EPA 600/R-13/185 j September 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
Literature Review of
Protocols for Processing
Soils Contaminated with
Bacillus anthracisSpores
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-13/185
September 2013
Literature Review of Protocols for
Processing Soils Contaminated with Bacillus anthracis
Spores
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center
Threat and Consequence Assessment Division
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Disclaimer
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and managed the literature review described herein under an Interagency Agreement with
the Defense Technical Information Center through the Battelle/Chemical, Biological,
Radiological, and Nuclear Defense Information and Analysis Center Contract No. SP0700-00-D-
3180, Delivery Order 0679/ Technical Area Task 886 and Delivery Order 0729/Technical Area
Task CB-11-0232).
This document has been subjected to the Agency's review and has been approved for
publication. Summaries of the literature found through this review are highlighted in this report.
However, any research that was not conducted under EPA's stringent Quality Assurance
Requirements could not be evaluated for accuracy, precision, representativeness, completeness,
or comparability of the results and therefore no assurance can be made regarding the quality of
the conclusions extracted from these publications. The contents of this document reflect the
views of the contributors and do not necessarily reflect the views of the Agency. Mention of
trade names or commercial products in this document or in the literature referenced in this
document does not constitute endorsement or recommendation for use. Questions concerning this
document or its application should be addressed to the EPA Task Order - Contract Officer
Technical Representative:
Erin Silvestri, MPH
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@epa.gov
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Table of Contents
Disclaimer i
Table of Contents ii
List of Tables iii
List of Figures iii
Acknowledgments iv
List of Acronyms and Abbreviations v
List of Trademarks vii
Executive Summary viii
1.0 Introduction 1
1.1 Characteristics of B. anthracis 2
1.2 Persistence of B. anthracis Spores in Soil 3
1.3 Purpose 4
2.0 Current State of the Science 5
2.1 Indirect Processing: Separating B. anthracis from Soil 5
2.1.1 Aqueous carrier media 5
2.1.2 Spore-soil disassociation 8
2.1.3 Physical separation of spores from soil 8
2.2 Direct Processing: DNA Extraction of Bulk Soils and Selective Culture Media 12
2.2.1 Selective culture media 13
2.2.2 Direct DNA extraction from bulk soils 17
2.2.3 Enrichment Steps 20
2.3 Purification Protocols 20
3.0 Conclusions 21
4.0 Quality Assurance 22
5.0 References 23
Appendix A - Table of Reviewed B. anthracis Soil Studies and their Design Elements 31
Appendix B - Table of Commercial DNA Kits Used for Direct Soil Analysis 47
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List of Tables
Table 1. Spore Separation Aqueous Carrier Media (Percent Recovery) 7
Table 2. B. anthracis Selective Culture Media 14
List of Figures
Figure 1. Classic B. anthracis natural lifecycle 2
Figure 2. Indirect soil processing flow diagram 5
Figure 3. Indirect soil processing steps 9
in
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Acknowledgments
The following individuals and organizations served as members of the Project Team and
contributed to the development of this project:
U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
National Homeland Security Research Center (NHSRC)
Erin Silvestri
Tonya Nichols
Frank Schaefer
Battelle, Contractor for the EPA
Pegasus, Contractor for the EPA
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List of Acronyms and Abbreviations
°C Degrees Celsius
|ig Microgram(s)
|im Micrometer(s)
ABA Anthrax Blood Agar ™
ATCC American Type Culture Collection
AZ dust Arizona Test Dust
BHI Brain heart infusion (medium)
BSA Bovine serum albumin
CDC Centers for Disease Control and Prevention
CEI Cereus Ident Agar™
CFU Colony forming unit(s)
ChrA R & F anthracis chromogenic agar
cm Centimeter(s)
CTAB Cetyltrimethylammonium bromide
DDAB Didecyldimethylammonium bromide
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme linked immunosorbent assay
EPA U.S. Environmental Protection Agency
fg Femtogram(s)
FITC Fluorescein isothiocyanate
FL Florida
g Gram(s)
GABRI Ground anthrax Bacillus refined identification
GEq Genomic equivalents
GFP Green fluorescent protein
HEPES 4-(2-Hydroxyethyl)-l-piperazineethanesulfonic acid
hr Hour(s)
HSGS High specific gravity separation
IgG Immunoglobulin G
IM-ECL Immunomagnetic-electrochemiluminescent
IMS Immunomagnetic separation
ITS Internally transcribed spacer (region)
/ Liquid medium
L Liter(s)
LB Luria broth
LC/MS Liquid chromatography/mass spectrometry
LOD Limit of detection
LRN Laboratory Response Network
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MATH Microbial adherence to hydrocarbons
MEP Mannitol-egg yolk-polymyxin B agar
min Minute(s)
mL Milliliter(s)
MN loam Minnesota loam
N Normal(ity)
NaOH Sodium hydroxide
ND Not determined
Nonidet P-40 Igepal, octylphenyl-polyethylene glycol
PBS Phosphate buffered saline
PBST Phosphate buffered saline amended with 0.5% TWEEN® 20
PC-PLC Phosphatidylcholine-specific phospholipase C
PCR Polymerase chain reaction
PCR-DGGE Polymerase chain reaction and denaturing gradient gel electrophoresis
PLET Polymyxin B, lysozyme, ethylenediaminetetraacetic acid, thallous acetate agar
PVPP Polyvinylpyrrolidone
qPCR Quantitative polymerase chain reaction
RBMS Reference background matrix soil
rDNA Ribosomal deoxyribonucleic acid
RFLP Restriction fragment length polymorphism (DNA analysis)
RNA Ribonucleic acid
rRNA Ribosomal ribonucleic acid
RV-PCR Rapid viability polymerase chain reaction
5 Solid agar medium
SASP Small acid-soluble proteins
SBA Sheep blood agar
SBD Synthetic building debris
SDS Sodium dodecyl sulfate
SHMP Sodium hexametaphosphate
TFA Trifluoroacetic acid
Triton® X-100 4-(l,l,3,3-Tetramethylbutyl)phenylpolyethylene glycol
TSA II Trypticase® soy agar with 5% sheep blood
TSB Trypticase® soy broth
TWEEN® 20 Polyethylene glycol sorbitan monolaurate
TWEEN® 80 Polysorbate 80
UV Ultraviolet
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List of Trademarks
Trademarked Name
Registration Holder
Anthrax Blood Agar™
ABA, Heipha, Germany
Bacto1M
Bacto Laboratories Pty Ltd, Mt. Pritchard, NSW, Australia
BioRobot™
Oiagen, Valencia, CA
Cereus Ident Agar 1M
Heipha, Eppelheim, Germany
Chelex®
Bio-Rad, Life Science Division, Hercules, CA
Nonidet™
Shell Chemical Co, Houston, TX
NucliSENS®
bioMerieux, Inc, Durham, NC
Nycodenz®
PROGEN Biotechnik, Heidelburg Germany
PickPen®
Bio-Nobile, San Diego, CA
PowerVac
Mo Bio Laboratories, Inc., Carlsbad, CA
PrepMan®
Applied Biosystsems, Foster City, CA
R& F®
R&F Laboratories, Downers Grove, IL
Sephadex®
Amersham Biosciences AB Corporation, Uppsala, Sweden
Stomacher®
Seward Limited, West Sussex, UK
Triton®
Dow Chemical Company, Midland, MI
Trypticase®
Becton, Dickinson and Company, Franklin Lakes, NJ
TWEEN®
ICI Americas, Bridgewater, NJ
Waring®
Conair Corporation, Stamford, CT
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Executive Summary
Bacillus anthracis (B. anthracis) spores are small, light in weight, and persistent. Many
organizations have sought to identify and quantify the presence of B. anthracis spores within the
environment. However, due to the number of other organisms and impeding chemical
constituents within soil, identifying virulent B. anthracis within soil is a difficult task. Regardless
of the detection assay, the initial sample must be processed efficiently to ensure that debris,
chemical components, and biological impurities do not obstruct downstream detection. Without
appropriate sample processing, the most sensitive detection assay will be ineffective. Therefore,
the objective of this project was to consolidate information regarding sampling and processing
protocols that have been investigated in the literature for multiple soil types. Open literature
searches were performed to collect and summarize over 100 pertinent documents, focusing
primarily on data gleaned in the last decade, regarding the processing of soils contaminated with
B. anthracis.
Soil sample processing protocols can be divided into two general types: direct and indirect. For
indirect processing, spores and soil particles are separated prior to downstream detection. Direct
processes utilize a soil sample without first separating the spores from the bulk sample. Direct
and indirect processing steps each have associated advantages and disadvantages. Indirect
sample separation steps increase the proportion of target spores within the final detected sample;
however, spore loss prior to detection also increases. For directly processed samples, there is
potential for background organisms to overwhelm detection technologies and prevent target
spores from being observed.
There are two requirements for successful indirect isolation of B. anthracis from soil samples:
dissociate the spores from the soil particles and physically separate the free spores from the soil
particles. Adding an aqueous carrier medium to a soil sample creates a sample slurry for easier
manipulation. While water has been utilized, chemical additives have often been included to aid
spore-soil dissociation. While some authors found that the carrier medium (or spore extraction
solution) was the most important factor influencing the extraction efficiency of spores from
wipes, others stated that the presence of a detergent in the aqueous carrier medium consistently
improved the separation of spores from soil particles over buffer or water alone. No consensus
on an optimum aqueous carrier medium could be determined from the reviewed literature.
Additional research focusing on the aqueous carrier medium for processing multiple soil types
under uniform dissociation and separation conditions is needed.
Centrifugation, high specific gravity separation (HSGS), immunomagnetic separation (IMS),
filtration, and settling have been used by various researchers to physically separate spores from
soil. To some authors, the utility of IMS for environmental samples was concerning, while
others presented several advantages to using IMS including ease of use, utility for large numbers
of samples, and shorter processing times compared to conventional protocols. Filtration showed
promise in being able to rapidly separate spores from diverse matrices. Future work that
combines an optimized aqueous carrier medium with the filtration may further increase recovery
rates.
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Direct soil processing falls under two principle types: culturing on B. anthracis selective agar
and bulk DNA extraction. Researchers have sought a B. anthracis specific agar medium that
deters background organisms and other non-anthracis Bacillus species and yet allows B.
anthracis propagation and identification. Several selective media for B. anthracis have been
developed: mannitol-egg yolk-polymyxin B agar (MEP), R & F® anthracis chromogenic agar
(ChrA) and Cereus Ident Agar (CEI). Additionally, anthrax Blood Agar™ (ABA) is a
nutrient medium containing sheep blood and supplements to inhibit many fast-growing
organisms. The medium that showed the most promise in the literature was Modified Polymyxin
B, lysozyme, ethylenediaminetetraacetic acid, thallous acetate agar (PLET) which includes
antibiotics and lysozyme to inhibit the growth of other Bacillus species.
Numerous kits are available from vendors that are specific for DNA extraction from soil
samples. There are many advantages to using a commercial kit for bulk DNA extraction.
Unfortunately, due to the difference in study designs and tested soil conditions, it is difficult to
determine an overall optimum DNA extraction kit from the currently available data. An
extraction kit optimization study using multiple soil types and uniform detection conditions is
needed to elucidate an ideal DNA extraction kit for multiple soils.
As shown through this literature review, an optimized soil processing protocol with a known
recovery rate and associated confidence intervals is needed. A reliable processing protocol would
allow for multiple technicians and laboratories to produce high quality, uniform results in the
event of a B. anthracis release. Recovery rates and confidence intervals would aid downstream
human health and consequence decisions.
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1.0 Introduction
Soil is a complex matrix with multiple components and a plethora of microbial activities. Soil as
defined by the US Department of Agriculture's National Resources Conservation Service is
comprised of solids, liquid, and gases that occur on the land surface and is characterized by
layers that are distinguishable from the initial material or by the ability to support rooted plants
in a natural environment (1). The properties of a soil fluctuate with time as weather patterns and
plant growth cycles directly affect soil conditions. For this reason, pH, soluble salts, organic
mass, flora, fauna, temperature, moisture, and the number of microorganisms all change with the
seasons and over extended periods of time (1). There are numerous types and conditions of soils
around the globe, each with specific components and compounds. The US Department of
Agriculture has supplied soil with its own taxonomic classification system, which designates the
following categories listed in decreasing rank: order, suborder, great group, subgroup, family,
and series. All 12 orders and 60 of the 64 suborders of soil are present within the surface area of
the United States and its territories (1).
One gram of soil reportedly contains up to 10 billion microorganisms and thousands of different
species (2). In addition, chemical constituents of soil: organics, humic acids, etc., can interfere
with the chemistry involved in downstream microbiological detection assays (3-10). An
understanding of the environmental distribution of bacterial pathogens and their fate over time in
nature is needed for multiple applications, including the determination of risk to wildlife,
livestock, and humans in any given area, and distinguishing between natural and anthropogenic
sources during an epidemic. However, due to the number of organisms and impeding chemical
constituents within soil, identifying a single virulent organism within a soil sample is a difficult
task.
Exposure of humans to Bacillus anthracis (B. anthracis) has been historically associated with
agricultural contact with infected animals. The most common route of exposure for humans is
through cutaneous exposure, while naturally occurring ingestion and inhalational exposures are
rare. As a Gram-positive spore-forming pathogen, B. anthracis spores can survive extreme heat
and drought for extensive periods. Global trade of goods and products has dispersed the
organism worldwide. Currently there are endemic anthrax foci on all continents except
Antarctica. Thus, B. anthracis is a naturally occurring organism in many soil environments (11).
Close relatives of B. anthracis can be collocated in the soil environments (12), making detection
of B. anthracis in soils even more complicated. There have been multiple reviews detailing the
various detection assays for B. anthracis (13-15); however, the added complexity of processing
soil samples for microbiological assessment is often neglected. The report herein will compile
soil sampling and processing information acquired from research conducted within the last
decade.
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1.1 Characteristics of B. anthracis
There are a number of theories regarding the lifecycle of B. anthracis in soil. The predominant
theory is that B. anthracis is an obligate pathogen with little propagation occurring directly
within soil, but rather, the soil acts as a holding site from which the hosts may ingest or inhale
the spores (16, 17). Within the classic B. anthracis lifecycle (Figure 1), vegetative B. anthracis
propagates in a host to concentrations in the millions of bacteria per milliliter of blood,
producing toxins that kill the host (18). Sporulation is initiated when predators (or other events)
open a carcass, allowing the bodily fluids to drain from the infected carcass, vegetative cells are
placed into the surrounding environment, and nutrients are depleted (19-23). There is a high level
of uncertainty regarding the factors leading to the initial case or cases of an anthrax epizootic.
Spores can persist in soil for years (18, 24), and yet there can be decades between outbreaks;
there is no clear understanding of the dormancy period. However, once a spore encounters
suitable environment, it will germinate, proliferate, and start the cycle again.
Vegetative Form
Spore form
INFECTED ANIMAL HOST
host disease
and death
V
carcass
V
SOIL
germination
<^=
ANIMAL HOST
CONTAMINATED
CARCASS
release of
—
anthrax with
fluids from
exposure
of animal
host to
spores
c>
sporulation
SOIL OR NATURAL
ENVIRONMENTAL
RESERVOIR(S)
Figure 1. Classic B. anthracis natural lifecycle.
