i

Detection and Differentiation of Anthrax and Environmental Bacillus

species at the Canberra Airport

Rory Scott Thomas

Bachelor Medical Science

National Centre for Forensic Studies

University of Canberra ACT 2601

A thesis submitted in partial fulfilment of the requirements for the degree of Bachelor of

Applied Science (Honours) at the University of Canberra

November 2013

v

Abstract

A bioterrorist attack, even a hoax attack, not only has the potential to cause mass fatalities but

can also have enormous economic, political and resource implications. Biological agents,

such as Bacillus anthracis, are able to form hardy and stable endospores, which can be

delivered easily to a large population over a large geographical area. B. anthracis occurs

naturally in the soils of the grazing regions of central Victoria and New South Wales. Given

the high traffic movements of people, luggage, mail, vehicles and livestock across Australia,

it is possible that low levels of bacterium could be present outside of this region. The

presence of the bacterium at transportation hubs is of particular importance, as the high traffic

environment of people arriving and departing these hubs means that diseases could be easily

disseminated over a large geographical area given only a short exposure time.

This study expands on previous work conducted by Rossi (2011) to determine the presence of

B. anthracis and closely-related potential hoax species at the Canberra Airport over a 12

month sampling period. In doing so, this study determined the baseline concentration of B.

anthracis and related species at the Canberra Airport so that background levels could be

differentiated from attack levels if there were ever an incident.

Sampling of six different locations throughout the Canberra airport over an eight month

period delivered a diverse sample set allowing conclusive results to be achieved. Analysis of

the sample extracts included three real-time quantitative qPCR assays being developed to

detect B. thuringiensis subsp. Israelensis, B. subtilis and the B. cereus group. Analysis also

included four qPCR assays designed by Rossi (2011) to detect B. anthracis PL3 genomic

marker, cya (pXO1 marker), capB (pXO2 marker) and B. thuringiensis subsp. Kurstaki.

Individual responses from each of the primer sets allowed for the determination of which

Bacillus subspecies were present in the samples. Detailed sample information with regards to

location and time of year (samples collected monthly) provided the ability to conclude on the

correlation of the subspecies present and possible causes for why they were present.

The results from this study indicate that there are trace amounts of non-pathogenic B.

anthracis at non-quantifiable levels present on surfaces at the Canberra Airport. The results

also show that trace amounts of B. thuringiensis subsp. Kurstaki are present.

vii

Acknowledgements

First and foremost, I would like to give thanks to my supervisors Drs Michelle Gahan, Dennis

McNevin, Michelle Nelson and Paul Roffey. Thank you all for your guidance and your

advice and for never giving up on me.

I would also like to thank everyone at the Canberra Airport and at the Australian Federal

Police who made going out to the airport every day for seven days each month for eight

months a thing to look forward to.

I would like to thank my fellow honours students, who made the experience a fun and often

entertaining adventure. In particular, I would like to thank James Grech for helping me with

all my formatting woes, expanding my taste in music and for steering me toward the right

YouTube videos to watch during my procrastination times. I would also like to give a special

thanks to Felicity Koens who gave her time to help me extract quite a bit of DNA. I know I

have made lifelong friends in the both of you.

A big thank you to my family as well, in particular, my dad, my step-dad, my sisters and

brother and my Aunty Cathy. Thank you for putting up with me and for being crutch over of

this past year.

Finally, I would like to thank my beautiful mother Julie Chevalier Hart. You saw me start

honours, but you didn’t get a chance to see me finish. However, although you’re no longer

with me, I have felt your strength urging me forward to the finish line every day. Everything

that I have ever done has been to make you proud. I have missed you every day since you left

us in March; more than I could ever hope describe in words. Your son, Rory.

ix

Table of Contents Chapter 1: Introduction .......................................................................................................... 1

1.1. Biological weapons and attacks .................................................................................. 1

1.1.1. Security-sensitive Biological Agents ................................................................... 2

1.2. Bacillus genus ............................................................................................................. 3

1.2.1. Bacillus cereus group ........................................................................................... 4

1.2.2. Bacillus subtilis group.......................................................................................... 7

1.3. Anthrax ........................................................................................................................ 8

1.3.1. Clinical manifestations....................................................................................... 10

1.4. Hoax attacks .............................................................................................................. 11

1.5. Identification of B. anthracis in a forensic sample ................................................... 12

1.5.1. Microbiological methods ................................................................................... 12

1.5.2. Immunological methods..................................................................................... 13

1.5.3. Molecular methods............................................................................................. 13

1.6. Differentiation of Bacillus species ............................................................................ 16

1.6.1. PCR inhibitors .................................................................................................... 16

1.7. Previous detection of B. anthracis and B. thuringiensis subsp. Kurstaki at the

Canberra Airport .................................................................................................................. 18

1.8. Aims of thesis ............................................................................................................ 18

Chapter 2: Materials and Methods ....................................................................................... 19

2.1. Bacterial strains ......................................................................................................... 19

2.2. Bacterial culture conditions ....................................................................................... 19

2.3. Collection of field samples ........................................................................................ 19

2.3.1. Location and timing ........................................................................................... 19

2.3.2. Sampling protocol .............................................................................................. 20

2.3.3. Pre-extraction processing of field samples ........................................................ 21

2.4. DNA extraction ......................................................................................................... 22

2.4.1. DNA extraction from field samples ................................................................... 22

x

2.4.2. DNA extraction from bacterial cultures ............................................................. 22

2.5. DNA quantitation ...................................................................................................... 23

2.6. qPCR protocol ........................................................................................................... 23

2.6.1. Oligonucleotides ................................................................................................ 23

2.6.2. qPCR protocol for field samples ........................................................................ 25

2.6.3. qPCR protocol for optimisation experiments .................................................... 26

2.7. Agarose gel electrophoresis ...................................................................................... 26

2.8. Data analysis ............................................................................................................. 27

2.8.1. Calculation of qPCR assay efficiency................................................................ 27

2.8.2. Calculation of copy number ............................................................................... 27

Chapter 3: Results ................................................................................................................ 29

3.1. In silico analysis of oligonucleotides ........................................................................ 29

3.2. Development of a singleplex qPCR assay for the detection and quantification of B.

subtilis .................................................................................................................................. 30

3.2.1. Determining the specificity of the 16S rRNA and aprE primers ....................... 30

3.2.2. Determining the cross-reactivity of the 16S rRNA and aprE primers against

other Bacillus species ....................................................................................................... 31

3.2.3. Determining the limit of detection of the aprE primer assay ............................ 33

3.3. Development of a singleplex qPCR assay for the detection and quantification of

B. thuringiensis subsp. Israelensis ....................................................................................... 34

3.3.1. Determining the specificity of the Cry4 primers ............................................... 34

3.3.2. Determining the cross-reactivity of the Cry4 primers against other Bacillus

species 35

3.3.3. Determining the limit of detection of the cry4 primers ..................................... 36

3.4. Development of a singleplex assay for the detection and quantitation of B. cereus . 37

3.4.1. Determining the specificity of hblC and gyrB primers ...................................... 37

3.4.2. Determining the cross-reactivity of the gyrB primers against other Bacillus

species 38

xi

3.4.3. Determining the limit of detection of the gyrB assay ........................................ 40

3.5. Determining the LOD of the B. anthracis str. Sterne assays .................................... 40

3.6. Determining the LOD of the of B. thuringiensis subsp. Kurstaki assay ................... 42

Chapter 4: Results ................................................................................................................ 43

4.1. Determining the presence of PCR inhibition in samples collected from the Canberra

Airport .................................................................................................................................. 43

4.2. Determining the presence and concentration of B. anthracis at the Canberra Airport

43

4.3. Determining the presence and concentration of B. thuringiensis subsp. Kurstaki at

the Canberra Airport............................................................................................................. 47

4.4. Determining the presence and concentration of B. thuringiensis subsp. Israelensis at

the Canberra Airport............................................................................................................. 49

4.5. Determining the presence and concentration of B. subtilis at the Canberra Airport . 49

4.6. Determining the presence and concentration of B. cereus at the Canberra Airport .. 49

Chapter 5: Discussion .......................................................................................................... 51

5.1. Selection of primers .................................................................................................. 51

5.1.1. Selection of primers for B. cereus...................................................................... 51

5.1.2. Selection of primers for B. subtilis .................................................................... 52

5.2. Development of a singleplex qPCR assay for the detection and quantification of B.

subtilis .................................................................................................................................. 53

5.3. Development of a singleplex assay for the detection and quantitation of B.

thuringiensis subsp. Israelensis............................................................................................ 56

