Persistence of Bacillus anthracis spores and Clostridium
botulinum and Destruction of Francisella tularensis and
Yersinia pestis in Municipal Solid Waste Landfill Leachates

EXTENDED ABSTRACT # 378

Wendy J. Davis-Hoover

National Homeland Security Research Center, Office of Research and Development, United
States Environmental Protection Agency, 5995 Center Hill Ave., Cincinnati, Ohio 452224

Mary Margaret Wade

Science and Technology Corporation, 500 Edgewood Rd. Suite 205, Edgewood, MD 21040
Ying Li

Science and Technology Corporation, 500 Edgewood Rd. Suite 205, Edgewood, MD 21040
Tracey Biggs

Edgewood Chemical Biological Center, AMSRD-ECB-RT-BP, Aberdeen Proving Ground, MD
21010

Philip G. Koga

Edgewood Chemical Biological Center, AMSRD-ECB-RT, Aberdeen Proving Ground, MD
21010

INTRODUCTION

The 2001 incident with five Bacillus anthracis contaminated letters resulted in contaminating 56
buildings in 10 US states and Washington, D.C. Items that were contaminated were such things
as: books, paper, wall hangings, staplers, telephones, furniture, computers, mail processing
equipment, carpeting, ceiling panels, wallboard, paneling, nail, trash, spoiled food, contaminated
decontaminate water, personal protective equipment and air scrubbing equipment. The ultimate
fate of "decontaminated" building materials, that is, the permanence of the disposal technique, is
of concern long after final disposal at a landfill site. As much of the decontaminated building
material will end up in municipal solid waste (MSW) landfills due to capacity problems with
incinerators and hazardous waste landfills, the United States Environmental Protection Agency's
Office of Research and Development's National Homeland Security Research Center (NHSRC)
in collaboration with the Department of Defense Edgewood Chemical Biological Center (ECBC)

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are evaluating the permanence of biological and chemical warfare agents in municipal solid
waste landfills. Decontaminated waste that can be verified as 100% free of residual biological
contaminant should present no problem in the landfill environment short of straining national
capacity. Materials that contain some residual active contaminants, however, may present a
different scenario and a different concern for the landfill operator even though leachate itself is
not sterile and dose and exposure risk must be considered. Exposure pathways for movement of
residual contaminants out of landfills are primarily leachate and air (fugitive emission and
collected gas); but the pathways may also include long-term potential for groundwater
contamination or even movement through solids (e.g., soil), both from movement through
leachate. If individual waste containers (sub-containment systems) become compromised in the
landfill, leachate may contain components of the disposed, and presumed decontaminated, waste.
Leachate is often discharged to wastewater treatment systems, and may pose a threat to human
health and the environment.

Study of the permanence of the final disposal of the inactivated or active agent of terrorism must
be examined by looking at the fate of various agents in the most likely pathway of escape. The
agents of terrorism to be reviewed are most likely to be biological because of multiplicity and
low (in most cases) infective doses, and biological agents may be the most economical to obtain
in effective quantity While approximately 209 million tons of MSW are generated annually in
the USA (Franklin Assoc. 1996), bacteria present in MSW are of great concern. Waste that then
might contain viable biological weapons or other pathogenic microorganisms is specifically of
concern especially if there is a chance that they could be transported to landfill leachate and
potentially infect groundwater. Some work has been done to isolate pathogens and other bacteria
in MSW and landfill leachate (Donnelly 1983). However, although various genera have been
identified in MSW such as Clostridium, Bacillus, Pseudomonas, Citrobacter, Corynebacterium,
Aeromonas and Enterobacter (Nwosu and Ladapol999, Van Dyke and McCarthy 2002, Francis
et al. 1980), no studies are available as to the fate of biological warfare agents in landfill leachate
or MSW.

MATERIALS AND METHODS

The purpose of this research is to determine the permanence of disposal of weapons of warfare
that may be introduced into municipal solid waste (MSW) landfills with insufficiently treated
rubble from contaminated sites. Because of capacity problems with incinerators and hazardous
waste sites, it is more likely that we will need to depend on MSW landfills to contain the
uncontaminated building products. Our approach is to inoculate raw un sterilized MSW leachate
with known quantities of biological agents, developing leachate microcosms in a secure
laboratory, and analyzing the spiked leachates for viable agent through a 12-month time course.
The samples were handled as waste would be: initially aerobically, sealed and then allowed to
reach anaerobiosis. Landfill temperatures vary over their lifetime and in the waste mass. To look
at two worst case scenarios, 12 and 37 0 C incubations were used to simulate soil temperature
and optimum temperature for pathogens. Uninoculated samples serve as negative controls and
inoculated growth media (different per agent) serve as positive controls. Four bacterial agents
were tested (Table 1).