Figure adapted from (17).
Within the classic theory, vegetative B. anthracis does not survive in the environment (25), and
multiplication does not occur at carcass sites (18). However, there are other hypotheses that
explain persistence of B. anthracis. Some propose that B. anthracis spores can germinate and
multiply vegetatively, in the rhizosphere of grass (Festaca arundinaceae) (26) or in the gut of
earthworms (Eisenia fetida) (17, 27). Evidence of B. anthracis spores germinating, replicating,
and re-sporulating in co-culture with the soil-dwelling amoeba Acanthamoeha castellanii and
Hartmannella vermiformis under simulated moist soil environments has been presented within
the open literature (28). Other work postulates that soil biofilms may play a role in the B.
anthracis lifecycle (29) or that bacteriophage infection of B. anthracis may restore saprophytic
functionality necessary for replication and survival (17, 27). Each of these hypotheses remains
controversial.
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Regardless of how B. anthracis spores came to a soil, it is generally accepted that some soils are
more prone to harboring spores than others, and weather conditions influence the occurrence of
environmental anthrax cases. B. anthracis is most often found in dry conditions with soils that
are high in organic deposits and calcium and are relatively alkaline (above pH 6) (21, 22, 30).
Louis Pasteur conjectured that oral cavity trauma experienced during drought conditions
increases the chance of a grazing animal to acquire anthrax from spores retained within the soil
(31).
Spores are formed as a survival mechanism when B. anthracis vegetative cells experience
nutrient-limiting conditions. Spores are metabolically dormant and extremely resistant to
environmental stresses (32, 33). B. anthracis spores have a diameter of approximately 1-1.5 |im
(34). The spore is composed of a series of concentric layers; the innermost layer is the core,
surrounded by a peptidoglycan layer called the cortex and two protein layers known as the spore
coat and the exosporidium (outermost layer) (35). Each layer aids in protecting the viability of
the spore (35). The chromosome, along with tightly bound small acid-soluble proteins (SASPs),
are found at the center of the spore core (36). High levels of calcium dipicolinic acid and the
SASPs protect the core DNA from ultraviolet (UV) degradation, while the core membrane and
the cortex work together to keep the core dry (35, 37). The coat protects the core from foreign
materials entering, while the exterior exosporidium surface-proteins interact with the
environment (35, 38). Interestingly, Bacillus spp. directly purified from natural soil
environments have been shown to have higher intrinsic UV resistance than laboratory strains,
suggesting that sporulation physiology may play a role in determining spore UV resistance (39).
Fully virulent B. anthracis includes two large plasmids, pXOl and pX02. The pXOl plasmid
contains three genes (pag, lef, and cya) which code for three proteins (protective antigen, lethal
factor, and edema factor, respectively) that make up the anthrax toxin (13). The pX02 plasmid
carries the proteins required for encapsulation through the cap A, capB, and cap C genes (40).
Encapsulation is important for virulence; however, the mechanisms by which encapsulation
contributes to virulence have not been determined (41).
1.2 Persistence of B. anthracis Spores in Soil
Studies of laboratory-stored soils have shown that B. anthracis can remain viable for extensive
periods. Sinclair et al. (42) compiled a literature review of persistence of category A agents in
the environment and found several soil studies in which virulent B. anthracis remained viable in
soil samples for up to 68 years (43-46). The multiple protective layers surrounding individual
spores allow them to survive harsh environmental conditions for periods ranging from decades to
centuries, during which time spores are thought to migrate within the soil following the flow of
water (47). Hendriksen and Hansen (48) found vertical dispersal of B. thuringiensis in a field to
be significant. Over 50% of the B. thuringiensis spores within the topsoil migrated deeper into
the soil over a five-year period. However, the same study determined horizontal dispersion after
seven years to be limited. Similarly, Manchee et al. (49) described viable B. anthracis dispersed
on Gruinard Island to be within the top 10 cm of soil after 40 years, while the horizontal
dispersal pattern had not changed significantly from the original release locations (21).
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Blackburn et al. (50) developed an ecological niche modeling tool to predict the geographical
distribution of B. anthracis across the continental United States. The study depicts a significant
corridor of increased B. anthracis presence running north to south from Canada to Mexico.
Griffin et al. (51) were able to confirm the existence of B. anthracis isolates within a similar
transect of North American soils. Historically, the identified areas follow cattle trails (50). In
many instances, recent anthrax cases are associated with old graves of anthrax stricken animals
and adequate soil conditions (21, 51, 52). Many researchers have sought to identify and quantify
the presence of B. anthracis within the environment. However, due to the number of background
organisms and impeding chemical constituents within soil, identifying B. anthracis within soil is
a difficult task.
1.3 Purpose
A complete method for detection of B. anthracis spores in soil would likely include details
regarding soil collection, transport, processing, analysis, and quality assurance standards for each
step. A detailed method would allow for multiple technicians and laboratories to produce high
quality uniform results in the event of a wide area B. anthracis release. A fully developed
method would be useful for determining the presence of B. anthracis spores, their viability, and
the extent of contamination. Multiple protocols have been developed either to separate spores
from soil samples before microbiological assessment or to directly extract bulk DNA to identify
the initial organism(s) present within the soil. However, these studies have never been integrated
to determine the overall breadth of knowledge regarding the processing efficiency. Therefore, the
objective of this project is to consolidate information acquired from previous research, focusing
primarily on data gleaned in the last decade, regarding the processing of soils contaminated with
B. anthracis.
This review is intended to provide a summary of sampling and processing protocols that have
been investigated in the literature for multiple soil types. Open-literature searches of PubMed,
Google Scholar, and the Battelle Library using the search criteria "Bacillus anthracis," "soil,"
and "soil microbiology" were used to collect nearly 100 pertinent documents. In addition, a
reference list was supplied by EPA during the project. The table in the Appendix A outlines a
brief synopsis of each applicable study including the organism strain, soil type, sample
processing protocol, DNA extraction protocol, detection assay, and limit of detection (LOD)
determined during the various studies discussed within this review. Detailed discussions of the
study results are presented hereafter.
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2.0 Current State of the Science
Multiple reviews have focused on various B. anthracis detection assays (13-15). However,
previous reviews have not included an in-depth discussion of various soil sample processing
protocols for microbiological assessment. Regardless of detection assay, the initial sample must
be processed efficiently to ensure that debris, chemical components, and biological impurities do
not obstruct microbiological detection. Without appropriate sample processing, the most
sensitive detection assay will be ineffective. As pointed out in a review by Lim et al. (53) there is
a need for a universal sample processing protocol to separate, concentrate, and purify target
agents from any sample type. Recovery efficiency is a critical factor in determining an ideal
processing protocol. A careful balance must be attained between ensuring that the maximum
number of spores and a minimum amount of debris and chemical constituents are retained in the
final sample. In addition, spore viability may be of concern, especially in the cases where
confirmatory culturing or sample archiving is required. Unfortunately, recovery efficiency data
are lacking for many processing protocols. While there are many B. anthracis detection assays,
few of these assays can be utilized directly with an environmental soil sample. Therefore, sample
processing protocols are used to isolate and concentrate spores from a bulk soil sample. Soil
sample processing protocols can be subdivided into two general types: indirect and direct. For
indirect processing, spores and soil particles are separated prior to downstream detection.
Conversely, direct processes utilize a soil sample without first separating the spores from the
bulk sample.
2.1 Indirect Processing: Separating B. anthracis from Soil
Because spores have the potential to adhere to large soil aggregates (39), there are two
requirements for successful isolation of B. anthracis from soil samples: dissociate the spores
from the soil particles and separate the free spores physically from the soil particles. Protocols
for spore purification from soil particles involve steps to accomplish both of these objectives.
The most common types of processing protocols can be broken down into three steps with the
first two working together to disrupt spore-soil interactions. The three processing steps are: (1)
introduce an aliquot of soil to an aqueous carrier medium; (2) mix the soil with the liquid to aid
in chemical and physical disassociation of spores from soil aggregates; and (3) separate and
concentrate spores away from soil particulates (Figure 2). In some cases, additional steps are
taken to concentrate and purify the final spore sample further.
2.1.1 Aqueous carrier media
The hydrophobic exosporidium of B. anthracis interacts with solid soil particles and requires
treatment prior to efficient spore recovery (54, 55). Adding an aqueous carrier medium to a soil
Slurry
the soil sample
Dissassociate
spores from soil
Separate and
Concentrate
spores from soil
Figure 2. Indirect soil processing flow diagram.
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sample creates a sample slurry that can be manipulated easily. While deionized water has been
utilized, chemical additives (buffers, chelating agents, surfactants, salts, emulsifiers) are often
included to aid spore-soil dissociation. Chelating agents (e.g., ethylenediaminetetraacetic acid
[EDTA], Chelex® 100) and surfactants (e.g., Triton™ X-100, TWEEN® 20, TWEEN® 80,
sodium dodecyl sulfate [SDS]) promote desorption of spores from soil particles, whereas salt
solutions (sodium chloride, aluminum sulfate) form a complex and precipitate extracellular DNA
and humic acids present within the soil (56). In a study conducted by DaSilva et al. (57), the
carrier medium (or spore extraction solution) was the most important factor influencing the
efficiency of extracting spores from wipes.
Within the reviewed studies, there were many different aqueous media used to separate spores
from soil samples. The most common type of carrier medium was a buffered solution or a buffer
solution with a surfactant (Appendix A). As previously mentioned, recovery efficiency data are
lacking in many studies. Table 1 outlines 10 studies that included recovery efficiency
information. Among these 10 studies were 14 soil types and 12 aqueous carrier media. Studies in
which recovery efficiency data were lacking or which looked at aqueous carrier media for
matrices other than soil (58, 59) are briefly summarized in Appendix A.
Triton™ X-100, TWEEN® 20, TWEEN® 80, and Nonidet™ P-40 are nonionic detergents used
to disrupt hydrophobic interactions between the spores and soil particles. Dragon and Rennie
(60) compared Nonidet™ P 40 to Triton™ X-100, and concluded that Triton™ X-100 was the
better detergent for separating spores from soil particles. However, no statistical results were
presented to support this conclusion. Rastogi et al. (61) noted that a pre-study experiment
showed no statistical difference in spore recovery between Triton™ X-100, TWEEN® 20, and
TWEEN® 80; however, the results were not detailed within the report. Da Silva et al. (57), in a
study assessing spore separation from wipes, concluded that the extraction solution (carrier
solution) PBS was the worst of those tested but the addition of TWEEN® 80 significantly
improved recovery efficiencies. While no study provided statistical evidence for an optimized
aqueous carrier medium, the individual studies each concluded that the addition of a surfactant
aided spore recovery when compared to PBS or sucrose solutions alone (57, 60).
Dabire et al. (62) compared a weak 0.1 Normal (N) sodium hydroxide (NaOH) solution to sterile
deionized water. The NaOH solution was meant to disrupt aggregates of sandy clay and clay
soils through chemical interaction to release the Pasteuriapenetrans spores. The basic solution
increased the recovery rates but not by a significant amount. Similarly, two other studies tested
the efficiency of a weak salt solution (0.08% sodium chloride). Santana et al. (63) reported
acceptable results while Ehlers et al. (64) found that deionized water alone yielded better
recovery rates from tropical soil samples.
As Table 1 demonstrates, spore recovery efficiency varied depending on the soil type and
aqueous carrier medium. Hong-Geller et al. (65) noted differences between strains of B.
anthracis with avirulent Sterne strain being more easily separated from wipes than the virulent
Ames strain. A number of other parameters not detailed within the table may also have
influenced the overall extraction efficiency (i.e., sample age, sample amount, dissociation
protocol, detection assay). Determination of an optimum aqueous carrier medium from the
available information is therefore difficult.
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Table 1. Spore Separation Aqueous Carrier Media (Percent Recovery)
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2.1.2 Spore-soil disassociation
Microbial cells are tightly bound to soil colloids with clay and organic matter posing particular
challenges in spore-soil separation (10). In an experiment conducted by Nicholson et al. (39),
99% of the natural spores present in a sandy test soil were associated with the soil aggregates and
not within the aqueous carrier medium, indicating that additional steps are needed to dissociate
the spores from the soil. Chemical additives to the aqueous carrier medium are used to help
disassociate spores from soil; however, physical means are also utilized. Physical agitation has
taken the form of manual shaking, gentle agitation, use of Stomacher® laboratory blending
paddle, use of blenders, vortexing, sonication, and/or bead beating.
Dabire et al. (62) noted that more energetic dispersion protocols yielded greater spore recovery
efficiencies. Dissociation of large soil aggregates was suggested as the primary cause for the
increased spore recoveries. Other studies have confirmed that more energetic dispersion
protocols aid in overall recovery rates. Da Silva et al. (57) determined that vortexing was
statistically superior to sonication for separating B. anthracis from wipe samples. Similarly,
Courtois et al. (70) saw enhanced homogenization using a Waring blender over sonication or
chemical treatment alone. Lindahl and Bakken (71) noted that ultrasonication treatment and
shaking were inferior dispersion protocols when compared to using a Waring® blender. Even
with significant physical disruption, spore-soil interactions are powerful and may be only slightly
interrupted by physical agitation (39). An estimated 35% - 55% of the spores remained with
large stable aggregates following total soil disruption with agate marbles (62).
2.1.3 Physical separation of spores from soil
After spore-soil disassociation, spores can be separated physically from soil particles. While
some protocols do not require debris-free sample material for downstream detection assays
(culture, direct DNA extraction followed by molecular detection), many assays have higher
sensitivities with purified samples. High and low speed centrifugation, high specific gravity
separation (HSGS), immunomagnetic separation (IMS), filtration, and extended settling times
have each been utilized with varying success (Figure 3).
8
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B.
Aqueous Carrier
Medium
* *
* *
Soil Slurry
'D.
~\
®
I ~ \
•v J
fH.
A
y
#
*
4
-\ r
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y
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*
-*
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Solution j
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>
-
V
*
*
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(G.
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y
®
¦ ¦ ¦ H ¦ ¦ ¦ ¦ H
20|xm
0.45|xm
Figure 3. Indirect soil processing steps. A. Initial soil sample with soil-bound spores (~). B. Soil sample with added
aqueous carrier medium. C. Soil slurry with soil-bound spores and dissociated spores (®). D -1. Separation and Concentration
methods; Density Separation via: D. Low-speed centrifugation; E. High-speed centrifugation; F. High specific gravity
separation; G. Settling. H. Affinity capture using antibody-labeled magnetic beads (-*-). I. Filtration with 20 (im and 0.45 (im
pore size filters.
2.1.3.1 Density Separation
Low-speed centrifugation precipitates only dense soil particles leaving the more buoyant free
dissociated spores within the supernatant. Spores remaining bound to soil particles after
dissociation steps are removed with the soil particles. Spores within the supernatant can be
detected directly or concentrated through additional steps. Fitzpatrick et al. (67) and Roh et al.
(67, 72) used low speed centrifugation (123 x g and 2900 x g5 respectively) to separate soil
particles from the microbial cell fraction before DNA extraction. However, neither study
specifically targeted B. anthracis within the soil samples. Fitzpatrick et al. (67) recovered less
than 7% of the Coxiella burnetii present within the sandy soil samples, while Roh et al. (72)
9
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concluded that separation of cells prior to DNA extraction (indirect DNA extraction) yielded a
lower quantity of higher quality DNA extracts when compared to directly extracted soil samples.