5.4. Development of a singleplex assay for the detection and quantitation of B. cereus . 57

5.5. Determining LOD for the B. anthracis and B. thuringiensis subsp. Kurstaki assays

58

5.6. Detection of B. anthracis at the Canberra Airport .................................................... 58

5.7. Detection of B. thuringiensis subsp. Kurstaki at the Canberra Airport..................... 60

5.8. Detection of other B. cereus, B. subtilis and B. thuringiensis subsp. Israelensis at the

Canberra Airport .................................................................................................................. 61

xii

5.9. Conclusions ............................................................................................................... 61

5.10. Future directions .................................................................................................... 62

Chapter 6: References .......................................................................................................... 63

xiii

List of tables

Table 1.1: Security-sensitive Biological Agents (SSBAs), established in Australia under Part

3 of the National Health Security Act 2007, as published on the Department of Health and

Ageing’s website (Minister for Health and Ageing, 2008). ....................................................... 2

Table 1.2: Summary of each step of a single PCR cycle ......................................................... 14

Table 1.3: Commonly encountered PCR inhibitors and their sources ..................................... 17

Table 2.1: Location, descriptions and surface types of field samples collected at the Canberra

Airport and Australian Air Express ......................................................................................... 20

Table 2.2: Oligonucleotides used in this study ........................................................................ 24

Table 2.3: Primer concentrations ............................................................................................. 25

Table 2.4: DNA concentration of bacterial species used in optimisation experiments ........... 26

Table 2.5: Genome sizes of bacterial species used in this study. ............................................ 28

Table 3.1: In silico analysis of the cross-reactivity of primer sets using the Basic Local

Alignment Search Tool (BLAST) database ............................................................................. 29

Table 3.2: Cross-reactivity of B. subtilis aprE and 16S rRNA primers ................................... 31

Table 3.3: Cross-reactivity of the 16S rRNA B. subtilis assay at primer concentrations of 0.1

µM and 0.05 µM ...................................................................................................................... 32

Table 3.4: Cross-reactivity of the aprE B. subtilis assay at primer concentrations of 0.1 µM

and 0.05 µM ............................................................................................................................. 33

Table 3.5: Cross-reactivity of the Cry4 primers ...................................................................... 35

Table 3.6: Cross-reactivity of the Cry4 B. thuringiensis subsp. Israelensis assay at primer

concentrations of 0.1 µM and 0.05 µM ................................................................................... 36

Table 3.7: Cross-reactivity of the gyrB primers ....................................................................... 39

Table 3.8: Cross-reactivity of the gyrB assay at primer concentrations of 0.1 µM and 0.05

µM. ........................................................................................................................................... 39

Table 4.1: Samples positive for the B. anthracis PL3 target ................................................... 44

Table 4.2: Concentration of samples positive for the B. anthracis PL3 genomic marker ....... 45

Table 4.3: Samples positive for the B. anthracis capB target .................................................. 45

Table 4.4: Samples positive for the B. anthracis cya target .................................................... 46

Table 4.5: Summary of the overlap between samples testing positive for the PL3, cya and

capB genes ............................................................................................................................... 47

xv

List of figures

Figure 1.1: The "Anthrax Belt" of Australia, adapted from Durrheim et al. 2009 .................... 9

Figure 3.1: Standard curve for the aprE assay. ........................................................................ 34

Figure 3.2: Standard curve for the cry4 assay.......................................................................... 37

Figure 3.3: standard curve for the gyrB assay. ......................................................................... 40

Figure 3.4: Standard curve for the B. anthracis str. Sterne PL3 assay. ................................... 41

Figure 3.5: Standard curve for the B. thuringiensis subsp. Kurstaki Cry1 assay. ................... 42

Figure 4.1: Positive Cry1 samples by month. 1, 2, 3,4 5 = Pair-wise comparisons (p < 0.05);

# = significantly different from all other months (p < 0.05); * = significantly different from

all other months, except April. ................................................................................................. 48

Figure 4.2: Positive Cry1 samples by sampling location ......................................................... 48

1

Chapter 1: Introduction

1.1. Biological weapons and attacks

Infectious diseases, brought about by pathogenic microorganisms and toxins, pose

serious threats to public health, as well as animal health, agricultural productivity and

the economic stability of a country (Christopher et al., 1997, Inglesby, 2002, Wallin et

al., 2007). Sporadic outbreaks of naturally occurring disease such as plague, smallpox

and influenza have already demonstrated the catastrophic impact that disease can play

on the health of a nation when confronted with high incidences of mortality, morbidity,

panic, civil unrest and resource strains (Christopher et al., 1997, Wallin et al., 2007).

Equally as concerning as natural outbreaks are the planned and deliberate outbreaks

caused by the weaponisation of these biological agents.

The use of microorganisms as weapons (bioweapons) is not a new phenomenon, with

examples of biowarfare evident in history as early as the 14th

century (Christopher et al.,

1997, Wallin et al., 2007). One of the earliest examples of such an event was in the 14th

century siege against Kaffa, long before the discovery of microorganisms. During the

siege, the invading Tartars experienced an outbreak of plague, caused by the bacteria

Yersinia pestis. The Tartar’s used this to their advantage, catapulting the bodies of their

deceased comrades into Kaffa with the intention of causing a similar outbreak

(Christopher et al., 1997). Not only were the Tartars successful in their conquest of

Kaffa, but those who evacuated the city went on to disseminate the disease across

Mediterranean port cities, sparking the second wave of Black Death throughout Europe

(Christopher et al., 1997).

Another popular historical example is the use of smallpox, caused by Variola virus, by

the British army to eliminate the immunologically naïve Native Americans. On 24 June

1763, following a smallpox outbreak in Fort Pitt, Captain Ecuyer recorded in his journal

that he had given blankets and a handkerchief used by smallpox sufferers to a Native

American tribe (Christopher et al., 1997). These objects sparked an epidemic of

smallpox in Native American tribes all down the Ohio River valley.

The Geneva Protocol, which prohibited the use of chemical and biological weapons was

signed in 1925. Both Japan and the United States of America (USA) signed the

Protocol, but refused to ratify it into their own laws. The Biological and Toxin Weapons

Convention (BTWC) went into force in 1975 and was the first multilateral instrument to

2

ban an entire class of weapons (Millett, 2006). The instrument prohibited the

stockpiling, development, sale, acquisition and retention of biological and chemical

weapons (Millett, 2006). In spite of the BTWC, many countries continued their

bioweapon programs (Christopher et al., 1997, Macintyre, 2000, Wallin et al., 2007).

As an example, the state of Iraq was discovered in 1991 to have botulinum toxin,

weaponised anthrax and aflatoxin in their possession, though there is no evidence to

suggest they ever used any of it (Wallin et al., 2007).

The use of bioweapons in military warfare has decreased through the centuries (Wessely

et al., 2001). Bioweapons have since become synonymous with the word ‘terrorism’; a

fact which Wessely et al. (2001) speculate may be due to the relative ineffectiveness of

biological agents as military weapons. Bioweapons present a greater threat to civilian

populations, particularly within densely populated cities (Spencer, 2003). Wessely et al.

(2001) also assert that the union of the words ‘bioweapon’ and ‘terror’ in the media has

confirmed the quintessential nature of bioweapons as vehicles for terrorism.

The topic of terrorism is broad and multifaceted. Broadly, it can be defined as ‘the

deliberate acts of physical and/or psychological nature perpetrated on select groups of

victims” (Wallin et al., 2007). The overall goal of terrorism is to strike terror, cause

panic and bring about fear (Wallin et al., 2007). In achieving this goal, terrorists are

able to instigate change by taking control of public opinion in order to pressure the

decision makers.

1.1.1. Security-sensitive Biological Agents

Part III of the National Health Security Act 2007 established the list of Security

Sensitive Biological Agents (SSBAs). The list contains biological agents which are

concern to Australia’s security. Items on the list are subject to more strict regulations.

Biological agents were assessed against a risk management template relating their rates

of mortality and morbidity, against their ease of transmission, availability of treatment,

as well as the level of social disruption and panic that would arise from an outbreak

(Department of Health, 2013).

Table 1.1: Security-sensitive Biological Agents (SSBAs), established in Australia

under Part 3 of the National Health Security Act 2007, as published on the

Department of Health and Ageing’s website (Minister for Health and Ageing, 2008).

