uv light inactivation of bacterial biothreat agents

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2009, p. 29872990 Vol. 75, No. 90099-2240/09/$08.000 doi:10.1128/AEM.02180-08

UV Light Inactivation of Bacterial Biothreat Agents

L. J. Rose* and H. OConnell

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


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posure on TSA II required more than 40 mJ/cm2 for a2-log10 inactivation, and further exposure to UV light didnot inactivate the sample further (Fig. 1), as seen in thetailing off of the inactivation curve. In order to investigatethis tailing off further, B. anthracis spores produced in SSMwere also challenged with the same UV fluences and foundto require 40 mJ/cm2 for a 2-log10 inactivation as well, but

they continued to be inactivated to a slightly greater degreethan the spores produced on SEA (Fig. 1). An additionalexperiment was conducted in which spores produced onSEA were grown on two media (SSM with 1.7% agar andTSA II) after UV exposure, and no difference in recoverywas observed (data not shown).

The inactivation results for Y. pestis, F. tularensis, Brucellaspp., and Burkholderia spp. reflect findings similar to those ofother waterborne pathogenic organisms, such as Escherichiacoli, Shigella sonnei, Yersinia enterocolitica, and Campylobacterjejuni (3, 4). These reported values ranged from 1.8 to 6 mJ/cm2 for a 3-log

10inactivation (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 boundsmall acid-soluble protein suppresses the formation of pyrim-idine dimers (as seen in vegetative cells) when irradiated withUV 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 sporephotoproduct and nucleotide excision repair, restoring the twothymines (6, 18, 19). Variations in resistance to UV may beattributed to differences in sporulation conditions, such as theavailability of metal ions present during sporulation, or germi-nation conditions (10, 11, 13, 18).

The susceptibility of B. anthracis spores grown on SEA inthis study can be compared to the results found by Knudson(6), in which a fluence of 120 mJ/cm2 was not sufficient toachieve a 2-log10 reduction. However, Nicholson and Galeano(12) did not observe tailing off of the disinfection curve occur-ring after a 2-log

10reduction. We therefore produced spores in

the same manner as Nicholson and Galeano to determine ifthe difference in spore preparation could account for the dif-ferences in UV susceptibility. Though this study noted agreater 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) alsoreported tailing off of the inactivation curve in a similar studyusing Bacillus subtilis spores and were unable to determine if

TABLE 1. UV fluence required for given log10

inactivation of each organism

OrganismFluence (mJ/cm2) for log10 inactivation of:

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 62E. colic 3.0 4.8 6.7 8.4Cryptosporidiumc 2.5 5.8 12 22Giardiac 2.1 5.2 11 22

Virus

c

58 100 143 186a 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/cm

2.

FIG. 1. UV inactivation curves of B. anthracis spores. B. anthracisSterne was grown and sporulated on SEA and SSM, and B. anthracisAmes was grown and sporulated on SEA.

2988 ROSE AND OCONNELL APPL. ENVIRON. MICROBIOL.


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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 resultof the presence of spore aggregates in the suspension, and thedegree of aggregation is directly related to the hydrophobicityof the spores. The hydrophobicity of the spores used in thisstudy was tested in the same manner as in the study byMamane-Gravetz and Linden (7) and found to correlate with theinactivation curves in Fig. 1. The SSM-produced spores wereless 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 at79.6% (SD, 3.4%). These observations agree with the previouspublication (7) in that the more hydrophobic spores tend toaggregate together to a greater extent, shielding a greaternumber of spores from exposure to UV radiation, therebycreating a more pronounced tailing off of the inactivationcurve.

Since the finding that UV irradiation can control protozoamuch more effectively than chlorine, installation of UV tech-nology in water treatment facilities has been on the rise, withmore than 150 treatment plants in North America currentlyusing the technology or planning installations in the near fu-ture (22).

The latest Environmental Protection Agency surface watertreatment rules require drinking water systems to document theirability to provide a 2- or 3-log10 inactivation (for unfilteredsystems) of Cryptosporidium (depending upon source watermonitoring results and treatment practices in place at the fa-cility), a 3-log

10inactivation ofGiardia, 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 asfiltration, 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, wecan expect a facility following Environmental ProtectionAgency regulations to remove or inactivate 3 log

10 Giardia spp.and Cryptosporidium spp. and also to effectively inactivate thegram-negative bacterial biothreat agents Y. pestis, F. tularensis,B. mallei, B. pseudomallei, B. suis, and B. melitensis. If thecontaminating agent is B. anthracis in spore form, the facilitymay not eradicate spores with UV treatment alone, requiringcotreatment with other disinfection methods. However, it ispossible that the clumping of spores may increase the efficacyof the facilitys coexisting available treatment, such as floccu-lation and filtration. Further examination of these practiceswould be necessary.

In the event that a biothreat agent is intentionally releasedinto the distribution system after water treatment, and no dis-infectant residual (chlorine or chloramine) is provided by thetreatment 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 ofPOU and POE UV systems. The class A systems, designed todisinfect contaminated clear water, are required to deliver aminimum UV fluence of 40 mJ/cm2. The class B systems offer

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 Adevice would prove effective against B. anthracis spores pre-pared in this manner, though only in providing 2-log

10inacti-

vation.These data, along with previous investigations of the efficacy

of chlorine and monochloramine against bacterial biothreatagents (16, 17), provide public health officials and water treat-ment facility operators essential information to better preparefor protecting public health in the event of a water contami-nation incident.