As part of the isolation steps of the GABRI (ground anthrax Bacillus refined identification)
protocol, low speed centrifugation (657x g) of the soil sample is combined with incubation of the
supernatant (54°C for 20 min) prior to plating on agar (73). Using GABRI (followed by DNA
extraction and PCR), B. anthracis was isolated from 16/20 soil samples, but specific
performance data for the protocol were not available (73). A slightly modified version of the
GABRI method which used a 2000 rpm centrifugation speed, an incubation temperature of 64°C
for 20 min, and addition of 50 (_ig/(_iL of Fosfomycin to the supernatant, was able to isolate B.
anthracis from 100% of spiked and naturally contaminated soil samples in the study (74).
In contrast to low-speed centrifugation, high-speed centrifugation precipitates free spores along
with other microorganisms or soil particles present in the initial suspension. Therefore, high-
speed centrifugation is typically used to wash away humic acids and extracellular DNA within a
soil sample before further analysis (7). Seven studies herein utilized a high-speed centrifugation
step to aid in pre-washing the soil samples (5, 7, 65, 75-78). A maximum 1 g aliquot of soil was
utilized in these studies. In all but one study (77), soil particles were not separated from the
spores before lysis and DNA extraction. Jain et al. (77) found that additional soil pre-washing
before DNA extraction diminished PCR inhibition. Conversely, Gulledge et al. (7) determined
that pre-washed soil samples were not significantly different from soil samples placed directly
into the extraction kit process.
A settling period following vigorous shaking has been used in combination with other separation
procedures. In one study (79), a settling time was included after a vortexing step to separate 45 g
of dense sand particles from the freed Bacillus atrophaeus subsp. globigii spores within a
phosphate buffered saline amended with 0.5% TWEEN® 20 (PBST) solution. The supernatant
was then withdrawn before concentrating the spores with high-speed centrifugation. Therefore,
only spores dissociated from the sand by physical and chemical means and suspended in the
collected supernatant were carried through to DNA extraction.
The studies discussed in this review used four types of HSGS solutions: sucrose solutions (40,
60, 68, 80, 81), Nycodenz® density gradient medium (64, 70, 82-84), sodium bromide solution
(39), and two-phase liquid systems (85, 86). Irrespective of gradient medium, HSGS utilizes
differences in specific gravity to separate B. anthracis from other organisms and soil
components. Depending upon the sub-species, B. anthracis ranges in density from 1.162 - 1.184
g mL"1 (87) and is concentrated in the upper layers of most density gradient solutions post-
centrifugation. Sucrose and Nycodenz® solutions are utilized at densities of 1.22 and 1.3 g mL"1,
respectively, allowing spores to concentrate within the uppermost layer following centrifugation.
Two-phase liquid systems and sodium bromide include a wider range of liquid densities within a
single centrifugation tube (1.0 - 1.3 g mL"1) (39, 85). The spore-rich layer in these solutions is
midway within the tube; the uppermost layers with lower density cell debris must be removed
prior to spore collection. The added step of removing the uppermost layer significantly reduced
the spore yield within the final sample. Nicholson et al. (39) determined that the addition of
sodium bromide HSGS decreased indigenous spore yields from 2% - 4% to less than 0.1%.
However, even with the added step, Agarwal et al. (88) were able to recover 9% - 20% of B.
10
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anthracis Sterne within garden soil and over 50% from sand samples using a two-phase liquid
HSGS protocol.
The utility of Nycodenz® HSGS for recovering B. anthracis is unknown. Multiple researchers
have used Nycodenz® HSGS to separate bacterial cells from soil. However, no studies found for
this review used it to target spores specifically. Rather, the Nycodenz® density gradient medium
was used to prepare soil samples for total indigenous DNA extraction. Furthermore, there are
conflicting efficiency results for Nycodenz® HSGS. Lindahl and Bakken (71) recovered 24% -
42%) of the total indigenous cells within loam soil samples using Nycodenz® HSGS, while
Courtois et al. (70) determined that 85% of the cells quantified by direct microscopy counts were
lost after Nycodenz® HSGS separation.
Two comparative studies concluded that HSGS with 1.22 g mL"1 sucrose was the most effective
protocol for spore separation, though yields were not high (40, 60). Ryu et al. (40) found a
minimum LOD of 106 spores g"1 in Korean soils when spores were heat-lysed and detected
through PCR. In a similar study conducted by Dragon and Rennie (60), an LOD of
approximately 40 spores g"1 was determined for B. anthracis American Type Culture Collection
(ATCC) 4229 spores spiked (2-8 xlO5 spores) into field and wallow soils and extracted using
HSGS with 1.22 mg mL"1 sucrose and Triton X-100 solution. B. anthracis spores were detected
via culture after being spiked in field soil, wallow soil, and potting soil with recoveries of
approximately 4.5%, 5-8%, and 28%, respectively (60). In a third study, HSGS was evaluated
using Arizona test dust, Minnesota loam, potting soil, and sand spiked withl04-106 B. anthracis
Sterne 34F2 spores g"1. However, results were variable (68). The highest recoveries from culture
shown by the Bradley et al. (68) study were 9% (104 spores g"1), 5.8% (105 spores g"1), 5% (106
spores g"1), and 3.7% (104 spores g"1) of the spores spiked into potting soil, sand, Arizona test
dust, and Minnesota loam, respectively.
2.1.3.2 Affinity Capture
Bradley et al. (68) went on to compare sucrose HSGS to automated IMS. IMS utilizes antibodies
bound to magnetic beads to capture and concentrate B. anthracis. Following the addition of the
aqueous carrier medium and spore-soil dissociation, paramagnetic beads conjugated with
polyclonal B. anthracis antibodies are added to the soil sample suspension. Any spores present
within the sample bind to the antibodies. A magnetic rod is used to transfer the paramagnetic
beads with the antibody-bound spores to tubes with PBST solution. The PBST solution allows
the spores to be concentrated, washed, and released from the beads within a final sample tube. In
the final sample tube, the spores can be verified and quantified through a variety of assays,
including culture and PCR.
Bradley et al. (68) compared automated IMS recovery efficiencies for four different soil types
(Arizona test dust, Minnesota loam, potting soil, and sand). For all tested soils, the minimum
2 1
LOD was 10 spores g" of soil. Recoveries ranged from 17% - 51% among the four soils with
the Minnesota loam and potting soil being the most recalcitrant. The study did note that there
were a few microorganisms other than B. anthracis detected after culture with sand and potting
soil, and the authors hypothesized that aggregates containing magnetic soil particles and
11
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microorganisms were transferred through to the final sample. The Centers for Disease Control
and Prevention (CDC) Division of Bioterrorism and Preparedness Response tested antibody
specificity using time-resolved fluorescence. Results indicate that the B. anthracis antibody can
differentiate between closely related and nonrelated bacterial strains (only B. anthracis spores
were tested, not vegetative cells) (68). In an effort to improve the selectivity of IMS-treated soil
samples, Chenau et al. (89) directly extracted SASP-B from the spores for highly sensitive liquid
chromatography-tandem mass spectrometry detection. While selectivity was improved, the
added processing/detection steps decreased overall sensitivity to a LOD of 7 x 104 spores g"1 soil.
Yitzhaki et al. (90) were able to increase the adsorption of B. anthracis to immunoglobulin G
(IgG) labeled magnetic beads significantly with the addition of didecyldimethylammonium
bromide (DDAB) in pure laboratory standards. However, they also conjectured that adsorption
efficiencies would decrease by 20% - 40% for environmental samples. While IMS adsorption
efficiencies for environmental samples may be of concern, IMS does have the advantage of
being rapid. Fisher et al. (91) developed a rapid IMS-lateral flow protocol for identification of B.
anthracis in liquid samples within approximately 40 minutes. Bruno and Yu (92) also noted that
IMS was attractive for detecting B. anthracis in soil due to its simplicity, speed, and utility for
large numbers of samples. "Liquid-phase" immunoassays have been used for spore capture of
B. anthracis from dust by adding anti-5. anthracis antibodies to spore suspensions, incubating,
and further processing the sample as described by Hang et al. (93).
2.1.3.3 Filtration
Dabire et al. (62) and Isabel et al. (62, 69) utilized filtration to separate dissociated spores from
soil samples. Using a series of sieves to separate a soil sample into different particle size
fractions (>200 |im, 50 - 200 |im, 20 - 50 |im, and 0-20 |im), Dabire et al. (62) concentrated
Pasteuriapenetrans spores into the 0-20 |im sample fraction. However, a significant number of
spores were also associated with larger clay aggregates. Isabel et al. (69) used dual syringe filters
to establish rapid filtration separation-based sample processing. Their protocol utilized a 5-|im
pore-sized filter to separate spores from a variety of matrices including soil, dust, silica, and
bentonite and an additional 0.45 |im pore-sized filter to concentrate the freed spores. On average
for all matrices tested, 68% and 51% of the B. atrophaeus spores were recovered using the
capture filtration step only (0.45 |im pore-sized filter) and the dual filter protocol, respectively.
2.2 Direct Processing: DNA Extraction of Bulk Soils and Selective Culture Media
Direct processing protocols include direct culturing of soil and bulk DNA extraction. It has been
said that clinical identification of B. anthracis is not a problem; it is the presence of organic and
inorganic compounds and extraneous bacterial flora (particularly other spore- forming Bacillus
species) in environmental samples that interferes with B. anthracis detection and identification
(75). While selective media have been used to isolate other Bacillus species from soil (94) and
DNA extraction has been evaluated for isolation of B. anthracis from other matrices such as
food, powders, and clinical samples (95, 96) or for other bacterial organisms in soil (97), direct
processing of B. anthracis in soil requires more research. Extensive testing must be done to
12
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develop a selective culture medium that allows differentiation between B. anthracis and other
Bacillus spp. In addition, DNA obtained directly from soil samples must be purified carefully
and DNA signature specificity must be carefully selected to ensure species selectivity.
2.2.1 Selective culture media
Although culturing is time consuming and laborious for large sample sets, there are times when it
is critical to determine the quantity of viable B. anthracis within a sample or to assess the
antimicrobial susceptibility of an environmental strain (98, 99). Soils abound with diverse
species of microorganisms. Researchers have sought a B. anthracis specific agar-based medium
that deters background cultures and other Bacillus species, yet allows B. anthracis to flourish.
Sheep or horse blood is often included within a B. anthracis selective medium to evaluate
hemolysis. B. anthracis is non-hemolytic, and the agar will remain red surrounding the cultures.
Conversely, the near-neighbor bacterium B. cereus is hemolytic and produces an enzyme that
lyses red blood cells and changes the appearance of the agar surrounding B. cereus growth. This
review found six culture media selective for B. anthracis within the open literature (Table 2).
13
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Table 2. B. anthracis Selective Culture Media
Reference
Medium
(State)
Incubation
Temperature
°C
Incubation
Time (hr)
Remarks
Bradley et al.,
2011 (68)
TSA II (s) and
PLET (s)
35
24-48
PLET CFU were within 72% - 77% of the number of CFU found on non-selective TSA
II plates. PLET agar was recommended for recovery of B. anthracis from unknown
soils. Recoveries ranged from 1% - 51% depending on the soil and separation
protocol.
Dragon and
Rennie, 2001
(60)
SBA (s), PLET
(s) and PLET
supplemented
with 5%
defibrinated
horse blood (s)
37
24-48
SBA recovered significantly more spores of B. anthracis ATCC 4229 than PLET
medium. PLET allowed a few nori-anthracis Bacillus strains to grow. Supplemented
PLET allowed more nori-anthracis test strains to germinate and grow. However,
except for B. subtilis and B. pumilus, the riori-anthracis strains could be differentiated
from B. anthracis. Recoveries ranged from 4% - 28% depending on the soil and
separation protocol employed.
Fasanella etal.,
2013(74)
TSMP
37
24-48
Authors stated that TSMP has the same efficacy as PLET for isolating B. anthracis.
No recovery efficiencies were recorded.
Juergensmeyer
etal., 2006
(100)
ChrA (s)
35-37
24, 48
Due to a mutation in B. anthracis, the activity of PC-PLC is reduced compared to other
Bacillus species. Therefore, colonies of other Bacillus species turn teal after 24 hr, and
colonies of B. anthracis turn teal only after 48 hr allowing for species level
discrimination. No recovery efficiencies were recorded.
Jula et al., 2007
(101)
PLET (s) and
SBA (s)
37
24
Spores were concentrated using a 0.45 |jm filter. The deposit on the filter was heat
treated to lyse vegetative cells prior to plating. Approximately 1/3 of the B. anthracis-
like colonies on the PLET agar were actually B. anthracis. No recovery efficiencies
were recorded.
Luna et al.,
2005 (102)
MEP (s) and
ChrA (s)
30, 35
24, 24-48
Suspected B. anthracis isolates were cultured on the MEP agar or ChrA to aid in
distinguishing between B. anthracis and B. anthracis-WWe organisms. No recovery
efficiencies were recorded.
Luna et al.,
2009 (98)
Antibiotic
amended
PLET (/ or s)
30
24, 48, 72,
96
Selectivity of PLET was improved with sulfamethoxazole, trimethoprim, polymyxin B,
and lysozyme, and can select for 6. anthracis in agricultural, environmental, and
forensic investigations of B. anthracis isolates. No recovery efficiencies were recorded.
Marston etal.,
2008 (66)
SBA (s), PLET
(s) and ChrA
(s)
37
24-48
PLET agar is more sensitive than ChrA agar. Recovery ranged from 0.5% - 8%
depending on the soil.
Tomaso et al.,
2006 (99)
CEI (s) and
ABA (s)
37
24
Non-anthracis spp. turn turquoise on CEI agar, whereas B. anthracis does not. ABA
contains supplements to inhibit fast growing environmental organisms and sheep
blood to allow hemolytic differentiation between Bacillus spp. Percent recovery on
ABA and CEI was 72% and 71%, respectively.
14
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Vahedi ef a/., PLET (s) and 37 24-48 Spores were concentrated using a 0.45 |jm filter. The deposit on the filter was heat
2009 (103) SBA (s) treated to lyse vegetative cells prior to plating. Confirmatory biochemical tests were
conducted with all B. anthracis-WWe colonies. No recovery efficiencies were recorded
ABA - Anthrax Blood Agar'M
CEI - Cereus Ident Agar™
ChrA - R & F® anthracis chromogenic agar
CFU - Colony forming units
I - Liquid medium
MEP - Mannitol-egg yolk-polymyxin B agar
PC-PLC - Phosphatidylcholine-specific phospholipase C
PLET - Polymixin B, lysozyme, ethylenediaminetetraacetic acid, thallous acetate agar
SBA - Sheep blood agar
s - Solid agar medium
TSMP- Columbia blood agar with trimethoprim, sulfamethoxazole, methanol, and polymyxin
TSA 11 - Trypticase® soy agar with 5% sheep blood
15
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A compounding difficulty for spore culturing is the existence of superdormant spores of Bacillus
species (32, 33). Superdormant spores require elevated concentrations of germination
compounds and/or extended incubation periods before they germinate. While most spores
germinate within minutes once exposed to adequate growth conditions, naturally occurring
superdormant spores may require hours to days before germination occurs (104). Therefore, even
after a suitable processing or culturing protocol for most spores is employed, any superdormant
spores present within a sample might not germinate. Previous work has indicated that B.
anthracis superdormant spores might react in a manner similar to B. cereus and B. megaterium
superdormant spores. However, no studies were found that specifically outline how to process
soil-borne superdormant spores (33). Mannitol-egg yolk-polymyxin B agar (MEP) has been used
as a selective and differential medium (102). B. anthracis colonies on MEP are colorless with a
weak lecithinase production giving an opaque zone just beneath the colony, whereas other
organisms turn yellow with mannitol fermentation and are translucent without lecithinase
production. While MEP can distinguish B. anthracis from a number of Bacillus species, MEP is
not sufficiently reliable (102).
