www.epa.gov/research technical BRIEF BUILDING A SCIENTIFIC FOUNDATION FOR SOUND ENVIRONMENTAL DECISIONS Persistence of Categories A and B Select Agents in Environmental Matrices INTRODUCTION Persistence is the ability of an organism to remain viable over time under a given environmental condition. For bacteria, viability is the ability to remain alive, whereas for viruses, it is the ability to remain infectious. Understanding a biological agent's ability to persist in the environment would help authorities to properly assess and respond in the event of the agent's release. The purpose of this brief is to summarize persistence data documented in an EPA literature review (U.S. EPA, 2014a) entitled Persistence of Categories A and B Select Agents in Environmental Matrices (EPA/600/R-14/0741 Category A biological agents cause high rates of mortality and are easily disseminated or easily transmitted from person to person. Category B agents cause illness with low mortality and are moderately easy to disseminate. Assessment of persistence data is important to (1) allow informed emergency response and remediation decisions following a contamination incident, and (2) identify gaps in the current state of the science so research can be focused toward closing these gaps. This brief provides a short summary of the open literature findings published to date, specifically for biological agent persistence. These data cover numerous agents, as well as various environmental conditions and surface types. This brief reports the longest duration of persistence, which is from the time of inoculation to the last sampling interval that produced viable organisms. Several studies, however, did not continue until organism could no longer be cultured; in those cases, the pathogen's actual duration of persistence could not be determined. "Biological agent means any microorganism (including, but not limited to, bacteria, viruses, fungi, rickettsiae, or protozoa), or infectious substance, or any naturally occurring, bioengineered, or synthesized component of any such microorganism or infectious substance, capable of causing death, disease, or other biological malfunction in a human, an animal, a plant, or another living organism; deterioration of food, water, equipment, supplies, or material of any kind; or deleterious alteration of the environment." 42 Code of Federal Regulations Part 73 August 2015 EPA/600/S-15/218 ------- CONDITIONS AFFECTING PERSISTENCE The literature review documents the duration of persistence of biological agents and provides information on environmental conditions and species interactions that either favor or hinder persistence. The environmental media considered were aerosol, soil, water, orfomite (an object, surface, or substance that can transmit pathogens). The review also identified the gaps in the scientific literature that impede a thorough understanding of the persistence of these pathogens. This summary, based on the literature review, describes what is known and what is unknown about these conditions and interactions. As noted below, some species enter into a state where they are metabolically active, yet not culturable, with unknown viability. Ducret et al., 2014, concluded that the physiological significance of the state is unknown and controversial; it could be an adaptive state favoring long-term survival, it could be a deteriorating state leading to death, or it could be an injured state that prevents organisms from being cultured. The ability of an agent to subsequently cause infection or induce human disease following release into the environment was beyond the scope of the review. The importance of this metabolic state is an area in need of additional research. Bacillus anthracis B. anthracis is a spore-forming bacterial Category-A select agent, the causative agent of the disease anthrax. Under appropriate conditions, B. anthracis can form protective biofilms on glass and polyvinyl surfaces covered with nutrient media. B. anthracis strains involved in gut colonization have also formed biofilms. However, conditions and the overall persistence of the formed biofilms have yet to be explored. B. anthracis spores can persist in soil for many years. Many symbiotic relationships between Bacillus species and soil-borne organisms have been reported in the literature. B. anthracis has been found to geminate in amoeba, and to colonize the hindgut of earthworms. There is some evidence that the