We thank Joseph C. Carpenter, Centers for Disease Control andPrevention, and Eugene W. Rice, U.S. Environmental ProtectionAgency, for their valuable insight and advice.

The findings and conclusions in this report are those of the authorsand do not necessarily represent the official position of the Centers forDisease Control and Prevention.

REFERENCES

1. Atlas, R. M. 1996. Handbook of microbiological media, 2nd ed., p. 1267.CRC Press, New York, NY.

2. Bolton, J. R., and K. G. Linden. 2003. Standardization of methods forfluence (U.V. dose) determined in bench-scale U.V. experiments. J. Environ.Eng. 129:20092215.

3. Butler, R. C., V. Lund, and D. A. Carlson. 1987. Susceptibility of Campy-lobacter jejuni and Yersinia enterocolitica to UV radiation. Appl. Environ.Microbiol. 53:375378.

4. Chang, J. C. H., S. F. Osoff, D. C. Lobe, M. H. Dorfman, C. M. Dumais, R. G.Qualls, and J. D. Johnson. 1985. UV inactivation of pathogenic and indica-tor microorganisms. Appl. Environ. Microbiol. 49:13611365.

5. Kahn, A. S., D. L. Swerdlow, and D. D. Juranek. 2001. Precautions againstbiological and chemical terrorism directed at food and water supplies. PublicHealth Rep. 116:314.

6. Knudson, G. B. 1986. Photoreactivation of ultraviolet irradiated plasmid-bearing and plasmid-free strains ofBacillus anthracis. Appl. Environ. Micro-biol. 52:444449.

7. Mamane-Gravetz, H., and K. G. Linden. 2005. Relationship between physio-chemical properties, aggregation and UV inactivation of isolated indigenousspores in water. J. Appl. Microbiol. 98:351363.

8. Meinhardt, P. L. 2005. Water and bioterrorism: preparing for the potentialthreat to U.S. water supplies and public health. Annu. Rev. Public Health26:213237.

9. National Sanitation Foundation. 2004. NSF/ANSI 55. Ultraviolet microbi-ological water treatment systems. National Sanitation Foundation, Ann Ar-bor, MI.

10. Nicholson, W. L., and J. F. Law. 1999. Method for purification of bacterialendospores from soils: UV resistance of natural Sonoran desert soil popu-lations of Bacillus spp. with reference to B. subtilis strain 168. J. Microbiol.Methods 35:1321.

11. Nicholson, W. L., N. Munakata, G. Horneck, H. J. Melosh, and P. Setlow.2000. Resistance of bacillus endospores to extreme terrestrial and extrater-restrial environments. Microbiol. Mol. Biol. Rev. 64:548572.

12. Nicholson, W. L., and B. Galeano. 2003. UV resistance of Bacillus an-thracis spores revisited: validation of Bacillus subtilis spores as UV sur-

rogates for spores of B. anthracis Sterne. Appl. Environ. Microbiol. 69:13271330.

13. Nicholson, W. L., and P. Setlow. 1990. Sporulation, germination and out-growth, p. 391450. In C. R. Harwood and S. M. Cutting (ed.), Molecularbiological methods for Bacillus. John Wiley & Sons, Sussex, England.

14. Nuzzo, J. B. 2006. The biological threat to U.S. water supplies: toward anational water security policy. Biosecur. Bioterror. 4:147159.

15. Rice, J. K., and M. Ewell. 2001. Examination of peak power dependence inthe UV inactivation of bacterial spores. Appl. Environ. Microbiol. 67:58305832.

16. Rose, L. J., E. W. Rice, B. J. Jensen, R. Murga, A. Peterson, R. Donlan, andM. J. Arduino. 2005. Chlorine inactivation of bacterial bioterrorism agents.Appl. Environ. Microbiol. 71:566568.

17. Rose, L. J., E. W. Rice, L. Hodges, A. Peterson, R. Donlan, and M. J.Arduino. 2007. Monochloramine inactivation of bacterial select agents.Appl. Environ. Microbiol. 73:34373439.

18. Russell, A. D. 1982. The destruction of bacterial spores. Academic Press,London, United Kingdom.

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19. Setlow, P. 2001. Resistance of spores of bacillus species to ultraviolet light.Environ. Mol. Mut. 38:97104.

20. U.S. Environmental Protection Agency. 2006. National primary drinkingwater regulations: long term 2 enhanced surface water treatment rule. Fed.Reg. 71:653702.

21. U.S. Environmental Protection Agency. 2006. Ultraviolet disinfection guid-

ance manual for the final long term 2 enhanced surface water treatment rule.EPA 815-R-06-007. U.S. Environmental Protection Agency, Office of Water,Washington, DC.

22. Wright, H., D. Gaithuma, D. Greene, and M. Aieta. 2006. Integration ofvalidation, design, and operation provides optimal implementation of UVdisinfection. J. Am. Water Works Assoc. 98:8192.

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uv light inactivation of bacterial biothreat agents

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