R & F® anthracis chromogenic agar (ChrA) has also been used to distinguish B. anthracis from
other Bacillus species (66, 100). ChrA includes the substrate 5-bromo-4-chloro-3-indoxylcholine
phosphate, which converts to a water-insoluble blue dye in the presence of phosphatidylcholine-
specific phospholipase C (PC-PLC). Among Bacillus species, only B. anthracis, B. cereus, and
B. thuringiensis produce PC-PLC. For B. cereus and B. thuringiensis, the color change occurs
within 24 hours, whereas for B. anthracis, the color change is seen only after 48 hours due to a
nonsense mutation that reduces PC-PLC activity and eliminates its hemolytic activity (100) .
Juergensmeyer et al. (100) tested ChrA on spiked soil, sewage, paper, cloth, and blood samples.
Selective ingredients within the ChrA reduced the number of background soil flora capable of
growing on the ChrA to approximately 103 colony forming units (CFU) g"1. The color changing
properties of B. anthracis colonies on the ChrA allowed them to be distinguished easily among
the remaining background flora. B. anthracis colonies are harder to identify when B. cereus and
B. thuringiensis growth is overwhelming (100). Luna et al. (102) suggested that either MEP agar
or ChrA could be added to the Laboratory Response Network (LRN) protocol to help reduce the
number of suspected B. anthracis positive environmental samples requiring confirmational
testing (102).(The LRN, established by the CDC, is tasked with maintaining an integrated
network of laboratories that can respond to bioterrorism, chemical terrorism and other public
health emergencies.)
Tomaso et al. (99) examined the utility of Cereus Ident Agar™ (CEI) and Anthrax Blood Agar™
(ABA). CEI contains a chromatogenic substrate similar to ChrA. Only the turquoise coloration
of non-anthracis spp. can be used to discriminate B. anthracis from its near-neighbors (99). ABA
is a nutrient medium containing sheep blood and supplements to inhibit many fast growing
organisms. The hemolysin gene of B. cereus has been found within B. anthracis strains on a few
occasions, so hemolytic morphology is not a definitive assessment (99). B. anthracis could be
identified appropriately 71% and 72% of the time on CEI and ABA, respectively, when tested
against 92 environmental B. anthracis isolates and 132 other Bacillus spp. (99).
Polymyxin B, lysozyme, ethylenediaminetetraacetic acid, thallous acetate (PLET) is another
selective medium described in the literature. Bradley et al. (68) compared PLET agar to
16
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Trypticase® soy agar amended with 5% sheep red blood cells (TSAII) and determined the two
media to be comparable. After overnight growth, the PLET CFU were within 72% - 77% of the
TSA II CFU counts indicating adequate germination on the selective medium compared to the
non-selective medium. The overall recommendation was to use PLET agar for B. anthracis
recovery from unknown soil samples (68). However, little analytical support was given for this
suggestion. In a comparison of PLET to ChrA, Marston et al. (66) found that PLET was more
sensitive and more selective against other Bacillus and non-Bacillus species than ChrA.
However, PLET and ChrA had similar B. anthracis recovery rates for the bacteria when it was
spiked into Texas soil and Arizona test dust. Jula et al. (101) and Vahedi et al. (101, 103) used
selective PLET agar to differentiate B. anthracis colonies from other organisms. In each study,
they found that PLET was not specific for B. anthracis. After confirmatory biochemical testing
of multiple B. anthracis-like colonies, B. cereus, B. circulans, B. megaterium, B. subtilis and B.
sphaericus were all found on the original formulation of PLET agar. Only approximately 33% of
the B. anthracis-like colonies tested by Jula et al. (101) were in fact B. anthracis colonies.
Researchers have sought to improve the original 1966 formulation of PLET medium for better
selectivity (60, 98). In 2001, Dragon and Rennie (60) compared non-selective sheep blood agar
(SBA) to PLET and PLET amended with 5% defibrinated horse blood. Results demonstrated that
although the original PLET was more selective than PLET amended with horse blood, SBA
recovered significantly more B. anthracis than PLET. These findings led Dragon and Rennie
(60) to conclude that although PLET is selective for B. anthracis, PLET is not an ideal recovery
medium and may underestimate the number of spores within a sample. In 2009, Luna et al. (98)
sought to improve the utility of the original PLET medium further with the addition of lysozyme
(150,000 units L ') and the antibiotics sulfamethoxazole (38 jag inL '), trimethoprim (2 jag
mL-1), and polymyxin B (15,000 units L *). The modified PLET medium was tested against 283
environmental isolates, including 23 isolates of B. anthracis, and could be used in a liquid broth
or solid agar state. The additional antibiotics and lysozyme within the medium inhibited the
growth of other Bacillus species and delayed the appearance of resistant B. cereus. Work-safety
regulations in some countries prevent the use of PLET due to the high concentrations of toxic
thallium acetate (1.9 mg/L) within its composition (98, 99). Based upon the breadth of data
known regarding the specificity of modified PLET medium, modified PLET medium is the most
promising selective culture medium for B. anthracis documented within the literature.
2.2.2 Direct DNA extraction from bulk soils
Prior to performing PCR analysis, DNA must be extracted from the sample. For direct DNA
extraction, a small amount of soil (0.1 g - 10 g) is added to a DNA extraction buffer. Cells from
all organisms present in a sample are lysed through both chemical and physical means. DNA-
identifying reactions are used to seek, amplify, and detect the DNA segments of interest within
the total mass of extracted DNA. The DNA extraction protocol influences the quantity and
quality of template DNA available.
DNA can be extracted directly from bulk soils or from spores already removed from the soil.
Delmont et al. (82) and Roh et al. (72, 82) found that direct DNA extraction produced over 33
times more DNA per gram of soil than indirect HSGS separation and over 100 times more DNA
per gram of soil than low-speed centrifugation separation. While indirect DNA extraction had a
17
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reduced concentration of DNA, the overall quality of DNA was increased compared to direct
extraction protocols. Lombard et al. (56) estimated that as much as 40% of the total microbial
DNA contained within a soil sample is lost during direct DNA extraction, and an additional 30%
can be lost during downstream purification procedures. The initial soil conditions also have an
effect on the quality and quantity of the DNA extracts. Zhou et al. (10) found that as the carbon
content increased within the bulk soil sample, so too did the DNA yield; while Sjostedt et al. (9)
noted that organic content is directly proportional to humic acids, known PCR inhibitors.
Therefore, appropriate measures must be taken to reduce PCR inhibitors in soil DNA extracts.
2.2.2.1 DNA Extraction Kits
Numerous kits are available from vendors that are specific for DNA extraction from soil
samples. In addition, extraction kits commercialized for other sample types have been used for
environmental soils. Herein, details including cost, time requirements, sample size, and LOD of
28 extraction kits and one manual protocol utilizing liquid nitrogen are presented (Appendix B).
There are two critical steps to cellular DNA extraction: cell lysis and DNA separation. The
components of most kits are proprietary, but there are a few general types of lysis and DNA
separation protocols. Many extraction kits utilize a combination of chemical disruption
(detergents) and physical agitation (bead beating) for effective lysis of cellular membranes and
release of spore DNA. Kuske et al. (105) found that 40 freeze-thaw cycles with liquid nitrogen
were not sufficient to lyse B. atrophaeus spores, but a combination of chemical and physical
agitation showed promising lysing efficiency. Once released, DNA is often bound to silica filters
or magnetic beads for purification. Humic acids, polysaccharides, and urea show solubility
properties equivalent to DNA and are often co-extracted, especially at higher pHs (3, 106).
Washing steps are utilized to reduce the presence of co-extracted compounds post-lysis before
purified DNA is concentrated in an elution buffer. In particular, polyvinylpyrrolidone (PVPP) is
used to adsorb inhibiting phenols, including humic acids (106). The final elution buffer often
contains Tris and EDTA to protect the extracted DNA from nuclease activity over time (106).
2.2.2.2 Comparison of DNA Extraction Kits for Soil Samples
While there are a multitude of commercial extraction kits available for soil samples, determining
the overall best kit is difficult. This literature search found only three studies that directly
compared two or more extraction kits for analyzing B. anthracis in environmental soil samples.
Gulledge et al. (7) demonstrated the utility of a PLET enrichment step, but concluded that no one
kit from the five tested was superior. Bradley et al. (68) determined that the QIAamp® DNA
Blood Mini Kit (QIAGEN; Valencia, CA) was more efficient for Arizona test dust, while the
UltraClean® Soil DNA Isolation Kit (MO BIO Laboratories; Carlsbad, CA) was more efficient
for potting soil. The most comprehensive comparison looked at six commercial DNA extraction
kits and three soil types: sand, clay, and loam. In this assessment, Dineen et al. (6) determined
that the FastDNA® SPIN Kit for Soil (Qbiogene; Solon, OH) yielded significantly higher
amounts of spore DNA from each of the three tested soil types.
18
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Other researchers have sought an optimum extraction kit for detecting other organisms from soil.
Whitehouse et al. (107) compared extraction kits for Francisella tularensis in multiple soils. F.
tularensis is a non-sporulating gram-negative organism and is easier to lyse than B. anthracis.
Whitehouse et al. (107) concluded that of the five commercial kits assessed, the UltraClean® Soil
DNA Isolation Kit outperformed the other kits in the quantity and quality of purified F.
tularensis DNA, having an LOD of 20 CFU g"1 of soil in all three tested soil types. The next best
was the PowerMax® Soil DNA Isolation Kit (MO BIO Laboratories; Carlsbad, CA) with an
LOD calculated at 100 CFU g"1 for all tested soil types. Interestingly, PCR inhibition was seen
only in samples extracted from the commercial potting soil with the QIAamp DNA Stool Mini
Kit (QIAGEN; Valencia, CA). A study by Fitzpatrick et al. (67) analyzed the effect of combining
extraction kits using Coxiella burnetii in sandy soil by comparing the QIAamp DNA Stool Mini
Kit to the QIAamp DNA Mini Kit (QIAGEN; Valencia, CA) and the UltraClean® Soil DNA
Isolation Kit when used singly and in sequence. Results showed that utilizing two kits in series
nearly eliminated the presence of inhibition within final PCR reactions; however, the additional
kit also reduced the overall DNA yield. Using C. burnetii spiked soil samples, they saw a
maximum genomic equivalent yield of 4.3% using the QIAamp DNA Stool Mini Kit alone. The
addition of a second extraction kit reduced the yield to less than 2%, demonstrating a significant
trade-off between DNA purity and DNA yield.
Two studies were found that compared DNA extraction kits using spiked household powders.
Though these studies did not utilize soil as a sample matrix, the sample media do provide insight
into the ability of the extraction kits to eliminate inhibition. Dauphin et al. (76) compared five
commercial kits using B. anthracis Ames spores in baking soda, talcum powder, and cornstarch.
Of the five tested kits, the UltraClean® Microbial DNA Isolation Kit (MO BIO Laboratories;
Carlsbad, CA) yielded the only DNA extract without viable spores, thereby significantly
reducing the risk to laboratory personnel. In a similar study setup, Rose et al. (108) spiked
multiple household materials with B. globigii. Their assessment found the PrepFilter™ Forensic
DNA Extraction Kit (Applied Biosystems; Foster City, CA) to be the best kit for extracting DNA
from powder samples; however, when including the sampled liquids and solids, the best overall
kit was the UltraClean® Microbial DNA Isolation Kit.
The most commonly used commercial extraction kits for soil samples found in the literature
search were the UltraClean® Soil DNA Isolation Kit and the Powersoil® DNA Isolation Kit, both
produced by MO BIO Laboratories (Carlsbad, CA). Both kits require approximately 90 minutes
for bead-beating lysis followed by a silica spin filter to concentrate the extracted DNA. While the
UltraClean® Soil DNA Isolation Kit can process a larger quantity of soil (1.0 g versus 0.25 g),
the primary difference between the two kits is the presence of an Inhibitor Removal Technology®
within the Powersoil® DNA Isolation Kit. In addition, each kit has a large volume companion
that uses the same technology to process 10 g samples. Whitehouse et al. (107) compared the
technologies for two kits; the UltraClean® Soil DNA Isolation Kit with a sample volume of 0.1 g
of soil and the PowerMax® Soil DNA Isolation Kit with a sample volume of 10 g of soil. The
UltraClean® Soil DNA Isolation Kit outperformed the PowerMax® Soil DNA Isolation Kit;
however, the differences were minimal (107). The soil conditions apparently have a pronounced
effect on the quality and quantity of extracted DNA.
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Care should be taken when using different lots of DNA extraction kits. Bushon et al.(109),
studied variability in DNA extraction of B. anthracis, F. tularensis, and Vibrio cholerae using
three different lots of the MO BIO Powersoil® DNA extraction kits and found significant
differences between the lots for all three organisms. The authors suggested that if different lots
of extraction kits are to be used, the lots should be checked for consistency, quality control
measures should be used, and new standard curves should be run with each new lot (109).
2.2.3 Enrichment Steps
Enrichment steps have been added to processing protocols to help improve recovery of spores
from samples that contain a low density of spores (5, 7, 9, 110). Addition of an enrichment
medium to the sample allows both germination of spores and growth of vegetative cells. As
nutrients are depleted, spore-forming bacteria begin sporulation, while the proportion of
vegetative cells and other non-spore forming bacteria decreases or are killed (110). Incubation
and heat treatment can be used other kill remaining vegetative cells (110). Patel et al. (110)
evaluated the recovery of B. thuringiensis spores from 58 soil samples that included enrichment
with glucose yeast extract salt medium as part of sample processing and were able to recover 55-
75% of the B. thuringiensis spores from the samples. The use of selective enrichment agar
significantly lowered the detection limits in three studies (5, 7, 9). In particular, Gulledge et al.
(7) found that a PLET enrichment step lowered the detection limits by as much as six orders of
magnitude. The relatively new process of rapid-viability PCR (RV-PCR) also incorporates an
enrichment step between two PCR reactions to determine the presence of germinated B.
anthracis spores rapidly within a collected sample (111, 112). Currently, no soil samples have
been analyzed using RV-PCR; however, optimization of this assay for soil could help reduce the
time required to determine both the quantity and viability of B. anthracis in soil.