Tier 1 Agents Tier 2 Agents

Abrin African swine fever virus

3

Bacillus anthracis (Anthrax – virulent

strains)

Capripoxvirus (Sheep pox virus and Goat

pox virus)

Botulinum toxin (reportable quantity

0.5mg) Classical swine fever virus

Ebolavirus Clostridium botulinum (Botulism; toxin-

producing strains)

Foot-and-mouth disease virus Francisella tularensis (Tularaemia)

Highly pathogenic influenza virus,

infecting humans Lumpy skin disease virus

Marburgvirus Peste-des-petits-ruminants virus

Ricin (reportable quantity 5mg) Salmonella typhi (Typhoid)

Rinderpest virus Vibrio cholera (Cholera; serotypes O1 and

O139 only)

SARS coronavirus Yellow fever virus (non-vaccine strains)

Variola virus (Smallpox)

Yersinia pestis (Plague)

1.2. Bacillus genus

The genus Bacillus is a large heterogeneous collection of rod-shaped, endospore-

forming bacteria, belonging to the Bacillaceae family. The genus represents one of the

largest, most variable and environmentally diverse genera of bacteria (Goto et al.,

2000). At least 83 unique species have been classed in this genus (Blackwood et al.,

2004). The high variability within this genus is evidenced by the wide range of DNA

G+C content within and between species (33 to 65%); a figure which greatly contradicts

the generally accepted taxonomic principle that species within a genus should not differ

by more than 10-12% (Blackwood et al., 2004).

A common feature between species within the Bacillus genus is their ability to produce

endospores (spores). Spores are dormant structures formed when the bacteria are

exposed to dry, low-nutrient or generally adverse environmental conditions. Spores

currently represent the hardiest known form of life on earth and are resistant to heat,

pressure, antimicrobial chemicals, desiccation, ultraviolet (UV) light and ionising

radiation. Spores are known to be able to survive in physically extreme conditions that

humans and vegetative bacteria otherwise couldn’t (Nicholson et al., 2000).

4

1.2.1. Bacillus cereus group

The B. cereus group, more formally titled B. cereus sensu latu, is a subdivision of the

genus Bacillus containing a cluster of six closely related species: B. weihenstephanesis,

B. mycoides, B. pseudomycoides, B. cereus, B. anthracis and B. thuringiensis (Kolstø et

al., 2009).

Members of B. cereus group collectively share the same ecological niche and are

ubiquitous in various types of soils as well as the guts of soil-dwelling invertebrates

across the world (Stenfors Arnesen et al., 2008). A common feature between members

are their complex genomes, with each species having unique chromosomal interspersed

sequence repeats and chromosomal prophages (Kolstø et al., 2009). Additionally,

members typically have a rich and diverse plasmid profile, with different species and

strains reported to carry between 1 – 12 plasmids of anywhere between 2 – 600

kilobases (kb) in size.

Despite the overall heterogeneous nature of the B. cereus group, many of the species are

unable to be differentiated from one another genetically and are often intermixed on the

same branches of phylogenetic trees (Stenfors Arnesen et al., 2008). B. anthracis, B.

cereus and B. thuringiensis are the most closely related, both in their gene content and

synteny (gene order). In addition, the 16S RNA sequences of these three species are

over 99% identical. It has been frequently suggested in the literature that B. cereus and

B. thuringiensis should be re-classified as a single species (Helgason et al., 2000, Rasko

et al., 2005, Vilas-Bôas et al., 2007, Stenfors Arnesen et al., 2008, Kolstø et al., 2009).

In addition to the high degree of chromosomal similarity between B. anthracis, B.

thuringiensis and B. cereus, differentiation of these species can be further complicated

by the horizontal transfer of plasmids. Horizontal transfer has been frequently noted in

the literature, with the detection of both pXO1 and pXO2 in near neighbours of B.

anthracis (Helgason et al., 2000, Hoffmaster et al., 2004).

1.2.1.1. Bacillus anthracis

B. anthracis is a gram-positive aerobic spore forming rod and the only obligate

pathogen within the B. cereus group. It is the causative agent of anthrax, a potentially

highly fatal zoonosis. B. anthracis is highly monomorphic, with only limited genetic

variation between strains isolated from opposite points of the globe (Kolstø et al.,

2009).

5

Fully pathogenic B. anthracis strains contain two large virulence plasmids, termed

pXO1 and pXO2, which code for the bacterium’s “anthrax toxin” and antiphagocyctic

capsule, respectively (Blaustein et al., 1989). Anthrax toxin refers collectively to three

separate proteins which are coded within a 44.8kb pathogenicity island (PAI) on the

pXO1 plasmid (184.5 kb) – lethal factor (LF; 83 kDa), oedema factor (OF; 89 kDa) and

protective antigen (PA; 85 kDa) (Blaustein et al., 1989). The anthrax toxin follows the

traditional AB-type model, with a single receptor-binding B-moiety (PA) and two

catalytic A-moieties (LF and OF) (Scobie et al., 2006).

Following infection and germination within the host, the three proteins are released

from the bacterium. PA binds selectively to cell surface receptors known to be widely

distributed through human tissues, ANTHRX1 (anthrax toxin receptor/tumour

endothelial marker 8; ATR/TEM8) and ANTHRX2 (capillary morphogenesis gene 2;

CMG2) (Scobie et al., 2006). Seven PAs associate with one another to form a ring on

the surface of the target cell. OF or LF bind to the PA ring, forming the receptor-PA-OF

or the receptor-PA-LF complex, which is then internalised into the target cell via

receptor-mediated endocytosis (Scobie et al., 2006). The low pH of the endosome

enhances the channel-forming properties of PA, which allows the PA heptamer to form

a pore within the phospholipid bilayer of the endosome (Blaustein et al., 1989, Scobie et

al., 2006). OF and LF disassociate and transverse the endosome into the cytosol through

this pore. LF inactivates mitogen-activated protein kinase kinase, which leads to the

release of tumour necrosis factor α and interleukin-1β and widespread cytokine

deregulation.

Plasmid pXO2 (95 kb) codes for the biosynthesis of the B. anthracis poly-γ-D-glutamic

(polyglutamate) acid capsule. The capsule aids in the evasion of the immune system by

protecting the vegetative cells from being engulfed and destroyed by macrophages

(Mock and Fouet, 2001).

Fully pathogenic strains of B. anthracis must contain both the pXO1 and pXO2

virulence plasmids (Mock and Fouet, 2001, Spencer, 2003). The loss of either plasmid

causes the attenuation of the bacterium, which can then be used as a vaccine. The Stern

strain has been cured of pXO2 (pXO2-), but still retains pXO1 (pXO1

+). The Stern

strain has been licenced in Australia for use a veterinary vaccine against anthrax

(Australian Pesticides and Veterinary Medicines Authority, 2012). The Pastuer strain is

pXO1- and pXO2

+ and is also used as a vaccine, though it is not licenced for use in

Australia.

6

1.2.1.2. Bacillus thuringiensis

B. thuringiensis is a ubiquitous inhabitant of soil, with a genome size of 5.4 million base

pairs (Mbp) (Carlson et al., 1994). B. thuringiensis is regarded globally for its

insecticidal properties, in fact, accounting for more than 90% of biopesticides

worldwide (Bavykin et al., 2008). In Australia, 22 products containing Bacillus

thuringiensis have been licensed as pesticides for commercial sale by the Australian

Pesticides and Veterinary Medicines Authority (APVMA) (Australian Pesticides and

Veterinary Medicines Authority, 2012). A few of the more common market names of

these biopesticides include ‘Dipel Nature’s Way Caterpillar Killer’, ‘AquaBac’

Biological Mosquito Larvicide and ‘VectoBac’ Biological Mosquito Larvicide

(Australian Pesticides and Veterinary Medicines Authority, 2012).

The insecticidal properties of B. thuringiensis primarily affect those insects in the orders

of Lepidoptera, Diptera and Coleoptera, with the spectrum of activity varying between

the 82 identified serotypes (Rasko et al., 2005).

The insecticidal crystal toxins produced by B. thuringiensis are coded on Cry genes,

located on a large transmissible plasmid. This plasmid is so common in the B.

thuringiensis species that it is now considered to delineate the species from the rest of

the B. cereus group. Spores of B. thuringiensis contain large crystalliferous protein

inclusions, known as parasporal bodies. These parasporal bodies are aggregates of a

protoxin (130-140 kDa), which when taken up by susceptible insect larvae, work to

immobilize and lyse the gut, eventually killing the insect from septicaemia (Rasko et

al., 2005).

1.2.1.3. Bacillus cereus

B. cereus, more formally known as B. cereus sensu stricto, is a ubiquitous soil dweller,

though can also be found in the gut of invertebrates as a symbiont (Bottone, 2010). B.

cereus is the only other member of the group considered to be of medical importance. It

is frequently implicated as a food poisoning agent, as well as an important opportunistic

and nosocomial infection (Rasko et al., 2005). B. cereus is known to cause at least two

distinct gastrointestinal syndromes; emetic and diarrheal (Rasko et al., 2005).