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Table 1. Listing of Strains and Growth Characteristics of Bacterial Cells.

Bacterial
Type

General Media (G)

Culture Temp.
& Time

Selective Media (S)

Clostridium
botulinum

(G) ATCC medium 1053 Clostridial (Oxoid CM149)

35-37°C

for
24-48 hrs

(S) PEA (phenylethyl alcohol agar) and egg yolk agar (EYA)

Francisella
tularensis

(G) ATCC medium 192
Cysteine heart agar

35-37°C

for
4-7 days

(S) Chocolate agar (CA), Thayer Martin (TM) and Buffered
Charcoal yeast extract (BCYE)

Bacillus
anthracis

(G) ATCC medium 254
Heart infusion agar

35-37°C

for
24-48 hrs

(S) Sheep blood agar (SBA) and PLET will be used (Polymyxin-
lysozyme EDTA-thallous acetate)

Yersinia
pestis

(G) ATCC medium 3
Nutrient agar

28°C
for
2-4 days

(S) Yersinia selective agar (YSA).

Media are listed as the general medium for culture maintenance and the selective medium used
for recovery from among leachate microflora.

Inoculated leachates (triplicate samples) were tested for quantities of viable biological agent
weekly for the first 2 months, then twice a month for 5 months, then monthly for 5 more months
(unless data indicated otherwise) or until no detects are observed in all replicates for 2
consecutive sampling periods. This would identify the termination of the experiment for that
agent. Tests of viability were performed using approved culture methods.

Numerous assumptions were made with this research, some of which are:l. Results obtained
from a MSW landfill's leachate will be representative of the results we may expect to see with
leachate from other landfills. We are analyzing leachates from only one site and may or may not
be able to extrapolate to what would occur at a different landfill. This landfill is closed,
synthetically covered, and has waste that is between 5-15 years old. However, using the one site
is a start and future studies may include multiple landfill sites. 2. Triplicate microcosms will
allow us to better understand the real-world situation. 3. Three ml microcosms will mimic
anaerobic conditions of landfills. 4. Temperatures vary greatly in landfills. Looking at two
temperatures mimics the world (average soil temperature (12° C) and higher incubation
temperature optimized for pathogens (37° C)). 5. The agents will always encounter undiluted
leachate in the landfill before any release is possible.

Bacterial cells were first streaked for isolation on appropriate media (General medium). A single
colony was then grown in broth culture (General medium). Glycerol stocks were prepared at -
80°C for long-term storage. An overnight broth culture was grown fori7, tularensis, Y pestis
and C. botulinum in appropriate media and incubated under appropriate aeration and temperature
conditions. The cells were harvested by centrifugation, washed with phosphate buffered saline

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(PBS) and resuspended in a minimal volume of PBS. The cells were used to inoculate landfill
leachate samples. B. anthracis spores were produced utilizing the MAP protocol for spore
preparation and used to inoculate leachate samples.

Viruses are propagated in either the mammalian cell line Vero or BSC-40 cells, and grown in
standard cell culture media. Viruses will be collected in cellular supernatant and used to
inoculate leachate samples (or can be stored indefinitely at -80°C).

Inoculum Enumeration

Bacterial cells and spores were serially diluted in PBS and enumerated by plating 100-|iL
aliquots in triplicate on plates. Virus stocks will be enumerated by standard plaque assay (in
quadruplicate) to determine concentration in plaque forming units per milliliter (pfu/ml).

Spiking of Inoculum

Bacterial cells and spores were added to appropriate aliquots of leachate such that the final
concentration of the target organism was approximately 107 cfu/ml. Viruses will be added to
appropriate aliquots of leachate such that the final concentration of the target organism is
approximately 106 pfu/ml.

Test Matrix

Landfill leachate samples were received from the Sandy Hill Landfill (Bowie, MD) and
processed within 24 hours of collection of samples. After spiking appropriate aliquots of
leachate with each bacterial target organism, 3 ml aliquots were dispensed to 5 ml anaerobic
culture vials until a total of 150 tubes for each target bacterium were reached. 72 tubes were
then incubated at 37°C and 72 tubes incubated at 12°C for each bacteria. The remaining six
inoculated tubes were incubated (three at 37°C and three at 12°C) and monitored for aerobic
status (aerobic controls). An anaerobic (oxidation-reduction) indicator dye, resazurin (which
turns from blue to pink to colorless as conditions go from aerobic to anaerobic), was added to the
aerobic controls and their color was monitored periodically to provide a measure of the anaerobic
status of the corresponding samples. Tubes were labeled (using pre-printed labels) with a control
number comprised of a 6 digit start date (mmddyy), 2-3 letter organism code (from Table 2), 2
digit + C temperature (37C or 12C), and a dash (-) followed by a 2 digit tube number. Aerobic
controls had AC for the organism code. Un-inoculated leachate was included as a negative
control (NC for organism code), while inoculated growth media (agent specific) served as a
positive control (organism code + PC). The same test matrix will be used for virus testing once
testing begins.