2.3 Purification Protocols
Because endospores of B. anthracis are highly resistant to unfavorable environmental conditions
in comparison to vegetative cells (34, 60), purification protocols such as heat treatment and
treatment with ethanol are used to help improve recovery of spores from soil and may be used
during either direct or indirect processing of the sample. Heat treatment is a method of
purification that has been used as part of the soil processing protocol to kill off vegetative cells in
soil samples while leaving viable spores (7, 77, 101, 103). Dry heat treatment (incubation in a
dry oven at 80 °C) of soil samples containing B. thuringiensis was evaluated by Santana et al.
(63), who found that isolation of B. thuringiensis from soil was improved after a five-hour dry-
heat treatment, although a more recent study by Patel et al. (110) was not able to achieve similar
results. Bacillus spores have been shown to be resistant to ethanol, so ethanol has alternatively
been used for removing vegetative cells from the sample (60). Dragon and Rennie (60)
compared spore stock samples of B. anthracis, vegetative B. cereus, and vegetative
Pseudomonas aeruginosa treated with both heat (incubation for 20 min in 63 °C water bath) and
50% ethanol and found that both treatments were equally effective in removing vegetative cells
from the stock while maintaining viability of the spores.
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3.0 Conclusions
Developing an ideal protocol for processing soil samples before microbiological assessment is
challenging. As evident through this review, a significant amount of work has been done to
ascertain the most efficient protocol for processing soil samples for B. anthracis detection. Direct
and indirect protocols for sample processing were reviewed in detail. Direct processing utilizes
bulk sample aliquots without first separating spores from soil particles, while indirect processing
uses multiple steps to separate spores from other organisms and particles prior to detection.
Direct and indirect DNA processing steps have associated advantages and disadvantages.
As described, multiple indirect soil processing protocols have been used to separated, anthracis
from soil particles. Indirect sample separation steps increase the proportion of target spores
within the final detected sample; however, spore loss prior to detection also increases. The
presence of a detergent in the aqueous carrier medium was consistently found to improve the
separation of spores from soil particles. However, no consensus on an optimum aqueous carrier
medium could be determined from among the reviewed works. Future research focusing on the
aqueous carrier medium for processing multiple soil types under uniform dissociation and
separation conditions would help fill this gap.
Spore/soil separation is a critical step in determining the overall recovery efficiency of indirect
processing protocols. IMS is an attractive option for separating B. anthracis in soil due to its
simplicity, speed, and utility for large numbers of samples, but continued work on IMS and its
ability to bind B. anthracis selectively at low concentrations is needed. The overall utility of
HSGS as a separation protocol needs to be determined before HSGS is applied within large-scale
projects. Although novel dual syringe filtration has shown promise for being able to separate
spores rapidly from diverse matrices, future work that combines an optimized aqueous carrier
medium with the dual filter steps may be needed to increase recovery rates further.
Direct soil processing falls under two principal types: culturing B. anthracis on selective agar
and bulk DNA extraction. When samples are directly processed, there is a potential for
background organisms to overwhelm the detection assay and prevent target spores from being
observed. Researchers have sought a B. anthracis-specific medium that deters background
cultures and other Bacillus species and yet allows B. anthracis propagation and identification.
Several B. anthracis-selective media have been developed. Based upon the amount of specificity
testing, modified PLET medium was identified as the most promising selective culture medium
for B. anthracis documented in the literature. The use of selective enrichment agar during
sample processing might improve recovery of spores from soil samples with low spore density.
To date no studies have utilized modified PLET agar as an enrichment step prior to B. anthracis
detection. Future recovery efficiencies could be dramatically increased with such an effort.
There are commercial kits available to extract DNA directly from bulk soil samples, and allow
for automated processing, reducing human exposure within the laboratories. While there are
numerous advantages to using a commercial kit for sample processing, unfortunately, due to the
difference in study designs, it is difficult to determine an overall optimum DNA extraction kit
from the currently available data. An optimized soil DNA extraction kit is needed; there has yet
21
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to be a soil DNA extraction study that compares multiple soil extraction kits uniformly across
multiple soil types to determine their overall DNA recovery.
The type of sample processing employed, direct or indirect, depends upon the desired
downstream applications (71). For DNA detection assays, direct bulk DNA extraction with
suitable DNA purification steps may be more appropriate. However, indirect processing might be
more appropriate if viability testing is required.
Regardless of whether direct or indirect processing protocols are employed, the overall recovery
rates and confidence intervals are critical pieces of information for downstream human health
and consequence decisions. As shown through this review, an optimized soil processing protocol
with a known recovery rate and associated confidence intervals is needed. Calculations for
recovery rates should be included in future studies. A reliable processing protocol would allow
for multiple technicians and laboratories to produce high quality uniform results in the event of a
B. anthracis release.
4.0QuaIity Assurance
This literature review was conducted under an approved quality assurance and quality control
plan. The only minor deviation from the QA/QC plan was a change to the title of the report.
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30
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Appendix A — Table of Reviewed H. anthracis Soil Studies and their Design Elements
31
-------
Appendix A: Table of Reviewed B. anthracis Soil Studies and their Design Elements
First Author,
Year
Organism(s)
Soil Type
Soil
Amt.
(g)
Soil
Processing
Aqueous Carrier
Solution
Spore/Soil
Disassociation
Spore/Soil
Separation
Lysis and DNA
Extraction Protocol
Detection Assay
Spore
Spike
(spore
g"1 soil)
LOD
(spores
g1
soil)
°/o
Recovery
Remarks
Agarwal, 2002
(88)
B. anthracis
Sterne
Sterile garden
soil,
sterile sand
Not
known
Indirect
PBS with polyethylene
glycol 4000
Vigorous
homogenization
Centrifugation
into two
phases of the
polymer
system
None
Immunofluorescence
microscopy with
fluorescein
isothiocyanate (FITC)-
conjugated antibody
against formalin-
inactivated spores of
B. anthracis Sterne
103 - 107
soil:
14000
sand:
5600
Soil: 9 - 20
Sand: 51 -
59
Agarwal attributed
recovery differences
between sand and
garden soil are to
flocculation/adsorptio
n of spores to soil
particles. Sonication
or other mechanical
disruption may aid in
disrupting this bond.
Balestrazzi,
2009 (3)
B. subtilis
Medium
textured
loamy sand
5
Direct
None: direct lysis
None
None
2% SDS, 1%
cetyltrimethylammonium
bromide (CTAB), 60 °C
2% SDS, 4% CTAB, 60
°C
2% SDS, 1% CTAB, 60
°C, liquid N2 grind
3% SDS, 1.2% PVP,
microwave thermal
shock
microwave thermal
shock, 3% SDS, 1.2%
PVP, liquid N2 grind
PCR southern blot and
PCR for swrAA gene
104 - 10s
2000
2000
2000
>2 x 10s
2x 10s
ND
Microwave based
approaches were not
effective and led to
~tenfold less spore
disruption.
Beyer, 1999 (4)
B. anthracis
Former
tannery sites
100
Direct
Trypticase 81 soy broth
(TSB) enrichment
medium
None
None
Invitrogen Easy-DNA™
Kit
PCR-Enzyme linked
immunosorbent assay
(ELISA) for pXOl,
pX02, and
chromosome
0.01 - 1
0.1
ND
Samples are enriched
twice before DNA
extraction.
Bielawaska-
Drozd, 2008
(75)
B. anthracis
34F2, 211
Sandy, forest,
wetland
0.1
Direct
TSB enrichment
medium
None
None
PLET enrichment nested
PCR PLET enrichment
PCR-ELISA
0.1 g soil boiled in TSB,
Nested PCR targeting
pag and cap genes
10 - 10s
10
10 - 100
ND
ND
Compared three
spore isolation
protocols for B.
anthracis in soil: (1)
double incubation in
TSB followed by DNA
centrifuged and washed extraction and a
in distilled water nested PCR
amplification, (2)
non-selective pre-
enrichment in TSB
followed by DNA
extraction and PCR-
32
-------
ELISA, (3) thermal
protocol where soil is
boiled for 10 minutes
(min) before DNA
extraction. Protocols
1 and 2 gave the best
results, and no
observed differences
were found between
the three types of soil
evaluated.
Bradley, 2011
(68)
B. anthracis
Sterne 34F2
Sand,
AZ dust,
potting soil,
MN loam
Indirect
PBS: Tween® 20,
Sucrose: Triton™ X-
100 solution
End-over-end
mixing
IMS
sucrose HSGS
(1.22 g mL1)
AIMS, UltraClean-1 Soil
DNA Isolation Kit
AIMS, QIAamp DNA
Blood Mini Kit
PLET culture
10 - 104
102 - 103
104 - 107
IMS -
sand: 51
AZ dust:
29 Potting
soil: 17
MN loam:
17
HSGS-
sand: 5.8
AZ dust: 5
Potting
soil: 9
MN loam:
3.7
Optimization of the
automatic IMS
protocol revealed that
separation of the B.
anthracis from soil
was best
accomplished by
preprocessing the soil
slurry samples by
sonicating and
vortexing (three min
each) to disrupt
clumps, filtering
through a 30 |jm pore
size filter, allowing
the slurry to settle,
and removing the
liquid from the top of
the sediment and
placing it in the IMS
tray.
Bruno, 1996
B. anthracis
Dark brown
0.1
Indirect
PBS
Not described
IMS
None
Immunomagnetic-
0 - 106 Sterne:
ND
Though there was a
(92)
Sterne, Ames,
Vollum
soil,
light
yellowish
sandy soil
electro-
chemi luminescent
(IM-ECL) detection
with polyclonal goat
antiserum
100
Ames:
104
Vollum :
105
loss of sensitivity
once soil was added,
the authors still liked
IM-ECL because of its
speed, simplicity, and
ease of use for large
sample sets.
Chenau, 2011
B. anthracis
Soil,
0.01
Indirect
4-(2-Hydroxyethyl)-l-
Vortex
IMS -
Immunocapture step
Immunocapture -
103 - 10s 7xl04
ND
Only one soil matrix
(89)
Sterne and 4
others, nine
other Bacillus
species
milk
piperazineethane
sulfonic acid/bovine
serum albumin
(HEPES/BSA) solution
Immuno
globulin G
(IgG) labeled
beads
followed by 80%
trifluoroacetic acid (TFA)
protein extraction,
neutralization, and
digestion
Liquid
Chromatography/
Mass Spectrometry
(LC/MS) targeted at
SASP-B proteins
was tested to show
proof of principle.
33
-------
Cheun, 2003
(5)
B. anthracis
Pasteur II
Commercial
peat soil,
nine field
samples
Indirect
70% ethanol
Gentle shaking
None
Wash, no enrichment
TSB, FastDNA® SPIN Kit
for Soil
Wash, single enrichment
TSB, FastDNA® SPIN Kit
for Soil
Wash, double
enrichment TSB,
FastDNA® SPIN Kit for
Soil
Nested and real-time 1
PCR targeting pag,
capA, and sap genes
103
1000
10
Indirect: Waring8'
blender
homogenization
Direct: none
ND 1 g of soil contains
103 - 106 spores of
different microbes,
therefore it would be
difficult to identify
one B. anthracis
spore g"1. Soil
samples are usually
heat-treated to kill
nonsporulated
bacterial cells, but
this study found that
heat treatment
generated false
positives.
Courtois, 2001
(70)
B.
thuringiensis
Sandy loam
Indirect & Indirect: 0.5 g soil into
direct 0.05 M pyrophosphate,
0.9% NaCI, or water,
Direct: none
Indirect:
HSGS -
Nycodenz'8',
Direct: none
Direct extraction:
manual chemical lysis
with and without bead-
beating
Indirect extraction:
Nycodenz'8' HSGS
followed by chemical
lysis
PCR targeting 16S None ND ND Bacteria present in a
rRNA and dot-blot soil sample depend
analysis upon the chemical
and physical
properties of the soil.
Percentage of
bacteria extracted
was not affected by
the buffer; however,
85% of the cells
detected by
microscopy cell
counts in the original
soil suspensions were
lost post Nycodenz®
HSGS.
Homogenization was
enhanced over
sanitation or chemical
treatments using a
Waring8' blender.
DaSilva, 2011 Green
(57)
fluorescent
protein (GFP)-
labeled B.
anthracis
Sterne
Wipes -
rayon, cotton,
polyester
None Direct water,
water with Tween'8' 80,
PBS,
PBS with Tween'8' 80
Vortex or sonicate
None
None
Direct culture on LB
agar plates
2x 105
ND
3-100 The addition of
Tween'8' 80 to the
carrier medium
significantly improved
the overall recovery
efficiency. Vortexing
physically separated
the spores from the
wipe material better
than sonicating.
Extraction efficiency
was dependant on
the extraction
solution and wipe
selected.
34
-------
Dabire, 2001
Pasteuria
Sandy clay,
10
Indirect
Distilled water or NaOH
End-over-end
Sieve bank
None
Malassez counting
106
ND
75-87
Increasing the energy
(62)
penetrans
clay
mixing
(200, 50, 20
Mm)
chamber microscopy
during washing steps
increased the %
recovery of the
inoculated spores.
Dauphin, 2009
B. anthracis
Baking soda,
0.025
Indirect
PBS
Vortex
Low speed
NucliSENS- Isolation Kit
pXOl, pX02
101- 106
106
ND
Spores were spiked
(76)
corn starch,
talcum
powder
centrifugation
- supernatant
used for DNA
extraction
QIAamp DNA Blood Mini
Kit
UltraClean® Microbial
DNA Isolation Kit
chromosome
106
107
into 0.025 g of soil
and washed before
using each kit. The
UltraClean1- Kit had
no viable spores in
the extraction
product.
Delmont, 2011
All soil
Park grass
Indirect
Indirect &
Indirect: 60 g soil into
Indirect: Waring81
Indirect:
Indirect: processed
PCR targeting the
None
ND
ND
Although the direct
(82)
organisms
silty clay,
loam
: 60
Direct:
0.5
direct
0.9% NaCI,
Direct: none
blender
homogenization
Direct: none
Nycodenz-1
HSGS,
Direct: none
sample into FastPrep
Lysing system
Direct: 0.5g soil into MP
Biomedical FastPrep
system
intergenic spacer
region between 16S
and 23S ribosomal
sequences
extraction protocol is
less time consuming
and uses less soil,
indirect DNA
extraction reduces
the proportion of
eukaryotic sequences
and increases the
DNA length of the
recovered DNA
strands.
Dineen, 2010
B. cereusl
Sand,
0.1 -
Direct
None: kit
None
None
Powersoil1- DNA
qPCR targeting the
107 -109
107- 10s
11-35
The selection of an
(6)
strain
clay,
loam
1.0
Isolation Kit
Soil Master™ DNA
Extraction Kit
EZNA® Soil DNA Kit
ZR Soil Microbe DNA
Kit™
FastDNA® SPIN kits for
phosphatidylinositol-
specific phospholipase
C gene of B. cereus
(Pl-PLQ
for sand
and
loam,
107 or
below for
clay
appropriate kit
depends upon the
initial soil conditions
and the downstream
applications. The
FastDNA®1 Spin Kit
gave the highest yield
of DNA while the
EZNA® Soil DNA and
PowerSoil® DNA Kits
were more efficient at
removing inhibitors.