The emetic syndrome was first recognised in 1970 following an outbreak in the United

Kingdom caused by eating cooked rice. The incubation period of this disease is between

0.5 – 6 hours, with typical symptoms including nausea and emesis (Stenfors Arnesen et

al., 2008). The disease is self-limiting, with a duration of usually less than 24 hours.

7

The emetic syndrome is known to be caused by a single non-ribosomally synthesised

dodecadepsipeptide, known interchangeably as either emetic toxin or cereulide (<5

kDa) (Kolstø et al., 2009). The toxin is resistant to heat, low pHs, proteolytic enzymes

and is encoded on a large pXO1-like plasmid, termed pCER270 (Rasko et al., 2005).

Cereulide-producing strains of B. cereus are few, and are known to represent only a

small cluster of genetically clonal strains (Kolstø et al., 2009). Cereulide has also been

identified in specific B. weihenstephanesis strains (Kolstø et al., 2009).

The diarrheal syndrome results from the action of enterotoxins in the illeal loop of the

small intestine, following the ingestion of viable vegetative cells or spores (Stenfors

Arnesen et al., 2008). The incubation period varies, though the average is 12 hours.

Similar to the emetic syndrome, the infection is self-limiting and typically dissipates

within 24 hours. To date, three have been identified; two related three-component

complexes and a single protein, termed non-haemolytic enterotoxin (NhE), haemolysin

BL (HBl) and cytotoxin K, respectively (Ryan et al., 1997; Stenfors Arnesen et al.,

2008). In addition to these enterotoxins, a number of other degradative enzymes,

haemolysins and cytotoxins have been implicated in contributing to B. cereus’

pathogenicity, such as, cereolysin O, haemolysin II, haemolysin III, InhA2 and various

phospholipases C (Ryan et al., 1997; Lund et al., 2000; Stenfors Arnesen et al., 2008).

Of the identified toxins, NhE is the most prominent among B. cereus strains and is now

thought to be present in all species within the B. cereus group (Ngamwongsatit et al.,

2008). HBl and CytK are present in less than 50% of randomly strains, though are more

frequently observed in clinical and food samples (Stenfors Arnesen et al., 2008).

The plasmid profiles of B. cereus are rich and diverse. To knowledge, there is not a

single plasmid present in all strains of B. cereus that is able to delineate the species, like

there is in B. thuringiensis.

1.2.2. Bacillus subtilis group

The B. subtilis group houses eight closely related species: Bacillus subtilis subsp.

Subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus atrophaeus,

Bacillus mojavensis, Bacillus vallismortis, Bacillus subtilis subsp. Spizizenii and

Bacillus sonorensis (Wang et al., 2007).

8

1.2.2.1. Bacillus subtilis

The 4.2Mbp genome of B. subtilis was one of the first to ever be fully sequenced (Kunst

et al., 1997), contributing to its status as the most well defined and understood gram

positive bacteria. B. subtilis has become to model organism for gram positive bacteria.

B. subtilis is an inhabitant of soils, though is also commonly found as a contaminant of

food. It can also be found in the normal microflora of the gut and has since been found

to aid in its balance. This property has seen antibiotic resistant strains of B. subtilis

marketed as probiotics (Oggioni et al., 1998). B. subtilis is often regarded as lowly-

pathogenic or non-pathogenic (de Boer and Diderichsen, 1991), though has been noted

as a rare opportunistic infection of immuno-compromised people. B. subtilis can be

found in a number of probiotic formulas licenced in Australia for veterinary use

(Australian Pesticides and Veterinary Medicines Authority, 2012).

1.3. Anthrax

Anthrax is a disease of both historical interest and contemporary importance. The

appearance of anthrax has been noted throughout history and is in fact suspected to have

been the fifth and sixth plague alluded to in the biblical book of Exodus (Wallin et al.,

2007). The high pathogenicity of B. anthracis, its global distribution and the relative

ease associated with obtaining and drying its spores into a powder, has seen the

bacterium become a favoured and well-studied bioterrorism agent.

B. anthracis’ potential as a bioweapon has been the subject of study for more than 90

years (Inglesby, 2002). During World War 1 (WW1), German scientists deployed B.

anthracis against their opponent’s livestock in a campaign directed at sabotaging the

enemies’ meat supply (Wallin et al., 2007). During Japan’s occupation of China, the

Japanese military sanctioned the study of biological agents, including B. anthracis,

leading to the deaths of approximately 10,000 people (Wallin et al., 2007). The most

recent example of the use of B. anthracis as a bioweapon occurred after the World

Trade Centre terrorist attacks in the United States of America (USA). Following the fall

of the World Trade Centres, at least five letters containing B. anthracis endospores were

received in Florida, New York and Washington DC between October 2001 and January

2002. These attacks caused 22 confirmed cases of anthrax, 11 of which were

inhalational and 11 cutaneous. Five people in total died from inhalational anthrax

(Leask et al., 2003). A 1993 US Congressional Office of Technology report estimated

that the release of 100 kilograms (kg) of B. anthracis spores along a 100 km aerial line

9

could lead to the death of between 95,000 and 130,000 people, which is comparable to a

hydrogen bomb (Wallin et al., 2007). Similarly, a study conducted by the World Health

Organisation (WHO) estimated that approximately 95,000 deaths would follow the

aerosolisation of just 50 kg of dried B. anthracis spores in a city of 500,000 inhabitants

(WHO, 2002). In addition to high fatalities, WHO asserted in their study that the

breakdown of the medical industry as well as civilian infrastructure and resources would

quickly follow these deaths . This would be due to the high influx of patients to

hospitals, the high demand for antibiotics (prophylactic and otherwise) as well as the

disposal of 95,000 bodies (Wallin et al., 2007).

In addition to deliberate attacks, there have also been a number of documented

outbreaks due to accidental release or occupational exposure. The accidental release of

B. anthracis spores occurred downwind of a Soviet military complex in Sverdlovsk in

1979, killing between 66 and 105 civilians from inhalational anthrax (Spencer, 2003).

Similarly, a number of inhalational outbreaks occurred in workers of a goat hair

processing plant in New Hampshire, USA, in 1957 (Jernigan et al., 2002).

Due to the persistence of B. anthracis spores in the soils and their global distribution

around the world, anthrax is usually regarded as an important disease of grazing

herbivores (Cieslak and Eitzen, 1999). Certain environmental and climatological

systems appear to foster the spores, generating what are known as “anthrax zones”

(Cieslak and Eitzen, 1999). Examples of such conditions include regions with soils rich

Figure 1.1: The "Anthrax Belt" of Australia,

adapted from Durrheim et al. 2009

10

in organic matter and dramatically changing climates (Cieslak and Eitzen, 1999). In

Australia, B. anthracis spores are concentrated in what is known as the “Anthrax Belt”,

which extends from central Victoria up into central NSW (Department of Agriculture,

Fisheries and Forestry 2008). The Australian ‘Anthrax Belt’ is depicted in Error!

Reference source not found..

Despite the spores’ persistence in Australian soils, cases of naturally-occurring anthrax

have become rare in past 4 decades (Durrheim et al., 2009). In general, only between 6 -

13 properties report cases of anthrax each year. Further, only 1-3 animals, namely

sheep, cattle and horses, are affected (Durrheim et al., 2009). Outbreaks of anthrax have

been noted. Following a 1-in-100 year rain event probably caused the near-concurrent

re-emergence of anthrax in the Hunter Valley, a region outside the Anthrax Belt

(Durrheim et al., 2009). The largest naturally-occurring outbreak of anthrax in recorded

history occurred in Zimbabwe, where over 10,000 people presented with human anthrax

(primarily the cutaneous form) between 1979 – 1985 (Spencer, 2003).

1.3.1. Clinical manifestations

B. anthracis infection is initiated when a sufficient quantity of the bacteria’s endospores

enter the body. Infection of the spores can occur via a number of routes, typically either

through the skin (cutaneous), the gut (gastrointestinal) or the lungs (inhalational)

(Spencer, 2003). The disease state caused by the bacteria is largely dependent on the

route of transmission, though fatal systemic anthrax is still a potential final outcome for

all states (Mock and Fouet, 2001).

Cutaneous anthrax occurs when spores enter the body via the skin, through events such

as abrasions, cuts and insect bites (Mock and Fouet, 2001). This form is the most

common but also the least fatal, manifesting at first as a small papule which later

ulcerates and dries to form a characteristic painless black escher (Spencer, 2003). When

properly treated with antibiotics, cutaneous anthrax will usually resolve with minimal to

no scarring within two weeks with fatality rates of less than 1%. Left untreated,

approximately 20% of cases develop secondary infections with the capability of leading

to sepsis and death (Spencer, 2003).

Gastrointestinal anthrax is initiated when spores access the body through the gut.