Performance Period and Experimental Design

Bacterial sampling was to occur every week for 2 months, then bi-weekly for 5 months and
finally monthly for 5 months, or until there was no detectable target in two consecutive sampling
periods. In order to assure that the absence of detectable growth was not attributable to
expiration of the specific recovery media, a fresh inoculum of the target BW agent into the

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specific recovery media was included in subsequent sampling periods once a "no detect" event
was observed. Continued absence of detectable target in two consecutive sampling periods with
positive growth observed in this second positive control was construed as evidence that the target
BW agent no longer persisted under the incubation conditions, and sampling for that target BW
agent was discontinued. A negative control was sampled at each time point. A positive control
was also sampled at each time point or until no growth was observed in two consecutive
sampling periods. The same sampling strategy will be employed for viruses.

Sampling and Analysis

At each time point for both incubation temperatures, microcosms (5 ml anaerobic culture vials)
of leachate spiked with each bacterial target were removed from incubation and analyzed for
viable bacteria. Samples were centrifuged, washed (pellets) and re-suspended in sterile
phosphate buffered saline (PBS using aseptic technique). Samples were serially diluted in PBS
and 100-|iL aliquots plated in triplicate on appropriate media using aseptic technique (Selective
media). Plates were labeled with the sample control number to which a dash and the 6 digit
(mmddyy) date of removal from timepoint incubation was appended followed by an A, B, or C
to indicate triplicate replicates. The inoculated and labeled plates were allowed to incubate at the
appropriate temperature, aeration conditions, and time, and then observed for growth of target
organisms . Growth of target organism indicated survival in leachate and the concentration was
determined. After confirmation, all plates were autoclaved and disposed.

For samples of leachate spiked with viral targets, samples will be removed over time from both
incubation temperatures and analyzed for the presence of virus, using aseptic technique at each
step. The spiked leachate sample will be filtered and the virus will be eluted from the filter and
added to cell culture-safe media. The virus-containing media will be serially diluted and each
dilution will be used to infect monolayers of mammalian Vero cells or BSC-40 cells. Infected
cell cultures will be incubated at the appropriate temperature for the appropriate amount of time
after which they will be stained and observed visually for viral plaque formation.

Microbial Identification System (MIDI) Analysis

Prior to beginning any experiments, an assessment of the native microflora present in raw
leachate was conducted using the MIDI. This microbial identification system compares the fatty
acid profile of the unknown sample with libraries containing fatty acid profiles of known
microbes in order to identify the sample. This analysis was performed to rule out the presence of
any harmful bacteria and to aid in the safety of the personnel on the project. It was determined
that leachate should be handled under BSL-2 conditions and posed no threat to personnel when
handled at this level correctly.

CONCLUSIONS

The characterization of the aerobic normal flora of MSW landfill leachate by methods of MIDI
are shown in Table 2 below. A Similarity Index (SI) of 0.5 or higher indicates a good library
comparison. An SI of 0.3 to 0.5 indicates it may be a good match, but just an atypical strain,
while an SI lower than 0.3 indicates that the species is not in the database. However in cases of

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SI lower than 0.3, the closest relative is given. Further information about the species are also
discussed below.

Table 2. MIDI Characterization of Aerobic Normal Flora of MSW Landfill Leachate

IDENTIFICATION

SI

CONCENTRATION
(CFU/ML)

Bacillus thuringiensis

0.547

>1000

Aeromonas hydrophila

0.573

1000

Pseudomonas putida

0.161

1000

Chryseobacterium indologenes

0.540

10-100

Aeromonas sobria

0.307

10

Nocardia otitidiscaviarum

0.511

10

QC (P. aeruginosa)

0.590



Bacillus thuringiensis: BSL 1 organism. It is a spore forming, aerobic gram-positive rod. B.
thuringiensis is an insect pathogen (and is not associated with any human diseases) and is used in
producing commercially available insecticides. B. thuringiensis is typically found in soils and
water.

Aeromonas hydrophila. BSL 2 organism. It is also non-spore forming and is a facultatively
anaerobic gram-negative rod.

Aeromonas species are ubiquitous inhabitants of natural waters and sewage. Human infections
can occur primarily by contact or ingestion of water or soil. Four types of disease can occur:
wound infection, septicemia, extraintestinal infections and diarrheal diseases with wound
infections occurring near or in water or diarrheal disease (due to drinking untreated water) being
the most common.

Pseudomonasputida. BSL 1 organism. It is a non-spore forming, aerobic, gram-negative rod. P.
putida is usually associated with soil, water and plants and animal sources. Although some
pseudomonads are important opportunistic pathogens, such as P. aeruginosa, P. putida is non-
pathogenic.