Soil
n 1-2-3™ Platinum Path
Sample Purification Kit
Dragon, 2001
B. anthracis
Potting soil,
2.5
Indirect &
Deionized water or
Shaken by hand
Low speed
HSGS, heat treatment
PLET culture,
Unknown 40
PS: 28,
"Although PLET is
(60)
ATCC 4229
field soil,
direct
sucrose solution
centrifugation
SBA culture
for both
field: 6,
selective for B.
wallow soil
- supernatant
HSGS, ethanol treatment
carrier
wallow:
anthracis, it is not an
used for
solutions
4.5
optimal recovery
sucrose HSGS
Deionized water, heat
medium and may
(1.14 -1.22 g
treatment
miss anthrax spores
mL"1)
in a sample." Ethanol
Deionized water, ethanol
purification proved as
treatment
effective as heat
35
-------
purification.
Ehers, 2008
Mixed
Ferralsol -
10
Indirect
Water or 0.8% NaCI
Waring® blender
Nycodenz-1
None
Quantified by acridine
3.7 xlO9
ND
water:
Water carrier solution
(64)
community
tropical soil
high in iron
and
aluminum
solution
homogenization
HSGS
orange direct counts
10.6
NaCI: 4.6
with pH amendment
to 7.5 gave the
greatest soil bacteria
yield after gradient
separation; however,
water without pH
modification gave
highest soil species
richness. Using 0.8%
NaCI with pH
amendment gave the
best purity.
The selection of
extraction protocol for
soil samples should
depend on the
purpose of the study.
EPA, 2012
B. globigii
Sand
Indirect
Indirect &
Indirect: PBS -Tween-1
Indirect: vigorous
Indirect:
Indirect: 45 g PBST
qPCR targeting recF
106
104
<1
Significant difference
(79)
: 45,
Direct:
0.25
direct
20
Direct: none
mixing
Direct: none
supernatant
from sand
settling high-
speed
centrifuged to
precipitate
spores,
Direct: none
wash, 0.25 g Powersoil-1
DNA Isolation Kit
Direct: 0.25 g
Powersoil-1 DNA
Isolation kit
gene
106
between 0.25 g and
45 g soil sample
aliquots. Presumably,
the 45 g samples
included a much
higher concentration
of spores; therefore,
DNA was above the
LOD.
Fasanella, 2012
B. anthracis
Soil from
7.5
Indirect
Sterile distilled water
Shaken
Low-speed
Supernatant incubated
Plated on
None
ND
ND
Ground anthrax
(73)
contaminated
farm
with 0.5% Tween-1 20
centrifugation
54 °C for 20 min.
Phosphomycin tryptose
soya broth added to
supernatant.
trimethoprim
sulfamethoxazole
polymixin5% sheep
blood agar
Bacillus refined
identification (GABRI)
protocol used to
recover B. anthracis
from Bangladesh soils
at outbreak site.
Fasanella, 2013
B. anthracis
Soil from
7.5
Indirect
Sterile distilled water
Vortexing for 30
Centrifugation
Tryptose Phosphate
Plated on Columbia
Spiked
ND
ND
The modified GABRI
(74)
contaminated
farm and
garden soil
spiked with B.
anthracis
with 0.5% Tween-1 20
min
at 2000 rpm
for 5 min.
Supernatant
incubated at
64 °C for 20
min.
Broth with 50 |jg/|jL
Fosfomycin added to
supernatant.
blood agar with
trimethoprim
sulfamethoxazole,
methanol, polymixin
samples
spiked
with 500
spores
per 7.5 g
sample
method was able to
isolate B. anthracis
from 100% of both
naturally
contaminated and
artificially
contaminated soil
samples.
36
-------
Fisher, 2009
(91)
B. anthracis
ATCC 14185
Milk,
water
10 mL Indirect
PBS
None
IMS
None
Lateral-flow immune-
chromatographic
device for
visualization of
various antigens
106 CFU
mL"1
5x 105
CFU mL"1
85-95 Not a soil protocol,
but rather a fluid milk
/water protocol for
food testing.
Fitz patrick,
2010 (67)
Coxiella
burnetii
20 soils from
across U.S.
Indirect
PBS
Vortex
Low-speed to
separate soil
followed by
high-speed
centrifugation
of
supernatant
to concentrate
spores
UltraClean-1 Soil DNA
Isolation kit
QIAmp DNA Minikit
QIAamp DNA Stool
Minikit
PCR for IS1111 gene
from C. burnetii
800 - 106 ND Max 4.3 C. burnetii is Gram-
negative. However,
the kits compared are
relevant for B.
anthracis detection.
Combining two kits
eliminated any seen
inhibition; however,
combining kits also
reduced DNA
(maximum yield was
4.3%) yield. The
precipitated spores
from the high-speed
centrifugation were
used to compare DNA
extraction kits.
Frostegard,
1999 (106)
B. anthracis
Sterne
vegetative
cells
Five French
sandy, clay
soils
1 Australian
sandy clay
0.2 Direct None: direct in situ
lysis
Waring® blender
grinding,
sonication,
vortexing
None
In situ freeze thaw with
DNA extraction in buffer
ranging in pH from 6.0 -
10.0
Dot blot hybridization 107 -109
ND ND For all soils tested,
DNA yield increased
with pH of the buffer.
However, larger
amounts of humic
materials were
released at higher pH
as well.
Griffin, 2009
B. anthracis
U.S. soils
0.25
Direct
None
None
None
1 g UltraClean-1 Soil DNA
PCR targeting the None
170
ND
LOD study done with
(51)
Isolation Kit
rpoB gene for Bacillus
cells not spores.
genus,
0.25 g Powersoil® DNA
PCR targeting rpoB
4
Isolation Kit
gene specific for B.
anthracis
37
-------
Gulledge, 2010
(7)
B. anthracis
Pasteur and
Sterne
FL sand, TX
sand, and
commercial
garden soil
(Peat)
0.1- Indirect & Pretreatment solution:
0.5 direct sodium pyrophosphate,
EDTA, Tris-CI
Vortex
None
UltraClean-1 Soil DNA
Isolation Kit
Soil Master™ DNA
Extraction Kit
FastDNA® SPIN Kit for
Soil
BioRobot™ M48
Workstation
PLET enrichment,
UltraClean-1 Soil DNA
Isolation Kit
PLET enrichment,
Soil Master™ DNA
Extraction Kit
PLET enrichment,
FastDNA® SPIN Kit for
Soil
Hybridization and PCR 10 -107 106
for capC, pag, and ief
genes
106 sand,
Peat>107
107
>107
105
sands,
Peat>107
100
100
ND Overnight enrichment
with PLET broth
lowered the detection
limits of four of the
five protocols by
several logs (2 - 6
logio). No significant
difference between
the untreated and
pretreated soils
(direct kit lysis and
indirect wash before
kit lysis). No one kit
gave superior DNA
recovery, and soil
type and organic load
should be considered
before selecting the
appropriate kit.
PLET enrichment,
MagNA Pure® LC
>107
PLET enrichment,
BioRobot M48
Workstation
100
Hang, 2008
(93)
B. anthracis
Sterne
Office
vacuum dust
Indirect
PBS with Tween-1 20
and BSA
Vortex
IMS
Liquid-phased
immunoassay
Sandwich and liquid-
phased immunoassay
103 - 107 4 x 104
Spores spores
mL"1 mL"1
ND Spores were spiked
into wipe samples
after removing dust
from the wipe. Brain
heart infusion
(medium) (BHI) broth
induced spore
germination within
five minutes.
Hong-Geller,
2010 (65)
B. anthracis
Sterne and
Ames
Yersinia pestis
A1122 and
C092
Swabs and
wipes off
glass,
stainless
steel, vinyl
and plastic
None
Indirect
PBS - Tween® 20
Vortex
None
FastDNA® spin kit for
soil
qPCR targeting pXOl 107
ND
Sterne:
>90 ;
Ames 2-75
No significant
difference was found
between swab and
wipe for B. anthracis.
Sterne spores were
easier to recover than
Ames spores. Spores
were recovered with
higher efficiency from
hydrophilic surfaces.
38
-------
Irenge, 2010
304 bacterial
14 soils
2
Direct
None
None
None
PowerMax-1 Soil DNA
qPCR targeting
104- 107
25 fg
ND
Sought to find B.
(113)
strains, 37 B.
anthracis
strains, Ames,
Sterne,
Vollum, Delta-
Sterne (soil
spikes, Ames)
Isolation Kit
phosphate (ptsl) and
adenylosuccinate
synthetase (purA)
genes
anthracis specific
primers.
Isabel, 2012
B. atrophaeus
23 common
0.2
Indirect
PBS
Mixing
Filtration (5
BD GenePhm Lysis Kit
qPCR targeting the
5000
5000
51
Assessed the utility of
(69)
powders
including
garden soil
Mm)
atpD gene
a syringe prefilter and
wash protocol.
Developed the DARE
procedure - dual-filter
for applied recovery
of microbial particles
from environmental
and powdery
samples. One filter is
used to separate
spores from soil, and
the next filter is used
to concentrate
spores.
Jacobsen, 1992
Pseudomonas
Sandy loam
50
Indirect
Chelex-1 100 in
Manual and orbital
Low speed
Manual DNA extraction
Dot blot, southern
2.5 x 107
ND
ND
An early study looking
(97)
cepacia
buffered solution
shaker
centrifugation
blotting, hybridization
CFU g"1
at non-sporulating
Gram-negative
Pseudomonas and
how to extract it from
soil samples.
Jain, 2011
B. anthracis
Field soil,
0.1
Indirect
PBS with Triton™ X-
Vortex
Low speed
Spore pellet 100 °C heat
Real-time LAMP
20 - 10s
50
ND
Real-time LAMP
(77)
talcum
powder
100
centrifugation
lysis
detecting pa# gene
detection was 2,000
times more sensitive
than traditional PCR
in this analysis
Juergensmeyer,
B. anthracis
Soil,
Not
Direct
Water
Vortex
Settle
None
Cultured on ChrA
107
10 - 103
ND
ChrA can distinguish
2006 (100)
multiple strains
sewage,
blood,
paper,
cotton
known
plates
between B. cereus, B.
thuringiensis and B.
anthracis with the
rate of color change
in the colonies after
48 hours.
Jula, 2007
B. anthracis
668 Iranian
Not
Direct
Distilled water
Mixed
None
Freeze thaw lysis
PLET and blood agar
None
ND
ND
21 of the 668 soils
(101)
soils
known
culture
contained virulent B.
anthracis isolates.
Spores in settled
supernatant were
filtered to
concentrate.
39
-------
Kane, 2009
B. giobigii
AZ dust on
0.5
Indirect
pH 9.5 buffer with
Vortex
Filtration
Heat-treated to lyse qPCR recFgene
102 - 104 200
ND
Inhibition at 103 and
(112)
ATCC 9372
wipes
Tween-1 80
vegetative cells. Spores
heat lysed in PCR plate
95 °C for 20 min before
PCR. Incubated in TSB
for 16 hrs
104 spores with 0.5 g
of AZ dust. Protocol
able to detect only
germinated spores.
Samples filtered to
concentrate spores,
not to separate soil
from the spores.
Kuske, 2006
(12)
B. anthracis,
Francisella
tularensis, Y.
pestis,
Clostridium
perfringens
129 U.S. soil
samples
0.5
Direct
None
None
None
Bead beating ethanol
precipitation with spin
Sephadex® G-200
column cleanup
PCR targeting pag
gene
None ND ND 0.1 pg template DNA
represents 17-46
genomic equivalents (
GEq), no work done
to determine the
extraction LOD;
extracted 0.2-146 |jg
of DNA g"1 soil.
Leishman, 2010
(59)
B. anthracis
Water,
whole milk,
orange juice
3 mL
Indirect
Hexadecane solution
Vortex
Spores
separate in
hexadecane
layer due to
hydrophobic
:ies
None
Microbial adherence
to hydrocarbons
(MATH) with culturing
on TSA plates
103
Spores
mL"1
ND
Hexadecane
separation protocols
were not effective.
Lindhal, 1996 B. subtiiis, Gamma
(83) Escherichia coii sterilized
agricultural
clay loam
6 or 60 Indirect
20 g soil into 0.05 M
pyrophosphate pH 8.0
solution or water
Waring® blender
homogenization
Nycodenz-1
HSGS
Physical disruption and
chemical disruption of
cells from soil particles
Fluorescent
microscopy
enumeration by
acridine orange direct
counting
109 ND 24 - 42 Method of cell-soil
disruption depends on
the purpose of the
cell extraction.
Pyrophosphate
solution more
efficient than water.
Luna, 2009
283 species,
5 from FL, 5
0.5
Direct
Modified PLET broth
Vortex
None
None
Modified PLET agar
104
ND
ND
Modified PLET
(98)
162 B. cereus
group (23 B.
anthracis
strains), 50
other Bacillus
species
from TX
with antibiotics
selectivity against
Bacillus species.
Selectivity against
Bacillus species 100%
at 24 hours (hr) and
96.8% at 48 hr at 30
°C.
Maarit Niemi,
2001 (78)
Environmental
Clay top soil,
sandy soil
Indirect
Crombach buffer
Stomacher
homogenization
High-speed
centrifugation
Five DNA extraction
protocols with varying
amounts of SDS and
guanidine isothiocyanate
and a MoBio Soil DNA
Isolation Kit
PCR-denaturing
gradient gel
electrophoresis
(DGGE) targeting 16S
rDNA V3 variable
region
None ND ND Different isolation and
purification protocols
resulted in different
bacterial profiles from
a soil sample.
40
-------
Marston, 2008
16 B. anthracis
TX soil,
1
Indirect
PBS -Tween-1 20
Vortex
Settling time -
None
Culture on PLET and
107
ND
0.5 - 7.7
7.7% of the spiked
(66)
strains
AZ dust
supernatant
cultured
ChrA
spores were
recovered from the
TX soil sample using
PLET and ChrA, while
only 0.5% was
recovered from AZ
dust. Overall, PLET is
more sensitive and
selective than ChrA.
Naclerio, 2009
B. subtilis
pyroclastic
Not
Direct
Buffered peptone-
Vigorous vortexing
None
None
Vegetative cells lysed
1010
ND
ND
Soil column
(54)
topsoil
known
water
by heat and before
culturing on LB plates
experiments were
conducted to
ascertain the
interaction between
B. anthracis and soil.
Key finding was that
exosporium does not
play a role in B.
anthracis spore
retention with the
studied soil type.
Nicholson, 1999
Environmental
Three
100
Indirect
Chelex-1 100 in buffer
Vortex
Low speed
None
Culture on nutrient
None
ND
1.4-4.3
Authors suggested
(39)
B. anthracis
Sonoran
desert soils
centrifugation
with
supernatant
filtration -
some samples
further
processed
with NaBr
HSGS (1.0 -
1.5 g mL"1)
sporulation medium
(NSM)
post
Chelex-1
cleaning;
<1 post
NaBr HSGS
that a majority of the
spores within the
tested soils were
unrecoverable as the
spores remained
attached to the large
soil particles. Their
HSGS protocol
significantly reduced
spore yields.