Infection in this state usually occurs from eating undercooked meats prepared from

animals that have been exposed to the spores. Gastrointestinal anthrax is characterised

by escher formation on the lining of the gastrointestinal tract. Symptoms, whilst

11

dependent on the location of the eschers, are typically non-specific and include nausea,

vomiting and fever (Spencer, 2003). In the absence of medical intervention, the primary

escher becomes necrotic, leading to widespread oedema and local lymphadenopathy

(Inglesby, 2002; Spencer, 2003). The mortality rates of this form of anthrax are highly

variable, with case-fatalities rates anywhere between 4-50% (Sirisanthana and Brown,

2002).

Inhalational anthrax is the most lethal form of the disease. Generally it occurs after the

exposure to airborne spores. Mild flu-like symptoms often proceed, followed by the

insidious onset of serious complicating issues, which are normally penultimate to death

(Mock and Fouet 2001). Though only very rare, inhalational anthrax has been described

to have mortality rates of up to 95% (Spencer, 2003).

1.4. Hoax attacks

Although physically harmless, hoax attacks fulfil the definition of terrorism through

their intended aim of causing widespread panic and terror. The induction of mass

sociogenic illness and hysteria from hoax attacks have not only been noted in the past

but also represent a danger to mental health comparable to a real attack (Wessely et al.,

2001). In addition, a hoax attack can be just as disruptive to normal society as a normal

attack, as normal operations need to be halted in order to deal with contaminated areas

and exposed individuals (Ellerbrok et al., 2002).

Following the 2001 anthrax postal attacks in the USA, numerous hoax attacks involving

white powders were been reported globally, with the NSW Police alone responding to

over 6000 anthrax hoaxes since October 2001 (Crighton et al., 2012). On October 12,

2001 Australia received its first publicised “white powder incident” after an airport

attended reported to the police an item of luggage covered in white powder (Leask et al.,

2003). It was determined later the powder did not contain B. anthracis spores. Although

Australia has not experienced a legitimate anthrax attack, white powders are still treated

with suspicion, most of them ordinary household products such as talcum powder,

sugar, laundry powder and starch (Leask et al., 2003).

The spores from B. thuringiensis and other members of the B. cereus group have the

potential to be used as convincing hoax powders. B. thuringiensis spores in particular

are available commercially as a pesticide, making them easy to acquire.

12

1.5. Identification of B. anthracis in a forensic sample

As outlined in the section above, the deliberate release of anthrax has the potential to be

catastrophic to individuals, the public health system and society at large. In the event of

a “white powder incident” it is vital that the presence of B. anthracis is able to be

rapidly and accurately determined to ensure appropriate prophylactic treatment and

decontamination methods are able to be administered in a timely manner.

It is equally important to be able to distinguish between B. anthracis, its near

neighbours and other closely related bacteria. An effective and ideal identification

system detects low copy numbers, does not cross-react with other bacterial species, has

a short assay time and is cost effective (Rao et al., 2010). Various methods have been

designed to identify B. anthracis in a sample and broadly include phenotypic analysis

(microbiological methods), antigenic analysis (immunological methods) and nucleic

acid and protein analysis (molecular methods).

1.5.1. Microbiological methods

Conventional microbiological and biochemical methods represent the gold standard for

the identification of B. anthracis in a forensic sample (Inglesby, 2002, Spencer, 2003,

Janse et al., 2012). Similar to other members of the B. cereus group, B. anthracis grows

at 37°C, reduces nitrate to nitrite, hydrolyses starch and is catalase positive (Spencer,

2003). In addition, B. anthracis is non-motile, susceptible to lysis by gamma phage,

non-haemolytic on sheep’s blood agar (SBA) and sensitive to penicillin (Spencer, 2003;

Janse et al., 2012). Selective media for B. anthracis includes polymyxin-lysozome

EDTA-thallous acetate (PLET) agar, with incubation under either aerobic or anaerobic

conditions. B. anthracis forms white, oval-shaped, granular colonies on SBA, which are

“characteristically tacky” when teased with a loop (Spencer, 2003).

Whilst these conventional techniques are considered the gold standard, there are a

number of problems and shortfalls associated with them. One such problem is the

stringent biosafety regulations restricting where these procedures and tests can be

performed. Due to the high pathogenicity of B. anthracis and the associated handling

risk, culture is restricted to specially equipped Physical Containment Level 3 (PC3)

laboratories, of which there are limited numbers in Australia. In addition, culture

methods are labour-intensive and time consuming, with results only available following

lengthy incubation period of up to 48 hours. In short, whilst microbiological methods

13

provide the gold standard of confirmatory results, they are too slow for practical first-

response use in a bioterrorism scenario.

1.5.2. Immunological methods

Antibodies are created by the immune system in response to antigens. Immunological

methods for the detection of pathogens exploits an antibodies affinity to bind

specifically to its target antigen. In theory, any pathogen capable of eliciting an immune

response, such as B. anthracis, has a target antigen that has a corresponding antibody.

Antigens and antibodies bind together to form the antigen-antibody complex, which can

visualised through a number of labels, including fluorescent, chemiluminescent, dye,

enzyme conjugates and gold particles (Rao et al., 2010). The visualisation utilising one

of these labels confirms the presence of complex, which in turn suggests the antigen or

the antibody in question is also present (Rao et al., 2010).

In general immunological detection methods represent a rapid method for the

presumptive detection of B. anthracis in a sample (Rao et al., 2010). Antigen-targeted

detection can be rapid, though in general lacks specificity, which is often the result of

co-contaminant near neighbours of B. anthracis (Rao et al., 2010). Antibody-targeted

detection is only applicable in a clinical setting after exposure to the pathogen.

However, antibodies for toxins and capsule components are often not detectable until

the late stages of infection after it is too late for medical intervention. This has led a few

to suggest that current immunological detection methods represent more of a research

tool than a practical and deployable detection method (Rao et al., 2010). Furthermore,

most immunoassays struggle to reach an acceptable sensitivity level, with many having

a threshold of 100- to 1000-fold above the infectious dose of B. anthracis.

1.5.3. Molecular methods

1.5.3.1. Real-time PCR (qPCR)

The polymerase chain reaction (PCR) technique developed by Kary Mullis in the

1980’s is a technique which has revolutionised the field of molecular biology (Valasek

and Repa, 2005). At its very simplest form, the PCR method is used to amplify specific

sections of DNA more than a billion-fold (Valasek and Repa, 2005). In doing so, DNA

becomes easier to detect and manipulate. It’s applications in the field of molecular

biology are manyfold and are exemplified by the indispensable part it played in

sequencing the human genome (Valasek and Repa, 2005).

14

As mentioned above, PCR is a method used to amplify DNA. This is achieved through

the exploitation of DNA polymerases which have the ability to synthesize new strands

of DNA from a template. Oligonucleotide primers are needed for the reaction to take

place. These primers are designed to bind to their complimentary sequence on the

segment of DNA that is to be amplified. Following the binding of these primers to the

DNA, the DNA polymerase synthesizes a new complimentary strand of DNA. The

reaction occurs under a number of heat-controlled steps, which are summarised in Table

2.

Table 1.2: Summary of each step of a single PCR cycle

PCR step Temperature (°C) Description

Denaturation 94-98

This temperature disrupts the hydrogen bonds

between complimentary strands of DNA;

“melting” the strands apart.

Annealing 50-65 The temperature is lowered to allow the primers

to anneal to the template strand.

Elongation 75-80

This temperature is largely dependent on the

DNA polymerase being used, though optimal

activity of DNA polymerases sits within this

temperature range.

Real-time PCR adds the advantage of “real-time” monitoring of products as they are

generated (Valasek and Repa, 2005). Real-time monitoring is achieved through the

addition of a fluorescent detection chemistry, which allows for greater fluorescence of

DNA products as they are generated.

Detection chemistries for qPCR can be separated into two broad categories: non-

specific and specific (Lim et al., 2005). Non-specific detection chemistries are primarily

in the form of intercalating dyes, such as SYBR Green and LC Green. Intercalating dyes

bind to double stranded DNA (dsDNA), which typically causes them to fluoresce much

more brightly; SYBR Green fluoresces 1000x greater when bound to dsDNA (Valasek

and Repa, 2005). As more copies of DNA are produced in PCR, the more dsDNA

becomes available for intercalating dyes to bind to. A disadvantage to intercalating dyes

is that they are not specific to the target sequence being amplified and will bind to any

dsDNA in the reaction, which has the potential to cause false positive in the event of

primer dimer formation or non-specific amplification (Valasek and Repa, 2005).

