation of UV tech-
nology in water treatment facilities has been on the rise, with
more than 150 treatment plants in North America currently
using the technology or planning installations in the near fu-
ture (22).
The latest Environmental Protection Agency surface water
treatment rules require drinking water systems to document their
ability to provide a 2- or 3-log10 inactivation (for unfiltered
systems) of Cryptosporidium (depending upon source water
monitoring results and treatment practices in place at the fa-
cility), a 3-log10 inactivation of Giardia, and a 4-log10 inactiva-
tion of viruses (21). No two treatment facilities are alike, but
these requirements can be met by physical removal such as
filtration, flocculation, and settling and/or by various disinfec-
tion methods such as use of chlorine, monochloramine, chlo-
rine dioxide, ozone, or UV irradiation (20, 21).
Should water be contaminated with biothreat agents up-
stream from a water treatment facility with UV capability, we
can expect a facility following Environmental Protection
Agency regulations to remove or inactivate 3 log10 Giardia spp.
and Cryptosporidium spp. and also to effectively inactivate the
gram-negative bacterial biothreat agents Y. pestis, F. tularensis,
B. mallei, B. pseudomallei, B. suis, and B. melitensis. If the
contaminating agent is B. anthracis in spore form, the facility
may not eradicate spores with UV treatment alone, requiring
cotreatment with other disinfection methods. However, it is
possible that the clumping of spores may increase the efficacy
of the facility’s coexisting available treatment, such as floccu-
lation and filtration. Further examination of these practices
would be necessary.
In the event that a biothreat agent is intentionally released
into the distribution system after water treatment, and no dis-
infectant residual (chlorine or chloramine) is provided by the
treatment facility, a point-of-use (POU) or point-of-entry
(POE) UV system may prove to be effective. NSF/ANSI stan-
dard 55 (9) establishes the requirements for two classes of
POU and POE UV systems. The class A systems, designed to
disinfect contaminated clear water, are required to deliver a
minimum UV fluence of 40 mJ/cm2. The class B systems offer
2989
supplemental reduction in pathogens and are required to de-
liver a UV fluence of 16 mJ/cm2. Both class A and B POU/
POE devices would be effective in providing a 4-log10 inacti-
vation of the gram-negative organisms tested. Only the class A
device would prove effective against B. anthracis spores pre-
pared in this manner, though only in providing 2-log10 inacti-
vation.
These data, along with previous investigations of the efficacy
of chlorine and monochloramine against bacterial biothreat
agents (16, 17), provide public health officials and water treat-
ment facility operators essential information to better prepare
for protecting public health in the event of a water contami-
nation incident.
We thank Joseph C. Carpenter, Centers for Disease Control and
Prevention, and Eugene W. Rice, U.S. Environmental Protection
Agency, for their valuable insight and advice.
The findings and conclusions in this report are those of the authors
and do not necessarily represent the official position of the Centers for
Disease Control and Prevention.
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