Panning, 2007
B. cereus, B.
50
0.1
Direct
None
None
None
Pre-extraction: 100 |jL
qPCR for pag gene of
200 - 2 x
200 CFU
ND
The study concluded
(96)
anthracis
Sterne
environmenta
1 and clinical
samples
sample with Gentra™
systems cell lysis
solution Lysozyme and
Proteinase K
pXOl
105 CFU
mL"1
mL"1
that in light of the
sensitivity and safety
seen, the QIAamp
Viral RNA Mini Kit and
Gentra Puregene® Blood
the MagAttract DNA
Mini M48 Kit were
Kit
optimal for spore DNA
extraction in low and
QIAamp DNA Mini Kit
high throughput
settings, respectively.
Viral RNA Mini Kit
MagAttract®1 DNA Mini
M48 Kit
MagAttract Viral RNA
M48 Kit
41
-------
Parachin, 2010
Environmental Garden soil
5
Indirect
BactXtractor-M or
Vortex
2-phase liquid
Manual DNA extraction
Nonspecific 16S-rRNA None
ND
ND
The environmental
(85)
soil organisms
BactXtractor-H
homogenization
HSGS
or MO BIO PowerMax-1
Soil DNA Isolation Kit
amplification
DNA extracted after
gradient flotation was
comparable in yield
and purity to the
direct commercial
PowerSoil® Kit
extracts.
Patel
2013(110)
B.
thuringiensis
53 soil 1
samples from
diverse
geographical
regions in
India
Indirect Enrichment with sterile Shaker and heat
glucose yeast extract treatment
salt
Low speed None
centrifugation
Luria-Bertani broth
agar plates
None ND 55-75% The enrichment
protocol recovered a
higher percentage of
spores than treatment
of the samples with
heat and sodium
acetate treatments
performed as
described by (63) and
Pillai, 1991 (80) Rhizobium
Pima clay 1
Indirect
Calcium chloride
Vortex
Sucrose HSGS
No specific DNA
PCR targeting the Tn5 107 - 10s
1 -10
ND
Spores were not
leguminosarum
loam, brazito
sandy loam
solution
(1.33 g mL"1)
extraction - cell solution
directly added to PCR for
heat lysis
insertion mutant
CFU
tested in this study.
The first supernatant
fraction following
SHMP wash with low-
speed centrifugation
and supernatant
filtration is sufficient
to quantify and
extract bacterial cells.
Their protocol
included low-speed
centrifugation,
supernatant filtration,
high-speed
centrifugation, and
final cell pellet
separation with
Nycodenz-1 HSGS.
Rastogi, 2009
Plasmid-free
Carpet,
1.7 cm2 Direct
Bacto™ buffered
Sonicate and vortex None
None
Culture on tryptic soy 106 - 10s
ND
25
Study sought the
(61)
strain of B.
anthracis
ceiling tile,
concrete,
steel,
wallboard,
wood
peptone water with
Tween-1 80
agar plates
decontamination
effects of chlorine
dioxide gas and
vaporous hydrogen
peroxide. A pre-study
experiment showed
that Tween-1 80,
Tween-1 20, and
Triton™ X-100
showed no statistical
difference in spore
Pote, 2010 Environmental Lake 100
(84) sediments
Indirect 2% sodium
hexametaphosphate
(SHMP)
Vortex
Low-speed PowerMax-1 Soil DNA
centrifugation, Isolation Kit
supernatant
filtration,
high-speed
centrifugation
and pellet
Nycodenz-1
HSGS
DNA quantified
through
spectrophotometry
None
ND
ND
42
-------
recovery.
Roh, 2006
(72)
Environmental
German soil,
sediment,
activated
sludge
0.1 Indirect & Indirect: 0.1 g soil into
direct buffer at pH 8.0, buffer
with surfactant; or
Chelex® 100
Direct: none
Indirect: 1 -10 hr
shake or
homogenized in
blender,
Direct: none
Indirect: low
speed
centrifugation,
Direct: none
Indirect: manual DNA
extraction
Direct: manual
extraction microwave
lysis, bead beating,
freeze-thaw lysis, or
Soil Master™ DNA
Isolation Kit
PCR targeting various
phylogenic groups
and restriction
enzyme digestions
None ND ND 0.1 g sample size
insufficient for
indirect extraction
protocols as shown
through ~hundredfold
increase of DNA yield
for direction
extraction.
Rose, 2011
(108)
B.
Biological
wash powder,
skimmed milk
powder,
flour,
talcum
powder,
spackling
powder
0.1 mL Direct
None
None
None
Instagene™ Matrix
UltraClean® Soil DNA
Isolation Kit
Extract-N-AmpT'
and Seed Kit
Plant
IT 1-2-3™ QFlow Kit
QuickGene DNA Tissue
Kits
PrepFilter™ Forensic
DNA Extraction Kit
PCR for Bg B-type 10s -1010 ND ND Study sought to find a
SASP gene single DNA extraction
protocol for liquids,
solids, and powders
in a BSL3 setting. The
Ultraclean-1 Microbial
DNA Isolation Kit was
statistically best
overall, and the
PrepFilter™ Kit was
best for the tested
powders.
MasterPure™ Complete
DNA and RNA
Purification Kit
Ryu, 2003
(40)
13 Bacillus
species
including 4 B.
anthracis
strains
Random soil
collected in
Korea
0.1 Indirect Indirect: sterile water,
10% Triton™ X-100 in
PBS or 1.22 g mL"1
sucrose plus Triton™
X-100 in PBS
Suspended and
centrifuged multiple
times
Low speed
centrifugation
Soil slurries incubated
for 20 min in
germination buffer, heat
lysed during initial
denaturation step of
PCR
Multiplex PCR
targeting pag, cap,
and sap genes
104 - 10s 106 -
>108
ND Sucrose/Triton™ X-
100 proved to be a
simple and effective
protocol as it was the
only one that gave
results at 106 spores
g"1. Hypothesized that
B. anthracis adheres
to a variety of solid
matrices with
hydrophobic
interactions;
therefore, solutions
with non-ionic
detergent and a high
concentration of
sucrose disrupt
hydrophobic
interactions and lift
the freed spores.
Sensitivity of
43
-------
germination
treatment was
reduced compared to
pure spore solutions.
qPCR system showed
identification of B.
anthracis at 104 spore
g"1 in three hr of
arrival at the
laboratory.
Saikaly, 2007
(55)
B. atrophaeus
spores and
cells
Synthetic
building
debris (SBD),
leachate
0.5
Direct
None
None
None
PowerSoil® DNA
Isolation Kit
qPCR targeting 16S-
23S rRNA ITS region
and recA gene
101- 107
Bg
spores
leachate
101,
SBD 102
ND
Amplification
efficiency for recA in
SBD was 87% for the
B. atrophaeus spores.
Santana, 2008
(63)
B.
thuringiensis
Venezuelan
soils
1
Direct
Dry heat followed by
saline solution
Vortexing
None
None
Spread plate LB agar
None
ND
60
Isolation of B.
thuringiensis from soil
better with a dry
preheat step.
Sjostedt, 1997
(9)
B. anthracis
Sterne and
Pasteur
Litter,
swamp,
meadow,
cultivated soil
0.1
Direct
None
None
None
Manual freeze thaw,
phenol/chloroform and
glass milk beads
PCR cap and ief
genes, southern blot
confirmation
107
103 - 104
ND
Detection was seen
only after enrichment
in Heart Infusion
Broth due
presumptively to
inhibiting compound
within the soil
samples.
Strati lo, 2012
(81)
Environmental
Soil from
Wood Buffalo
National Park
2.5
Indirect
Sucrose solution
Shaken by hand
Low speed
centrifugation
- supernatant
used for
sucrose HSGS
(1.14 -1.22 g
mL"1)
Suspected colonies were
processed with
PrepMansample
preparation reagent
PLET culture
None
ND
ND
Processing protocol
followed steps from
Dragon and
Rennie(60).
Tims, 2004 (58)
B. anthracis
Ames
Talcum
powder,
corn starch,
powder
sugar, baking
soda
0.001
Direct
PBS
Five min incubation
None
None
Biosensor assay
105
3.2 x 105
ND
Samples were spiked
with 105 spores and
tested.
Travers, 1987
(94)
B. anthracis,
B.
thuringiensis
WY soil
0.5
Direct
Sodium acetate
buffered LB broth
Shaker and heat
treatment
None
None
Culture on LB agar
plates
106
<100
ND
While B. thuringiensis
was the target of this
study, B. anthracis
was also removed
from the soil samples.
Vahedi, 2009
(103)
Environmental
Iranian soil
Not
known
Indirect
PBS
Overnight
incubation
Settling time -
supernatant
concentrated
Heat inactivation, freeze
thaw lysis, centrifugation
PLET cultures
followed by PCR
targeting B. anthraas
chromosome,
protective antigen,
and capsule
None
ND
ND
Soil samples were
cultured and positive
cultures were
confirmed with PCR.
Samples were filtered
to concentrate spores
in the settled
44
-------
supernatant.
Whitehouse,
2007
(107)
F. tularensis
Silt loam,
clay,
potting soil
0.1-10 Direct
None
None
None
Gentra Puregene-1 DNA
Purification Kit
QIAmp DNA Stool Mini
Kit
Soil Master® DNA
Extraction Kit
UltraClean® Soil DNA
Isolation Kit
PowerMax® Soil DNA
Isolation Kit
PCR for fopA gene
10 - 105
spores
2 x 102-
2x 104
500
102 -103
20
100
ND
UltraClean® and
PowerMax®1 Soil DNA
Isolation Kits were
the most consistent
and sensitive kits for
extracting F.
tularensis from soil.
Wielinga, 2011
(95)
B. anthratis,
B.
thuringiensis
corn meal,
whey
powder,
wheat flour,
soybean
flour,
corn grain,
Irish milk
0.1
Direct
None
None
None
NucliSENS8' lysis buffer
and NucliSENS® DNA
Magnetic Extraction
Reagents
Lysis buffer-soil slurry
cultured on BHI agar;
qPCR for B. anthracis
and B. thuringiensis
3x10" ND <1-60 Sampling matrix can
influence the DNA
extraction efficiency.
Yitzhaki, 2006
(90)
B subtilis, B.
thuringiensis,
B. anthracis
None
None
Indirect
PBS
Sonicate or shake
IMS
None
Electron microscopy
and flow cytometry
Zhou, 1996
(10)
Pseudomonas Loam,
sp. strain B13 sandy loam,
sandy clay
loam
Direct
None
None
None
Direct manual lysis with
CTAB extraction buffer,
SDS and proteinase K
PCR targeting 16S
rRNA, restricted
fragment length
polymorphism (RFLP),
southern blotting
Unknown ND 40 - 90 Cationic surfactant
aided in linking the
spores to the silica
magnetic particles
(increasing from 40 to
90% with the addition
of DDAB). Overall
adsorption to the
magnetic particles
was low.
Unknown ND 27 - 80 Significant correlation
was observed
between crude DNA
yield and soil organic
carbon content, as
the carbon content
increased so too did
the DNA yield.
AZ dust - Arizona Test Dust
BHI - Brain heart infusion medium
BSA - Bovine serum albumin
CFU - Colony forming units
'®
ChrA - R & F anthracis chromogenic agar
CTAB - Cetyltrimethylammonium bromide
DDAB - Didecyldimethylammonium bromide
DNA - Deoxyribonucleic acid
EDTA - Ethylenediaminetetraacetic acid
ELISA - Enzyme linked immunosorbent assay
EPA - U.S. Environmental Protection Agency
fg - Femtogram(s)
FITC - Fluorescein isothiocyanate
FL - Florida
GEq - Genomic equivalents
HEPES - 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
HSGS - High specific gravity separation
IgG - Immunoglobulin G
IM-ECL - Immunomagnetic-electrochemiluminescent
IMS - Immunomagnetic separation
ITS - Internally transcribed spacer region
LB - Luria broth
LC/MS - Liquid chromatography-mass spectrometry
LOD - Limit of detection
MN loam - Minnesota loam
ND - Not determined
PBS - Phosphate buffered saline
PCR - Polymerase chain reaction
PCR-DGGE - Polymerase chain reaction and denaturing
gradient gel electrophoresis
PLET - Polymyxin B, lysozyme, ethylenediaminetetraacetic
acid, thallous acetate
PVPP - Polyvinylpyrrolidone
qPCR - Quantitative polymerase chain reaction
rDNA - Ribosomal deoxyribonucleic acid
45
-------
RFLP - Restriction fragment length polymorphism (DNA
analysis)
RNA - Ribonucleic acid
rRNA - Ribosomal ribonucleic acid
RV-PCR - Rapid viability-polymerase chain reaction
SASP - Small acid-soluble proteins
SBA - Sheep blood agar
SBD - Synthetic building debris
SDS - Sodium dodecyl sulfate
SHMP - Sodium hexametaphosphate
TFA - Trifluoroacetic acid
®
TSA - Trypticase soy agar
®
TSB - Trypticase soy broth
46
-------
Appendix B — Table of Commercial DNA Kits Used for Direct Soil Analysis
47
-------
Appendix B: Table of Commercial DNA Kits Used for Direct Soil Analysis
Manufacturer
Kit
Protocol
Sample
Mass
(g)
Cost/
Sample
($)
Processing
Time
(hr)
Consumables
Additional
Reagents
Additional
Equipment
Reference
Organism
Soil Type
PCR target
gene
LOD
(CFU
g1)
°/o
Recovery
Notes
Applied Biosystems,
Foster City, CA
PrepFilter™
Forensic DNA
Extraction Kit
Detergent lysis,
magnetic bead DNA
concentration
0.1
4.3
3
Ethanol,
Isopropanol
Magnetic
stand
Rose eta/.,
2011 (108)
Bacillus globigii
Household
powders
B-type SASP
gene
ND
ND
Statistically best kit overall for dry
powders.
BD Molecular
Diagnostics
Franklin Lakes, NJ
GeneOhm™ Lysis
Kit
Bead beating lysis, heat
lysis
0.2
0.2
Tubes
Heat block
Isabel eta/.,
2012 (69)
B. atrophaeus
Garden soil
atpD
5000
51
Dauphin et
al., 2009 (76)
B. anthracis
Ames
Baking soda
pXOl, pX02,
chromosome
106
Viable spores were seen in the final
Corn starch
106
ND
extracts; however, kit gave the
greatest total yield of B. anthracis
DNA.
Guanidine
Talcum
107
bioMerieux Inc.,
Durham, NC
NucliSENS®
Isolation Kit
thiocyanate/Triton™ X-
100/Tris HCI lysis, silica
bead DNA concentration
0.2
9.4
3
Ethanol, Acetone
None
Weilinga et
al., 2011 (95)
B. anthracis, B.
thuringiensis
Multiple food
matrices
pXOl, pX02,
chromosome
ND
<1 -60
Sampling matrix influences the DNA
extraction efficiency.
Bio-Rad
Laboratories,
Hercules, CA
InstaGene™ Matrix
Heat lysis, no DNA
concentration -
inhibitors are bound
0.1
0.5
0.5
None
None
Rose eta!,
2011(108)
B. globigii
Household
powders
B-type SASP
gene
ND
Roh eta/.,
2006 (72)
Environmental
German soil,
sediment,
activated sludge
16S rRNA
ND
ND
Direct extraction resulted in eDNA
fragments of about only 12kb in size
due to significant shearing
throughout the process.