15

Specific detection chemistries are primarily in the form of hydrolysis probes and

molecular beacons. Both of these chemistries work under a similar principle and offer a

solution to the problem of specificity presented by non-specific intercalating dyes (Rao

et al., 2010). Hydrolysis probes, better known by the proprietary name TaqMan probes,

are sequence-specific dually flurophore-labelled oligonucleotides (Valasek and Repa,

2005; Jian-Bing Fan et al., 2006; Rao et al., 2010). One flurophore gives the florescent

signal (the reporter), while the other absorbs it (the quencher). During PCR, the DNA

polymerase hydrolyses the 5’ end of the oligonucleotide, which separates the reporter

from the quencher, liberating the energy and producing a florescent signal (Valasek and

Repa, 2005; Rao et al., 2010). Molecular beacons work in a similar way, expanding on

the reporter-quencher theme, though have a different structure to TaqMan.

qPCR also allows for the quantification the quantification of DNA in a sample (Valasek

and Repa, 2005). Another advantage to qPCR over PCR is the elimination of any end-

point analysis, which reduces potential cross contamination of PCR products, which in

turn lowers the chance of false positives. In addition, qPCR can be multiplexed, which

allows rapid identification of multiple organisms in a single sample (Valasek and Repa,

2005). Whilst culture methods are far more sensitive than qPCR, the fast turnaround of

results (2 hours compared to 48 hours) makes qPCR a far more optimal identification

assay for crime scene response scenarios (Crighton et al., 2012).

A number of studies have successfully detected B. anthracis in a sample utilising the

qPCR technique (Qi et al., 2001; Ellerbrok et al., 2002; Bode et al., 2004; Hurtle et al.,

2004; Wang et al., 2004; Kim et al., 2005; Tomioka et al., 2005; Manzano et al., 2009;

Crighton et al., 2012). Frequently targeted genes include those on B. anthracis’

virulence plasmids and include lef, pag, cya (pXO1), capA, capB and capC (pXO2).

Commonly referenced chromosomal targets include gyrA, gyrB, ropB, BA813, plcR,

BA5345 and Pl3 (Blackwood et al., 2004; Bode et al., 2004; Rao et al., 2010). Each

target has been used with varying success. The gyrA gene was shown to be highly

specific for B. anthracis by Hurtle et al. (2004), with 100% sensitivity among the 171

organisms tested. The gyrA gene’s specificity for B. anthracis is based on a single

nucleotide difference, which greatly increases the chances of obtaining a false-positive

result (Bode et al., 2004). The ropB gene showed similar specificity to gyrA with only a

single strain cross-reacting out of the 175 Bacillus strains tested (Qi et al., 2001). A

study conducted by Zasada et al. (2006) later determined that the exact sequence of the

16

ropB gene as reported by Qi et al. (2001) matched that of at least two strains of B.

thuringiensis.

1.5.3.2. Multiplex qPCR

Multiplex qPCR expands on the qPCR technique through the use of multiple primer sets

to detect multiple targets simultaneously within the same reaction (Lim et al., 2005).

The technique is often used to detect multiple genes within the same organism, as well

as to detect multiple organisms within the same reaction.

Multiplex qPCR’s have been successfully designed and validated to distinguish virulent

from non-virulent strains of B. anthracis, though the simultaneous detection of a

chromosomal marker and a target on each of the virulence plasmids (Wang et al., 2004,

Tomioka et al., 2005). A multiplex qPCR was successfully designed to simultaneously

detect B. anthracis as well as Y. pestis and F. tularensis, a Tier 1 and Tier 2 SSBA,

respectively (Skottman et al., 2007). In addition, a multiplex qPCR for the detection and

differentiation of the B. cereus group was successfully designed (Kim et al., 2005). In

their study Kim et al. (2005) developed a highly specific qPCR assay targeting the lef

and capB genes, as well as sequence motif found within the spore structural gene

(sspE).

1.6. Differentiation of Bacillus species

Despite the huge similarities between B. cereus and B. thuringiensis a couple of studies

have reported being able to differentiate between the two using molecular methods.

Manzano et al. (2003) was able to distinguish the two species utilising PCR and agarose

gel electrophoresis, using primers specific to the B. cereus gyrA gene. No PCR products

were visualised in the lanes containing DNA from B. thuringiensis and the primers also

showed no cross-reactivity with a number of other Bacillus species including, B. subtilis

and B. mycoides. The PCR method developed by Manzano et al. (2003) was, however,

not validated robustly and only tested the primers for cross-reactivity against two strains

of B. thuringiensis. In addition, other studies have simply grouped the detection of B.

cereus and B. thuringiensis together with no attempt to differentiate the two, opting

instead to count their detection as a negative result for B. anthracis (Kim et al., 2005).

1.6.1. PCR inhibitors

Forensic samples, often from degraded or environmental sources, can present a special

challenge to the PCR technique (Alaeddini, 2012, Opel et al., 2009). The nature of

17

forensic samples often necessitate their collection in a variety of environments outside

of a clean and controlled laboratory setting (Volkmann et al., 2007). Forensic samples

collected from or exposed to soil, waterways, the human body, and the environment at

large have all been noted to affect the utility of the PCR reaction, primarily through the

co-extraction of powerful inhibitors. The identity of many PCR inhibitors have been

elucidated, and can either be part of the sample composition itself (e.g. haem, melanin)

or simply co-extracted from the site of the sample (e.g. humic acid) (Baar et al., 2011,

Alaeddini, 2012). When DNA copies are at acceptable levels for the PCR to work, the

most common cause of PCR failure is attributed to PCR inhibition (Alaeddini, 2012). A

list of commonly encountered and identified PCR inhibitors are shown at Table 1.3.

Table 1.3: Commonly encountered PCR inhibitors and their sources

PCR inhibitor Source of inhibitor

Humic and fulvic acid Soil and plant material

Melanin and eumelanin Hair and skin

Bile salts Faeces

Bilirubin Faeces

Haem Blood

IgG Blood

Haematin Blood

Tannic acid Leather and plant materials

Indigo Denim and other fabrics dyed with indigo

Theoretically, each component of the PCR can be affected or even completely

abrogated by an inhibitor, though the mechanisms underpinning such inhibition are not

fully understood. In many cases, the identity of the inhibitor is also not known

(Alaeddini, 2012).

In order to mitigate the effects of PCR inhibitors, the sample is either purified in an

attempt to remove inhibitors, or additives are combined into the reaction to facilitate the

PCR. Common facilitators include, bovine serum albumin (BSA) and T4 gene 32

protein (Alaeddini, 2012). Additionally, the sample can also be diluted or concentration

of DNA polymerase increased (Volkmann et al., 2007, Opel et al., 2009, Alaeddini,

2012). The use of genetically modified inhibitor-resistant Taq polymerases has also

been demonstrated to successfully mitigate the effects of PCR inhibition (Kermekchiev

et al., 2009, Baar et al., 2011).

18

1.7. Previous detection of B. anthracis and B. thuringiensis subsp.

Kurstaki at the Canberra Airport

A previous study conducted by Rossi et al. (2011), developed and validated a sampling

method and qPCR assay for the detection of B. anthracis and B. thuringiensis for use in

determining the background levels of the two species at the Canberra Airport. In

determining the background levels, the study aimed at establishing a baseline of the two

species. Given the high traffic movements of people, luggage, mail, vehicles and

livestock through transported hubs as well as the endemic status of B. anthracis in

Australia, the study hypothesised that it would be possible for trace amounts to be

present on surfaces at the Canberra Airport. The study, however, was unable to detect B.

anthracis at the Canberra Airport, though did detect low levels of B. thuringiensis.

Further, this study was limited in that only four months were able to be sampled and

analysed.

1.8. Aims of thesis

This study will aim to expand on the work conducted by Rossi (2011), by expanding the

sampling time frame by eight months so that 12 months of data can be analysed.

Additionally, new assays will be designed to target more potential false-positive-

causing and hoax agents closely related to B. anthracis. The aims of this project are to;

1. Develop and validate a singleplex real-time PCR (qPCR) assays, specific for

Bacillus thuringiensis subsp. Israelensis, B. subtilis and B. cereus;

2. Take field samples from the Canberra Airport between December 2011 and July

2012; and

3. Use the optimised methods to determine the background concentration of B.

anthracis, B. thuringiensis subsp. Israelensis, B. thuringiensis subsp. Kurstaki,

B. subtilis and B. cereus.

19

Chapter 2: Materials and Methods

2.1. Bacterial strains

B. subtilis, B. cereus and B. thuringiensis var. kurstaki were all supplied by the National

Centre for Forensic Studies, University of Canberra. B. anthracis str. Sterne, B.

mycoides and B. megatarium were kindly donated by the Australian Federal Police

Forensic and Data Centres. B. thuringiensis var. israelensis was kindly donated by Liam

O’Connor, Pathwest Laboratory Medicine.