interaction between earthworms and B. anthracis might be dependent on the presence of bacteriophages. It is unknown whether the colonies formed in the hindgut of earthworms and the spores that germinate in amoeba can result in increased numbers of spores in the environment or if these spores would be virulent. The B. anthracis lifecycle and the earthworm lifecycle in the soil have similarities. Both prefer alkaline, calcium rich, organic soils. In addition, anthrax outbreaks often occur following flooding, which is also when earthworms tend to migrate to the surface. The post-flooding anthrax outbreaks could be due to B. anthracis spores being carried upward to the soil surface and onto vegetation via colonized earthworm digestive systems. (For more information on the persistence of B. anthracis spores in soil, see U.S. EPA, 2014b, entitled Literature Review on Mechanisms that Affect Persistence of Bacillus anthracis in Soils.) ------- The literature search included persistence on surfaces, but little data on persistence on surfaces was found. Testing on fomites was not conducted at environmental conditions <25°C, <80% RH, or with simulated sunlight. Virulent B. anthracis contains two plasmids, pX01 and pX02. The pX01 plasmid has been found to be required for spore germination within the amoeba. For strains lacking pX01, bacteriophage infection of B. anthracis could restore functional gene activity necessary to survive and replicate in earthworms, rhizosphere, biofilms, and soil. Researchers have hypothesized that pX02 is lacking in multiple natural B. anthracis strains because it is not required for proliferation. However, there is little information in the literature on conditions that promote or inhibit proliferation. Some studies have found that spores can germinate and propagate in the rhizosphere of a common pasture grass, but do not germinate without the grass. Conflicting studies suggest that presence of grass does not increase survival or multiplication of B. anthracis in soil, but serves to promote transmission of B. anthracis to grazing hosts by attracting more hosts to the area. Further research is needed to determine if B. anthracis can take up the pX02 plasmid in the environment and thus regain virulence. Brucella species Brucella species are non-spore-forming bacterial Category-B select agents, which cause the disease brucellosis. Brucella species can enter a state where they are metabolically active, yet not culturable, with unknown viability. Brucella melitensis is typically associated with goats, Brucella suis is associated with swine, and Brucella abortus is often associated with cattle. All three species can infect humans. The persistence of B. melitensis and B. suis in water (<9 days at 5°C and 25°C) was considerably lower than the persistence of B. abortus in water (>57 days at 8°C and 20°C). It is uncertain if the difference is attributed to species-level differences or the study parameters. Under certain conditions (e.g., nutritionally deficient, low oxygen) B. abortus was found to aggregate and form biofilms, which can enhance the bacteria's tolerance to desiccation. 6. abortus appeared to survive better in moist soil than dry soil (66 days in wet soil compared to <4 days in dried soil). Brucella species can survive for several weeks in water, but the bacteria are sensitive to heat, with < I day survival at 37° C in lake water. Brucella species also appear to be adversely affected by exposure to sunlight. No data on the survival of Brucella species in aerosols was found during this review. Research is needed on the mechanisms that contribute to the environmental persistence of Brucella species including species-specific data. ------- Burkholderia mallei B. mallei is a non-spore-forming non-motile bacterial category-B select agent, which causes the disease glanders. B. mallei can enter a state where they are metabolically active, yet not cultivable, with unknown viability. B. mallei is primarily associated with horses and is not expected to survive outside a host for long durations. However, B. mallei "has an affinity for warm and moist conditions and may survive for up to 3 months in stable bedding, manure, feed and water troughs (particularly if heated), wastewater and equine transporters. Although three persistence studies were identified with B. mallei in water, the results were somewhat conflicting. No data on the survival of B. mallei in aerosols or on soil was found