Chryseobacterium indologenes (Flavobacterium): BSL 2 organism. It is a gram-negative rod. It
is aerobic and non-spore forming. Flavobacteria are ubiquitous in soil and water. F. indologenes
has rarely been associated with human infection.

Aeromonas sobria. BSL 1 organism. It is a non-spore forming, gram-negative rod. It is
facultatively anaerobic.

Nocardia otitidiscaviarum. BSL 2 organism. It is an aerobic actinomycete. It is non-spore
forming and stains gram-positive. N. otitidiscaviarum is considered a human pathogen; however,
it is not the most common species associated with human infection (pulmonary and systemic).

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Several actinomycetes can cause infection, but Nocardia species usually account for the majority
of reported cases with N. asteroides causing over 90% of all infections. Nocardia are inhabitants
of soil and water and infection in humans can occur after inhalation or inoculation through
breaks in the skin. It should be noted however, that human infections typically only occur in
immunocompromised individuals.

The characterization of the anaerobic normal flora of MSW landfill leachate by methods of MIDI
are shown in Table 3 below. Again a similarity Index (SI) of 0.5 or higher indicates a good
library comparison. Further information about the species are discussed below.

Table 3.MIDI Characterization of Anaerobic Normal Flora of MSW Landfill Leachate

IDENTIFICATION

SI

CONCENTRATION
(CFU/ML)

Aeromonas hydrophila

0.786

1000

Aeromonas sobria

0.346

10

Corynebacterium glucuronolyticum

0.117

10

Brevundimonas diminuta

0.510

10

QC (P. aeruginosa)

0.755



NOTE: Both Aeromonas species are discussed above. Since they are facultative organisms, they
were present under both anaerobic and aerobic conditions.

Corynebacterium glucuronolyticum: BSL2 organism. It is a gram-positive, non-sporeforming,
facultative anaerobic rod. The organism is usually associated with the human urinary tract. Other
members of the genus Corynebacterium are more notable such as C. diphtheriae, which causes
diphtheria. The genus includes other human pathogens and opportunistic species, but information
on C. glucuronolyticum is limited.

Brevundimonas (Pseudomonas) diminuta: BSL 1 organism. It is a gram-negative, non-spore
forming bacillus. Although most Pseudomonads are aerobic, some strains of B. diminuta may be
anaerobes. B. diminuta has been isolated from water, hospital equipment and human clinical
specimens. B. diminuta has been the cause of at least one reported case of septicemia, but clinical
significance of B. diminuta has not been determined.

Fate of BWA in MSW Landfill Leachate
Bacillus anthracis

At both 12 and 37 0 C, the MSW leachate allowed for consistent survival of B. anthracis for at
least 24 weeks.

Clostridium botulism

Clostridium botulism survived at both 12 and 37 0 C for at least 22 weeks. The survival was not

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as stable as it was for B. anthracis. This may suggest that it sometimes exists in an anaerobic
vegative state instead of a spore formation.

Yersinia pestis

At 37 0 C Y pestis was non viable at 2 weeks. At 12 0 C, it was non viable at 6 weeks. The cooler
soil-like temperature seems to protect it more.

Frattcisella tularensis

F. tularensis was non viable at 7 weeks at both 12 and 37 0 C in the MSW landfill leachate.

Thus the spore-forming agents B. anthracis and C. botulinum are surviving in the MSW leachate
for longer periods than the non- spore forming bacteria. Longer term studies will be performed
on these organisms. Incubation temperatures are not much of a variable for any of the agents as
the degradation rates were similar with different temperatures. Using raw leachate allowed for
simulation of competition by normal flora.

In conclusion, although it would be hoped that survival of biological weapons and pathogens
would not be insured in landfill leachates, the persistence of some of these agents must be
considered before deciding to place them into municipal solid waste landfills. However, landfills
are never sterile. Thus concentration of agent, infective and lethal doses, and likelihood of
exposure to a sensitive population must be taken into account.

REFERENCES

Franklin Associates, Ltd., Prairie Village, KS. 1994. In: Statistical Abstract of the United States,
p. 237 (1996).

Donnelly, J. A. 1983. Isolation, Characterization and Identification of Microorganisms from
Laboratory and Full-Scale Landfills. Ph. D. Dissertation. University of Cincinnati. Cincinnati,
Ohio.

Nwosu, V.C. and J.A. Ladapo. 1999. Current Microbiol. 39:249-253.

Van Dyke, M.I. and A.J. McCarthy. 2002. Appl. Environ. Microbiol. 68:2049-2053.

Francis, A.J., S. Dobbs, and B.J. Nine. 1980. Appl. Environ. Microbiol. 40:108-113.

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