2.2. Bacterial culture conditions

All bacterial strains were routinely cultured in nutrient broth or nutrient agar (Oxoid),

both of which were prepared according to the manufacturer’s instructions. Media was

sterilised by autoclaving for 20 minutes at 220 kilopascals (kPa) and 121°C. Inoculated

broth cultures were grown with aeration in a rotating Orbital Shaker Incubator (Bioline)

at 120-150 revolutions per minute (rpm) and 37°C. Agar plates were incubated

aerobically at 37°C. All strains were stored in nutrient broth containing 50% (v/v)

sterile glycerol (Unilab) at -80°C.

2.3. Collection of field samples

2.3.1. Location and timing

Field samples were collected from December 2011 until July 2012. Samples were

routinely collected for a period of seven consecutive days, in approximately the third

week of each month. Samples were collected from six different sites within the

Canberra Airport (1/2 Brindabella Circuit ACT 2609) each day Table 2.1.

Field samples were also collected from the Australian Air Express freight terminal (3

Rogan Place, Canberra International Airport ACT 2609). These samples were collected

alongside the airport samples on the same seven days in the months of December 2011

and January 2012. Australian Air Express moved all operations away from the airport in

early February 2012 so samples were not able to be collected between the months of

February 2012 and July 2012.

20

Table 2.1: Location, descriptions and surface types of field samples collected at the

Canberra Airport and Australian Air Express

Sample Code Description Location Surface Type

People OUT (PO) People departing

Canberra

Approximately 4 metres

away from the security

checking zone, level 2,

CA

Tiles (non-

porous)

People IN (PI) People arriving into

Canberra

Directly outside the

one-way door at the

base of the exiting

escalator, level 1, CA

Tiles (non-

porous)

Luggage OUT

(LO)

Luggage departing

Canberra

Check-in luggage

carousel for all domestic

flights, level 2, CA

Smooth rubber

(non-porous)

Luggage IN (LI) Luggage arriving

into Canberra

QANTAS baggage

claim carousel, level 1,

CA

Textured rubber

(non-porous)

People OUT

vacuum (POV)

People departing

Canberra – vacuum

sample

Approximately 2 metres

way from the security

checking zone, level 2,

CA

Carpet (porous)

Low level Control

(LC)

Low traffic area of

the airport

An area of low traffic

underneath the exiting

escalator, level 1, CA

Tiles (non-

porous)

Freight IN (FI) Freight arriving into

Canberra

Freight storage case,

Australian Air Express

Stainless steel

(non-porous)

Freight OUT (FO) Freight departing

Canberra

Freight security

scanning machine,

Australian Air Express

Textured rubber

(non-porous)

CA – Canberra Airport. Adapted from Rossi (2011). Note: With the exception of POV

all samples were collected using wipes.

2.3.2. Sampling protocol

Individual sampling packs, corresponding to the location to be sampled, were prepared

at the beginning of each sampling week. Each sample pack contained an individually

wrapped sterile 3 mL transfer pipette (Thermofisher Scientific), a pair of latex gloves

(Mediflex) and a pre-labelled sterile 50 mL tube (Greiner Bio-one). Each of these items

was packed into a re-sealable plastic bag. Fresh phosphate buffered saline (PBS)

(Amresco) was also prepared at the beginning of each sampling period and was

sterilised by autoclaving for 20 minutes at 220 kPa and 121°C.

At each non-porous sampling location (see Table 2.1), a 0.5 m x 1.0 m area was marked

out using a portable wooden measuring apparatus. Using the transfer pipette, a sterile

wipe was moistened with approximately 2 mL of PBS and streaked horizontally back

21

and forth across the whole area bordered by the measuring apparatus. The area was then

streaked vertically with the wipe and then horizontally once more, before being sealed

in the pre-labelled 50 mL tube.

For porous surfaces, a high-efficiency particulate air (HEPA) filter vacuum unit with a

sterile vacuum filter attachment (3M Trance Evidence Collection Filter) was used to

collect samples. The 0.5 x 1.0 m sampling site was marked out using the portable

wooden measuring apparatus. The sampling area was vacuumed horizontally back and

forth, vertically back and forth and then horizontally once more. The filter attachment

was removed from the vacuum, re-capped and then sealed in a sterile re-sealable plastic

bag.

To prevent cross-contamination of sampling sites, the measuring apparatus was sprayed

with 80% ethanol and wiped clean following the collection of each sample.

2.3.3. Pre-extraction processing of field samples

Immediately following collection, samples were taken to the Forensic Laboratory at the

University of Canberra for processing using a previously optimised method (Rossi,

2011). Each 50 mL tube containing a wipe sample was filled with 40 mL of PBS. For

the vacuum samples, the seal on the filter attachment was cut open with a sterile razor

blade and the internal filter was removed. The filter was placed into a beaker containing

30 mL of PBS and swirled gently. Using a set of tweezers, the filter was rolled up and

transferred to a 50 mL tube. The PBS in the beaker was poured into the tube. A further

10 mL of PBS was used to rinse the beaker and then this was added the 50 mL tube

containing the vacuum filter. All samples were then vortexed for three minutes using a

Vortex-Genie 2 (SI Scientific Industries) with a horizontal 50 mL 6-tube attachment.

Samples were left to rest for 5 minutes immediately following the vortexing step. The

wipe samples and filter were then removed from each tube using sterile tweezers, with

care taken to ensure that only a minimal amount of liquid was lost from the tube. The

remaining cells were then pelleted by centrifuging the 50 mL tubes at 4000 x g for 15

minutes at 4°C (Centrifuge 5810R, Eppendorf). The supernatant was discarded and the

pellet was resuspended in 250 microliters (µL) of PBS for the wipe samples and 1 mL

of PBS for the vacuum sample and transferred to a 1.5 mL tube. All samples were

stored at -20°C for further processing.

22

2.4. DNA extraction

2.4.1. DNA extraction from field samples

The extraction of DNA from field samples was performed using QIAamp DNA

extraction mini kits (Qiagen) and a modified protocol developed and validated by Rossi

(2011). A volume of 300 µL of ATL buffer and 100 µL of each sample were added to a

1.5 mL tube and mixed by pulse vortexing. The samples were incubated at 80°C for 20

minutes, before being transferred into individual 2 mL tubes containing 0.1 millimetre

(mm) glass beads (Precellys) and 1 µL of Antifoam Y (Precellys). The samples were

homogenised in a bead beater (Minilys, Precellys) at 5000 rpm for three cycles of 20

seconds with 15 second rest intervals. The samples were then centrifuged at 4000 x g

for three minutes. The supernatant from each sample was removed and transferred into a

1.5 mL tube, where it was combined with 20 µL proteinase K and incubated at 56°C for

60 minutes. Each solution was then incubated at 95°C for 15 minutes to inactivate the

proteinase K. An aliquot of 200 µL of AL buffer was added to each sample tube and

these were incubated for a further 10 minutes at 70°C. An aliquot of 200 µL of 96 –

100% ethanol was then added to each tube and these were mixed by pulse vortexing.

The sample mixtures were then transferred to individual QIAamp mini spin columns

which were centrifuged at 6000 x g for 1 minute. The columns were washed twice with

500 µL of AW1 buffer and three times with 500 µL AW2 buffer. They were centrifuged

at 6000 x g for 1 minute after each wash, with the exception of the final AW2 wash

where they were centrifuged at 16,000 x g for 3 minutes. To elute the DNA, 100 µL of

pre-heated 37°C ultrapure water was added to each column before incubation for 5

minutes. The columns were then centrifuged at 6000 x g for 1 minute. The eluent from

each column was collected in a maximum recovery 1.5 mL tube (Axygen). The elution

steps were repeated to give a final DNA volume of 200 µL. The extracted DNA was

stored at -20°C until further use.

2.4.2. DNA extraction from bacterial cultures

The extraction of DNA from overnight cultures was performed using the QIAamp DNA

extraction mini kit according to the manufacturer’s protocol. Briefly 1 mL of bacterial

cells from an overnight culture were pelleted by centrifuging at 2600 x g for 10 minutes.

The supernatant was removed and the pellet was re-suspended in 180 µL of ATL buffer.

An aliquot of 20 µL of proteinase K was added before the solution was incubated for 60

minutes at 56°C. The solution was then incubated at 90°C for 15 minutes. A volume of

23

200 µL of AL buffer was added before the solution was incubated for 10 minutes at

70°C. 200 µL of molecular grade ethanol was added and mixed by pulse vortexing. The

sample mixture was then transferred to a QIAamp mini spin column and centrifuged at

6000 x g for 1 minute. In the event that not all of the mixture passed through the

membrane, the column was centrifuged again at 8000 x g for 1 minute. The column was

washed with 500 µL of AW1 buffer and centrifuged at 6000 x g for 1 minute. The

column was subsequently washed with 500 µL of AW2 buffer and centrifuged for 3

minutes at 20,000 x g. To elute the DNA, 200 µL of pre-warmed 37°C ultrapure water

was added to the column which was incubated for 5 minutes before centrifugation at

6000 x g for 1 minute. The eluent was collected in a maximum recovery 1.5 mL tube

and stored at -20°C.