during this review. Information on B. mallei persistence on fomites found during this review was not supported with specific data or laboratory controlled studies. Burkholderia pseudomallei B. pseudomallei is a non-spore-forming motile bacterial category-B select agent, which causes the disease melioidosis. B. pseudomallei can enter a state where they are metabolically active, yet not culturable, with unknown viability. B. pseudomallei is associated with water and with soil. B. pseudomallei occurs in tropical and sub-tropical climates and is associated with decaying organic matter in the environment. B. pseudomallei can grow in anoxic (oxygen deficient) environments. Interactions with other organisms can influence persistence. B. pseudomallei is associated with the rhizosphere, roots, and above ground parts of various grasses, especially non-native grasses introduced for grazing animals. The relationship between B. pseudomallei and vegetation is not well understood. Other soil bacteria (e.g., Burkholderia multivorans) could inhibit B. pseudomallei growth. On the other hand, B. pseudomallei can form biofilms and survive in amoeba cysts and fungi. Survival in various media is not well understood. B. pseudomallei can be transported by aerosols and has been isolated from aerator spray associated with a water treatment plant. However, no data on the survival durations of B. pseudomallei in aerosols was found during this review. Only one study investigated the persistence of B. pseudomallei on fomites. However, the influence of different environmental conditions (temperature and humidity) on survival on fomites was not investigated. Environmental factors dictating the occurrence of 6. pseudomallei in soil and water are not well established. Similarly, survival in soil and the effect of sunlight is not well understood. Subsurface samples (e.g., at a 25-60 centimeter [cm] depth) are more likely to yield B. pseudomallei than surface soil. B. pseudomallei is infrequently found in surface soil, possibly because the organism might be adversely affected by sunlight. However, the review found no definitive studies on the effects of sunlight on B. pseudomallei persistence. It is known that B. pseudomallei were killed ------- by radiation from an UV lamp (465 microwatt [|jW]/square centimeter [cm2] for 7.75 minutes), but radiation associated with natural sunlight can be absorbed by other materials thereby limiting sunlight's killing effect. Other factors also affect persistence in soils. Optimal temperatures for B. pseudomallei growth in soil are 37°C to 42°C, although some strains were able to grow at 4°C. Optimal soil pH was 6.5 to 7.5. B. pseudomallei appears to benefit from moist soil, but can persist in soil that gradually dries. B. pseudomallei was not recovered following inoculation into dry soil, but was recovered from dry soil in farmed areas suggesting that factors other than moisture are needed to support B. pseudomallei growth. Under conditions of low water content, organic matter in the soil might enable B. pseudomallei to survive. Coxiella burnetii C. burnetii is an obligate intracellular bacterial category-B select agent, which causes Q fever. Commonly, barnyard dust from infected cattle, sheep, and goats transmits the disease to humans. C. burnetii can be aerosolized and transported by wind long distances. C. burnetii is not known to be strongly affected by high or low temperatures, drought, or humidity levels. No data on the persistence of C. burnetii in water was found during this review. There are a number of environmental reservoirs that could aid in the persistence and transmission of C. burnetii. C. burnetii can invade a variety of hosts including amoebae, ticks, birds, and mammals. It appears likely that these hosts facilitate the dissemination of the bacterium throughout the environment. C. burnetii was able to survive within amoeba for 6 weeks; soil amoeba could provide an intracellular niche for the survival of C. burnetii in a spore-like form. Epidemiological evidence suggests that C. burnetii may be carried over considerable distances on fomites. There are uncertainties and unknowns in the information on persistence for C. burnetii. The bacterium may exist as "large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC)" physiological forms, with SCV and SDC being the environmentally persistent forms. Persistence might vary by the form of C. burnetii excreted from the host, but the