Whitehouse
Francisella
tu/arensis
Silt loam
100
Kit removed inhibitors from all three
soil types tested.
and Hottel,
Clay
fopA
1000
ND
Hot detergent lysis,
inhibitor removing resin-
filled spin columns and
DNA concentration
2007 (107)
Potting soil
1000
Epicentre®
Madison, WI
Soil Master® DNA
Extraction Kit
Sand
107
0.59
0.1
3.9
2
Ethanol
None
Dineen eta/.,
2010 (6)
B. cereusl-
strain
Clay
Pl-PLC
10s
0.00
Loam extracts required dilution to
dilute inhibition.
Loam
10s
0.01
FL sand
106
TX sand
106
ND
Gulledge et
a/., 2010 (7)
B. anthracis
Pasteur
Garden soil
capC, pag,
and lef
ND
Inhibition seen at concentrations
greater than 107 spores g"1 soil.
48
-------
Manufacturer
Kit
Protocol
Sample
Mass
(g)
Cost/
Sample
($)
Processing
Time
(hr)
Consumables
Additional
Reagents
Additional
Equipment
Reference
Organism
Soil Type
PCR target
gene
LOD
(CFU
g1)
°/o
Recovery
Notes
MasterPure™
Complete DNA and
RNA Purification Kit
Hot detergent lysis, DNA
precipitation
concentration
0.1
Rose eta/.,
2011 (108)
B. globigii
Household
powders
B-type SASP
gene
ND
Epicentre-'
Madison, WI
0.003
1.45
0.5
None
None
Luna eta/.,
2003 (114)
B. anthracis
Pasture
Flour, baking
soda, talcum
powder,
cornstarch
chromosome
BaS13
4000
ND
These results were found after
germination, heat shock, sonication
and autoclaving prior to DNA
extraction.
Fuji Film
Corporation,
Tokyo, Japan
QuickGene® DNA
Tissue Kit S and
QuickGene-Mini80
Detergent lysis, vacuum
filter DNA concentration
0.1
0.5
Ethanol
None
Rose eta/.,
2011 (108)
B. globigii
Household
powders
B-type SASP
gene
ND
ND
Dineen etal,
2010 (6)
B. cereusl-
strain
Sand
107
0.06
Idaho Technology
Bead beating lysis, DNA
binds to magnetic
Clay
Pl-PLC
ND
ND
Salt Lake City, UT
IT 1-2-3™ Sample
0.5
11.25
0.25
None
PickPen®
Loam
107
0.00
Now Biofire
Diagnostics
Purification Kits
beads, inhibition wash,
DNA concentration
1-M
Rose etal.,
2011 (108)
B. globigii
Household
powders
B-type SASP
gene
ND
ND
Whitehouse
Silt loam
20
Kit removed inhibitors from all three
soil types tested.
and Hottel,
F. tularensis
Clay
fopA
20
ND
2007 (107)
Potting soil
20
Dauphin et
al., 2009 (76)
B. anthracis
Ames
Baking soda
pXOl, pX02,
chromosome
106
At a concentration of 106 spores mL"1
Corn starch
106
ND
no viable spores were seen in the
final extract, and the final extract
have very clean DNA.
Talcum powder
107
MO BIO
Laboratories
Carlsbad, CA
Bead beating lysis, silica
spin filter DNA
concentration
Vortex
Griffin etal.,
2009 (51)
Bacillus species
N-S US transect
rpoB
170
ND
UltraClean® Soil
DNA Isolation Kit
1
3.78
1.5
Ethanol
adapter,
PowerVac®1
manifold
Fitzpatrick et
at., 2010 (67)
Coxiella burnetii
Light sandy soil
IS1111
FL sand
ND
Gulledge et
al., 2010 (7)
B. anthracis
TX sand
capC, pag,
and lef
105
ND
Inhibition seen at concentrations
Pasteur
Garden soil
106
greater than 107 spores g1 soil.
Bradley et a/.,
2011 (68)
B, anthracis
AZ dust
LRN
102
Better at extracting DNA from
potting soil than AZ dust.
Sterne
Potting soil
primer/probe
103
ND
Rose etal.,
2011 (108)
B. globigii
Household
powders
B-type SASP
gene
ND
ND
Statistically best kit overall across
multiple sample types among the kits
evaluated in this study.
49
-------
Manufacturer
Kit
Protocol
Sample
Mass
(g)
Cost/
Sample
($)
Processing
Time
(hr)
Consumables
Additional
Reagents
Additional
Equipment
Reference
Organism
Soil Type
PCR target
gene
LOD
(CFU
g1)
°/o
Recovery
Notes
Pote eta/.,
2010 (84)
Environmental
Lake sediments
None
ND
ND
Extracted DNA was only quantified
though spectrophotometry.
Whitehouse
Silt loam
100
Kit removed inhibitors from all three
soil types tested.
and Hottel,
F. tu/arensis
Clay
fopA
100
ND
2007 (107)
Potting soil
100
MO BIO
Laboratories
Carlsbad, CA
PowerMax® Soil
DNA Isolation Kit
Bead beating lysis,
Inhibitor Removal
Technologysilica spin
filter DNA concentration
10
20.3
0.5
None
50 mL
centrifuge
Irenge eta/.,
2010 (113)
B. anthracis; B.
cereus
Unknown
ptsl and purA
4
ND
LOD for PCR was 25 fg
(corresponded to Ct values of
35.85-38.33). Lowest soil spike
concentration 104 spore g"1.
Parachin et
a/., 2010 (85)
Environmental
soil organisms
Garden soil
16S rRNA
region
ND
ND
The environmental DNA extracted
after gradient flotation was
comparable in yield and purity to the
direct commercial PowerSoil-1 Kit
extracts.
Maarit Niemi
eta/., 2001
(78)
Environmental
Clay top soil,
sandy soil
16S rRNA V3
variable
region
ND
ND
Griffin eta/.,
2009 (51)
Bacillus species
Gulf coast soils
rpoB
4
ND
LOD for PCR was 25 fg (Ct values of
Dineen eta/.,
2010 (6)
B. cereus 1-
strain
Sand
107
5.28
35.85—38.33).
Clay
Pl-PLC
109
0.00
MO BIO
Laboratories
Carlsbad, CA
Bead beating lysis,
Vortex
Loam
107
0.22
PowersoN181 DNA
Isolation kit
Inhibitor Removal
Technologysilica spin
0.25
4.44
1.5
Ethanol
adapter,
PowerVac
EPA, 2012
(79)
106
ND
0.25 g of sand were directly
extracted using the kit.
filter DNA concentration
manifold
B. globigii
Sand
104
ND
45 g of sand were washed and the
remaining pellet was processed
through the kit.
SBD
recA
102
87
Saikaly et a/.,
B. atrophaeus
SBD
16S US
region
101
104
Saikaly et at. added a heat
incubation step before the
2007 (55)
Leachate
16STTS
region
101
97
PowerSoil-1 kit protocol, 70 °C for 20
min with solution CI.
None
Manual
Freeze-thaw lysis, DNA
precipitation
5
6
All
All
Balestrazzi et
a!., 2009 (3)
B. subti/is
Loamy sand
swrAA
104
99
Spores were much harder to lyse
than cells.
50
-------
Manufacturer
Kit
Protocol
Sample
Mass
(g)
Cost/
Sample
($)
Processing
Time
(hr)
Consumables
Additional
Reagents
Additional
Equipment
Reference
Organism
Soil Type
PCR target
gene
LOD
(CFU
g1)
°/o
Recovery
Notes
Omega Bio-Tek
Norcross, GA
EZNA® Soil DNA Kit
Bead beating lysis, heat
lysis, inhibitor removal
reagent, silica spin filter
DNA concentration
1
1.98
2.5
Tubes,
isopropanol,
ethanol
None
Dineen eta/.,
2010 (6)
B. cereusl-
strain
Sand
Pl-PLC
107
0.39
Clay
107
0.00
Loam
107
0.30
Qbiogene
Solon, OH
Now MP
Biomedicals
FastDNA® SPIN Kits
for Soil
Bead beating lysis, silica
spin filter DNA
concentration
0.5
4.81
2
Tubes, ethanol
FastPrep bead
beater
Cheun eta/.,
2003 (5)
B. anthracis
Garden soil
pag, capA,
and sap
103
ND
After two rounds of soil sample
enrichment the LOD decreased to 10
spores g"1 soil.
Dineen eta/.,
2010 (6)
B. cereusl-
strain
Sand
Pl-PLC
107
17.24
Loam extracts required 100X dilution
to reduce inhibition. Highest
Clay
107
11.54
Loam
107
2.80
recovery rates.
Gulledge et
al., 2010 (7)
B. anthracis
Pasteur
FL sand
capC, pag,
and lef
ND
Inhibition seen at concentrations
greater than 107 spores g"1 soil.
TX sand
107
ND
Garden soil
107
Hong-Geller
et al., 2010
(65)
B. anthracis
Sterne and
Ames
Swabs and wipes
off of glass,
stainless steel,
vinyl, and plastic
pXOl
ND
Sterne:
>90 Ames:
2-75
Ames spore DNA was more difficult
to recover than the Sterne spore
DNA.
Delmont et
al, 2011 (82)
All soil
organisms
Park grass soil
ribosomal
spacer region
ND
ND
Extracted soil to determine soil
metagenome. Bacillus species found
after bead beating at 18 - 21 cm
depth.
QIAGEN
Valencia, CA
Gentra Puregene®
Yeast/Bacteria Kit
Detergent lysis, alcohol
DNA precipitation
1
1
3
None
None
Whitehouse
and Hottel,
2007 (107)
F. tularensis
Silt loam
fopA
2000
Clay
20000
ND
Potting Soil
200
QIAGEN
Valencia, CA
Gentra Puregene®1
Blood Kit
Detergent lysis, alcohol
DNA precipitation
1
1
3
None
None
Panning et
al, 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
103 CFU
mL"1
ND
Panning used a lysozyme, proteinase
K, and heat pre-extraction cleanup
protocol prior Kit DNA extraction.
QIAGEN
Valencia, CA
QIAamp DNA Stool
Mini Kit
Hot detergent lysis,
inhibitor removing resin-
filled spin columns and
DNA concentration
0.5
3.84
1
Ethanol
None
Whitehouse
and Hottel,
2007 (107)
F. tularensis
Silt loam
fopA
500
Inhibition was seen in the potting soil
samples- no inhibition from silt loam
or clay soils.
Clay
500
ND
Potting soil
500
51
-------
Manufacturer
Kit
Protocol
Sample
Mass
(g)
Cost/
Sample
($)
Processing
Time
(hr)
Consumables
Additional
Reagents
Additional
Equipment
Reference
Organism
Soil Type
PCR target
gene
LOD
(CFU
g1)
°/o
Recovery
Notes
QIAGEN
Valencia, CA
QIAamp DNA Stool
Mini Kit and
MagAttract DNA
Stool Mini Kit
Hot detergent lysis,
InhibitEX-' adsorption of
PCR inhibitors, silica
spin column DNA
concentration
0.5
7.1
1.5
Ethanol
BioRobot M48
workstation
Gulledge et
a!, 2010 (7)
B. anthracis
Pasteur
FL sand
capC, pag,
and lef
105
Inhibition seen at concentrations
greater than 107 spores g1 soil.
TX sand
104
ND
Garden soil
ND
QIAGEN
Valencia, CA
QIAamp DNA Blood
Mini Kit
Enzyme lysis, silica spin
filter DNA concentration
1
2.64
1
Ethanol
None
Panning et
al., 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
2000
CFU
mL"1
ND
Dauphin et
al, 2009 (76)
B. anthracis
Ames
Baking soda
pXOl, pX02,
chromosome
107
The final extracts contained clean
DNA and some viable spores.
Corn starch
106
ND
Talcum Powder
10s
Bradley et al,
2011 (68)
B. anthracis
Sterne
AZ dust
LRN primers/
probes
102
ND
Better at extracting DNA from AZ
dust than potting soil.
Potting soil
103
QIAGEN
Valencia, CA
QIAamp DNA mini
Kit
Enzyme lysis, silica spin
filter DNA concentration
0.25
2.92
1
Ethanol
None
Panning et
al, 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
104 CFU
mL"1
ND
Panning used a lysozyme, protease
K, and heat pre-extraction cleanup
protocol prior Kit DNA extraction.
QIAGEN
Valencia, CA
QIAamp Viral RNA
mini Kit
Enzyme lysis, silica spin
filter DNA concentration
0.25
4.4
1
Ethanol
None
Panning et
al, 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
103 CFU
mL"1
ND
Panning used a lysozyme, protease
K, and heat pre-extraction cleanup
protocol prior Kit DNA extraction.
QIAGEN
Valencia, CA
MagAttract DNA
Mini M48 Kit
Enzyme lysis, Magnetic
Bead separation and
concentration
0.25
3.26
1.5
Ethanol
BioRobot M48
workstation
Panning et
al, 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
103 CFU
mL"1
ND
Panning used a lysozyme, protease
K, and heat pre-extraction cleanup
protocol prior Kit DNA extraction.
QIAGEN
Valencia, CA
MagAttract Viral
RNA M48 Kit
Enzyme lysis, Magnetic
Bead separation and
concentration
0.25
3.68
1.5
Ethanol
BioRobot M48
workstation
Panning et
al, 2007 (96)
B. cereus, B.
anthracis Sterne
Environmental
samples
pag
105 CFU
mL"1
ND
Panning used a lysozyme, protease
K, and heat pre-extraction cleanup
protocol prior Kit DNA extraction.
Roche
Indianapolis, IN
MagNA Pure LC
DNA Isolation Kit III
Soil prewash and
centrifuge, bead-beat
lysis, magnetic bead
technology
0.5
2.19
1.5
None
MagNA Pure
LC System
Gulledge et
al, 2010 (7)
B. anthracis
Pasteur
FL sand
capC, pag,
and lef
ND
Inhibition seen at concentrations
greater than 107 spores g"1 soil.
TX sand
ND
ND
Garden soil
ND
Sigma-Aldrich,
St. Louis. MO
Extract-N-Amp™
Plant and Seed Kit
Liquid N2 lysis, no DNA
concentration
1
2.1
0.25
PCR grade water
None
Rose et al,
2011 (108)
B. globigii
Household
powders
B-type SASP
gene
ND
ND
Kit did not perform well with the
tested media.
Zymo Research
Irvine, CA
ZR Soil Microbe
DNA Kit™
Bead beating lysis, silica
spin filter DNA
concentration
0.25
3.05
1.5
Tubes
None
Dineen eta!,
2010 (6)
B. cereus 1-
strain
Sand
Pl-PLC
107
0.04
Only 1 of 3 clay extracts were
detected at 107, 3 of 3 detected at
10s.
Clay
ND
Loam
107
0.02
AZ dust - Arizona test dust RBMS - Reference background matrix soil
CDC - Centers for Disease Control and Prevention RNA - Ribonucleic acid
DNA - Deoxyribonucleic acid rRNA - Ribosomal ribonucleic acid
EPA - U.S. Environmental Protection Agency SASP - Small acid-soluble proteins
LOD - Limit of detection SBD - Synthetic building debris
LRN - Laboratory Response Network
ND - Not determined
PCR - Polymerase chain reaction
52
-------
SEPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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