specific forms excreted in milk, feces or placentas are unknown. In addition, much of the persistence data of C. burnetii (in aerosol and fomites studies, and in some soil studies) were based on environmental sampling associated with Q fever outbreaks and viability was not assessed. Also, the studies were not conducted in controlled laboratory settings, so recontamination/cross-contamination could have occurred. Francisella tularensis F. tularensis is a non-spore-forming bacterial category-A select agent, which causes tularemia, also called rabbit fever. F. tularensis can enter into a state where they are metabolically active, yet not culturable, with unknown viability. F. tularensis can infect mammals, arthropods, and ------- protozoans. Virulent strains of F. tularensis were shown to survive in the cysts of Acanthamoeba castellanii for at least 3 weeks post-infection. F. tularensis can survive in diverse environments, but the mechanism by which F. tularensis persists or establishes an environmental reservoir following a release is unknown. F. tularensis can survive and reproduce in water for relatively long periods of time (including waters with high nutrient levels and protozoan predation), although virulence may be lost. F. tularensis survived longer in brackish water than fresh water. The salt and sulfur content of the brackish water may have contributed survival; sulfur-containing amino acids, cysteine or cystine, are usually required for the cultivation of F. tularensis. Sodium chloride also enhances F. tularensis culture growth. By forming a biofilm in natural ecosystems, Francisella might be able to survive the environmental conditions of mud and waterways, and forming a biofilm could be a mechanism for persistence within the ticks. F. tularensis subspecies holarctica, which is found more widely in the northern hemisphere, is associated with water-borne disease and is transmitted by mosquitoes, ticks, and biting flies. No information is available on its persistence in soil. Unidentified constituents in the air (possibly olefins from oil refineries and dense car populations) may reduce F. tularensis viability. No information in the influence of solar radiation (UV light) on F. tularensis persistence. Viral Encephalitis and Hemorrhagic Fever Agents Encephalitis viruses (category B) and hemorrhagic fever viruses (category A) are viral agents. Viruses for which persistence was studied include viral hemorrhagic fever filoviruses (e.g., Ebola and Marburg) and arenaviruses (e.g., Lassa and Machupo), flaviviruses (Japanese encephalitis virus, St. Louis encephalitis virus, and yellow fever virus), bunyaviruses (hantavirus and Crimean-Congo virus), and alphaviruses (Venezuelan equine encephalitis [VEE]). Japanese encephalitis virus and VEE virus appeared to have inverse relationships with relative humidity (RH) in aerosols. Persistence data were only identified for viral hemorrhagic fever agents in aerosols and on fomites. Persistence data on viral encephalitis and hemorrhagic fever agents is sparse. No data were identified on the persistence of viral encephalitis or hemorrhagic fever agents in soil. Persistence studies on viral encephalitis and hemorrhagic fever agents in aerosols or on fomites were not continued until attempts to culture produced no viable organism. So, the longest duration that these agents could survive as aerosols or on surfaces remains unknown. Studies in water were limited to VEE virus. None of the studies identified for viral encephalitis and hemorrhagic fever agents assessed the impact of sunlight on persistence. ------- Yersinia pestis Y. pestis is a non-spore-forming non-motile bacterial category-A agent, which causes plague. Y. pestis can enter a state where they are metabolically active, yet not culturable, with unknown viability in water. Y. pestis is also possibly survives inside amoeba. Most persistence data on Y. pestis were associated with survivability in water. Y. pestis persistence in water ranged from days to years with no apparent relationships between environmental conditions and persistence. Variability associated with Y. pestis persistence in water might be attributed to differences in water chemistry, organism strain or growth phase, growth media for culturing and recovery, and inoculum levels. Few studies were found focusing on the impact of various environmental parameters (e.g., humidity, sunlight, and temperature) on persistence. Little data were identified on the persistence of Y. pestis in aerosols and fomites. RANGE OF PERSISTENCE The range of persistence durations are summarized by agent and medium in Table 1. The table also includes the environmental condition associated with each value and identifies agent/medium combinations where persistence data are lacking. Persistence was found to be highly variable between species and subspecies. Persistence was also found to be affected by various environmental media and factors (e.g., temperature, relative humidity (RH), and sunlight), preparation and application methods, and nutrient conditions. Persistence can increase in the presence of organisms that serve as hosts or symbionts (e.g., amoeba, earthworms); persistence can decrease in the presence of competing and/or predatory organisms. Differing analytical methods also affect comparison or interpretation of persistence results across studies (e.g., different culture media, incubation temperatures and times). Similarly, the estimates of persistence may differ depending on the technique used (molecular, culturing, counting, etc.). 7 ------- Table 1. Agent Persistence in the Environment Agent Bacillus anthracis* Brucella species Burkholderia mallei Medium Aerosol Fomite Soil Water Aerosol Fomite Soil Water Aerosol Fomite Soil Water Shortest Duration Reported Persistence Associated Environmental Duration Condition Reference* - 25°C, 80% RH on stainless steel 6 hours coated with silver and zinc zeolite paint n_ . . . Topsoil RH 46%, UV and no . . 96 hours(veg) K . ... U. 3 days Distilled water Galeano et al. (2003) S. EPA, 2014 Sinclair et al. (2008) - . , 22°C, 45% RH on painted joint 4 nours tape <4 days Dried soil <1 day 37°C Ryan (2010) Nicoletti(1980) Nicoletti(1980) - Environmental survival (specific 3 weeks fomites not identified) in wet, humid, or dark conditions - . , Dechlorinated municipal water, 1 aay 5°C and 25°C Dvorak and Spickler (2008) Gilbert and Rose (2012) Longest Duration Reported Persistence Duration Associated Environmental Condition Reference - 7 , . 37°C on polystyrene and glass as aaysj a biofi|m in BH| broth 40 years (spore) 120 hours (veg) 100 hours(veg) 6 days Lee et al. (2007) Manchee et al. [1994) Topsoil, RH 46%,no UV U Topsoil, RH 60% (approx.) simulated sunlight Water S. EPA, 201 4c S. EPA, 201 4c Sinclair et al. (2008) - 22°C, 40% RH on aluminum and 56 daysf glass; and 5°C, 30% RH on aluminum, glass, and wood 43 days 77 days Bison partition sites in Greater Yellowstone, identified in April Room temperature Calfee and Wendling (2012) Aune et al. (2012) Nicoletti(1980) - 3 months On stable bedding, troughs, and harness equipment Malik et al. (2012) - 28 days Tap water, room temperature Miller et al. (1948) 8 ------- Aerosol Fomite pseudomallei ^ Soil Water Aerosol Fomite Coxiella burnetii Soil Water Aerosol Fr3nr/se//3 tularensis Fomite Soil Water - 6 hours <10 days 60 minutes Applied in Butterfield buffer to glass, paper, polyethylene, and stainless steel; and applied in BHI broth to stainless steel Soil inoculated with antagonistic bacteria (e.g., B. multivorans) Water exposed to sunlight Shams et al. (2007) Linetal. (2011) Sagripanti et al. (2009) - - 20 daysf 20°C, 4°C, and -20°C Evstigneeva et al. (2007) - 29 minutes (Tgg value) § 20 minutes 50% RH, wet dissemination Desiccated on filter paper Sinclair et al. (2008); Cox (1971); Cox and Goldberg (1972) Faith et al. (2012) - <1 day Dechlorinated municipal water, 5°C and 25°C Gilbert and Rose (2012) - ,. , Applied in BHI broth to paper, a^s polyethylene, and stainless steel Soil stored in plastic bags at 30 months ambient temperature (1 3°C to 33°C). 16 yearsf Distilled water, 25°C Shams et al. (2007) Thomas and Forbes- Faulkner (1981) Pumpuang et al. (2011) - - 20 daysf 20°C, 4°C, and -20°C Evstigneeva et al. (2007) - 5 days gQ% R|_^ wgt dissernination 16 daysf 37°C, 0% RH on stainless steel Sinclair et al. (2008); Cox (1971); Cox and Goldberg (1972) Wilkinson (1966) - 34 daysf Brackish-water, 21 °C Berrada and Telford(2011) ------- Agent Medium Aerosol Viral encephalitis and Fomite hemorrhagic ,., .. fovo|- agents Water Aerosol Yersinia pestis Fomite Soil Water Shortest Duration Reported Persistence Duration 1 hourf 5 minutes Associated Environmental Condition 22°C, 50% RH Desiccated, 37°C Reference Smither et al. (2011) Fogarty et al. (2008) <60 minutes 34 minutes (Tgg value) 7 minutes (Tgg value) 24 daysf 1 day Tap water (1 mg L1 free available chlorine or 2 mg L1 total bromine), 21 °C 26°C, 87% RH 30°C, 52% RH on metal (stainless steel) Arizona soil during late October in an area with limited exposure to UV light Dechlorinated municipal water, 5°C Wade et al. (2010) Sinclair et al. (2008) Sinclair et al. (2008); Wilkinson (1966) Eisen et al. (2008) Gilbert and Rose (2012) Longest Duration Reported Persistence Duration 120 days (Tgg value) 50 daysf Associated Environmental Condition 21 °C, 23% RH 4°C, 55% RH in tissue culture medium on glass Reference Sinclair et al. (2008) Piercy et al. (2010) - 69 days (Tgg value) 57 minutes (Tgg value) 7 daysf 10 monthsf 3 yearsf 4°C 26°C, 50% RH 20°C, 50% RH on aluminum and painted joint tape 4°Cto8°C Autoclaved river water Sinclair et al. (2008) Sinclair et al. (2008) Ryan (2010) Sinclair et al. (2008) Pawlowski et al. (2011) BHI, brain-heart infusion; RH, relative humidity; uv, ultraviolet; veg, vegetative state. - Not tested/not reported. * This review focused on vegetative B. anthracis only. t The longest duration tested (i.e., the actual persistence duration could be longer). § The time required for the microbial count to decrease by 99% 10 ------- REFERENCES Aune, K. et al., 2012. J Wildlife Manage 76(2):253-261. Berrada, Z.L. and S.R. Telford III, 2011. Arch Microbiol 193(3):223-226. Calfee, M.W. and M. Wendling, 2012. Lett Appl Microbiol 54(6):504-510. Cox, C.S. and L.J. Goldberg, 1972. Appl Microbiol 23(1):1-3. Cox, C.S., 1971. Appl Microbiol 21(3):482-486. Ducret, A. etal., 2014. BMC Microbiol 14:3. Dvorak, G.D. and A.R. Spickler, 2008. J Am Vet Med A 233(4):570-577. Eisen, R.J. et al., 2008. Emerging Infect Dis 14(6):941-943. Evstigneeva, A.S. et al., 2007. Eurasian Soil Sci 40(5):565-568. Faith, S.A. et al., 2012. Front Cell Infect Microbiol 2:Article 126. Fogarty, R. et al., 2008. Virus Research 132(1-2): 140-144. Galeano, B. etal., 2003. Appl Environ Microbiol 69(7):4329-4331. Gilbert, S.E. and L.J. Rose, 2012. Lett Appl Microbiol 55(3): 189-194. Gilbert, S.E. and L.J. Rose, 2012. Lett Appl Microbiol 55(3): 189-194. Lee, K. etal., 2007. Microbiol 153(6):1693-1701. Lin, H.-H. etal., 2011. Microbiol Immun 55(9):616-624. Malik, P. et al., 2012. Veter Ital 48(2):167-178. Manchee, R.J. et al., 1994. Appl Environ Microbiol 60(11):4167-4171. Miller, W.R. etal., 1948. J Bacter55(1):115-126. Nicoletti, P. 1980. Adv Veter Sci Comp Med 24:69-98. Pawlowski, D.R. etal., 2011. J Bioterror Biodef 53:004. Piercy, T.J. et al., 2010. J Appl Microbiol 109(5):1531-1539. Pumpuang, A. etal., 2011. Trans R SocTrop Med Hyg 105(10):598-600. Ryan, S.P., 2010. U.S. Environmental Protection Agency, EPA/600/R-10/086. Sagripanti, J.-L. etal., 2009 Photochem Photobiol 85(4):978-986. Shams, A.M. et al., 2007. Appl Environ Microbiol 73(24):8001-8004. Sinclair, R. et al., 2008. Appl Environ Microbiol 74(3):555-563. Smither, S.J. etal., 2011. J Virol Methods 177(1):123-127. Thomas, A.D. and J.C. Forbes-Faulkner, 1981. Aust Vet J 57(11 ):535-536. U.S. EPA, 2014a. U.S. Environmental Protection Agency, EPA/600/R-14/074. U.S. EPA, 2014b. U.S. Environmental Protection Agency, EPA600/R-14/216. U.S. EPA, 2014b. U.S. Environmental Protection Agency, EPA/600/R-14/150. Wade, M.M. et al., 2010. Int J Microbiol 2010, Article ID 412694, 4 pages. Wilkinson, T.R., 1966. Appl Microbiol 14(3):303-307. CONTACT INFORMATION For more information: visit EPA's website (http://www2.epa.gov/homeland-security-research) Technical Contacts: Worth Calfee (calfee.worth@epa.gov) Erin Silverstri (silvestri.erin@epa.gov) General Feedback/Questions: Kathy Nickel (nickel.kathy@epa.gov) If you have difficulty accessing this PDF document, please contact Kathy Nickel (Nickel.Kathy@epa.gov) or Amelia McCall (McCall.Amelia@epa.gov) for assistance. U.S. EPA's Homeland Security Research Program (HSRP) develops products based on scientific research and technology evaluations. Our products and expertise are widely used in preventing, preparing for, and recovering from public health and environmental emergencies that arise from terrorist attacks or natural disasters. Our research and products address biological, radiological, or chemical contaminants that could affect indoor areas, outdoor areas, or water infrastructure. HSRP provides these products, technical assistance, and expertise to support EPA's roles and responsibilities under the National Response Framework, statutory requirements, and Homeland Security Presidential Directives. 11 August 2015 EPA/600/S-15/218 ------- |