2.5. DNA quantitation

The concentration of double-stranded DNA (dsDNA) in bacterial DNA stocks was

determined by fluorescence spectroscopy, using the Qubit® 2.0 Fluorometer and

Qubit® dsDNA BR assay kit (Invitrogen). Following the manufacturer’s instructions,

the Qubit® dsDNA BR reagent was diluted 1:200 with the Qubit® dsDNA BR buffer.

2.6. qPCR protocol

All qPCR experiments were performed on the 7500 Real-Time PCR System (Life

Technologies).

2.6.1. Oligonucleotides

Oligonucleotides were purchased from GeneWorks. Oligonucleotides used for the

analysis of field samples are shown in Table 2.2.

24

Table 2.2: Oligonucleotides used in this study

Oligonucleotide Sequence (5’ – 3’) Target Reference

Cry1 (F) CTG GAT TTA CAG

GTG GGG ATA T

Bt Cry1 crystal

protein (plasmid) (Jensen et al. 2002)

Cry1 (R) TGA GTC GCT TCG

CAT ATT TGA CT

Bt Cry1 crystal

protein (plasmid) (Jensen et al. 2002)

Cry4 (F) GCA TAT GAT GTA

GCG AAA CAA GCC

Bt Cry4 crystal

protein (plasmid)

(Ben-Dov et al.,

1997)

Cry4 (R) GCG TGA CAT ACC

CAT TTC CAG GTC C

Bt Cry4 crystal

protein (plasmid)

(Ben-Dov et al.,

1997)

PL3 (F)

AAA GCT ACA AAC

TCT GAA ATT TGT

AAA TTG

Lambda pro-phage

type 3 (genomic)

(Wielinga et al.

2011)

PL3 (R)

CAA CGA TGA TTG

GAG ATA GAG TAT

TCT TT

Lambda pro-phage

type 3 (genomic)

(Wielinga et al.

2011)

Cya (F)

AGG TAG ATT TAT

AGA AAA AAA CAT

TAC GGG

Edema factor

(pXO1)

(Wielinga et al.

2011)

Cya (R) GCT GAC GTA GGG

ATGGTA TT

Edema factor

(pXO1)

(Wielinga et al.

2011)

capB (F)

AGC AAA TGT TGG

AGT GAT TGT AAA

TG

Capsule synthesis

component B

(pXO2)

(Wielinga et al.

2011)

capB (R)

AAA GTA ATC CAA

GTA TTC ACT TTC

AAT AG

Capsule synthesis

component B

(pXO2)

(Wielinga et al.

2011)

aprE (F) TTT ACG ATG GCG

TTC AGC AAC

Subtilisin toxin

precursor (genomic)

(Sadeghi et al.,

2012)

aprE (R) GGA AGT GCC TTC

ATT TCC GGC T

Subtilisin toxin

precursor (genomic)

(Sadeghi et al.,

2012)

gyrB (F)

TTT CTG GTG GTT

TAC

ATG G

β-subunit DNA

gyrase (genomic)

(Manzano et al.

2003)

gyrB (R)

TTT TGA GCG ATT

TAA

ATG C

β-subunit DNA

gyrase (genomic)

(Manzano et al.

2003)

(F) = Forward primer; (R) = Reverse primer; Bt = B. thuringiensis.

2.6.1.1. In silico cross-reactivity analysis of oligonucleotides

To verify the specificity of potential primer sets, a search was run on the Basic Local

Alignment Search Tool (BLAST) database. The primer specificity stringency was set to

ignore targets that had five or more mismatches to the primer. Cross-reactivity was

checked against all species within the Bacillus genera database. Cross-reactivity against

25

Bacillus species, including the number of strains the target sequence appeared was

recorded for each primer set.

2.6.2. qPCR protocol for field samples

For field samples, qPCR reactions were prepared in 96-well plates. Each reaction was

made to a final volume of 25 µL and contained a variable concentration of DNA

template, 2.5x FastStart SYBR Green Master Mix (Roche), 400 ng BSA and 0.05 – 0.2

µM of 5’ – 3’ primers made up with DNase free water. The final primer concentrations

depended on the bacterial targets and are summarised in Table 2.3. The FastStart SYBR

Green Master Mix contained reaction buffer, FastStart Taq polymerase, dNTPs and

SYBR Green I intercalating dye. Reaction conditions included an initial activation step

of 95°C for 10 minutes, following by 40 repeating cycles of denaturation (95°C; 30

seconds), annealing (54°C; 30 seconds) and extension (72°C; 30 seconds). Data was

collected in the extension component of each qPCR cycle.

Table 2.3: Primer concentrations

Oligonucleotides Concentration in

qPCR protocol (µM)

PL3 0.2

Cya 0.2

capB 0.2

Cry1 0.2

Cry4 0.05

gyrB 0.1

aprE 0.05

Results from qPCR assays were obtained in the form of an amplification plot, where

cycle threshold (CT) values were measured and reported. CT values were calculated

from the point at which amplification intersected a manually defined threshold.

Thresholds were set within the exponential phase of fluorescence, as low as possible

without capturing any background noise. In most cases, thresholds were set at 10,000.

All reactions were performed in duplicate (n = 2). A no template control (NTC) was

also included in each qPCR assay performed.

An inhibition control was run on each of the field samples collected, utilising the Cry1

assay. The inhibition control comprised of the reaction mixture described above spiked

with a known concentration of standard B. thuringiensis template DNA. A positive

control using the same known concentration of B. thuringiensis subsp. Kurstaki was

26

also subjected to qPCR protocol alongside the inhibition control samples. The CT values

of the spiked Canberra Airport samples and the positive control were compared and if

no difference was observed, the sample was considered not to be inhibited.

2.6.3. qPCR protocol for optimisation experiments

All qPCR experiments were performed on the 7500 Real-Time PCR System. For

optimisation assays, qPCR reactions were set-up manually in 96-well plates. Each

reaction was made to a final volume of 25 µL and contained 2.5x FastStart SYBR

Green Master Mix, 400 ng BSA and 0.05 – 0.2 µM of 5’ – 3’ primers made up with

DNase free water. Concentrations of DNA added to each reaction differed between

species. The concentrations of DNA used for optimisation experiments are summarised

in Table 2.4. Reaction conditions included an initial activation step of 95°C for 10

minutes, following by 40 repeating cycles of denaturation (95°C; 30 seconds), annealing

(54°C; 30 seconds) and extension (72°C; 30 seconds). Data was collected in the

extension component of each qPCR cycle. All reactions were performed in triplicate

(n = 3). A no template control (NTC) was also included in each qPCR assay performed.

Table 2.4: DNA concentration of bacterial species used in optimisation

experiments

Bacterial species Concentration

B. subtilis 13.5 ng

B. cereus 7.82 ng

B. anthracis str. Sterne 4.69 ng

B. thuringiensis subsp. Kurstaki 19.9 ng

B. thuringiensis subsp. Israelensis 0.902 ng

Results from qPCR assays were obtained in the form of an amplification plot, where CT

values were measured and reported. CT values were calculated from the point at which

amplification intersected a manually defined threshold. Thresholds were set within the

exponential phase of fluorescence, as low as possible without capturing any background

noise. In most cases, thresholds were set at 10,000.

2.7. Agarose gel electrophoresis

PCR products were subjected to agarose gel electrophoresis in order to view the size of

the amplified fragments. All gels were made to a 1% concentration by combining

27

500 mg of agarose (Bioline) and 50 mL of 1 x tris-acetate-EDTA (TAE) buffer. The

solution was mixed and heated in a microwave until the solution appeared clear. A 1 x

concentration of SYBR safe DNA gel stain (Life Technologies™) was added to the

solution, before it was poured into a mould with a comb and left to set for 20-25

minutes. The gel was placed in tank, which was then filled to the maximum level with 1

x TAE buffer. 10 µL of the PCR product was then combined with 2 µL of 5 x loading

buffer (Bioline) and pipetted into one of the wells in the agarose gel. PCR products

were electrophoresed at 100 volts (v) for 50 – 60 minutes. The PCR products were

visualised under UV light using a Gel Doc™ XR+ transilluminator (Bio-Rad). The sizes

of the fragments were compared with those in the Hyperladder™ 50bp DNA standard

ladder (Bioline).

2.8. Data analysis

Statistical analysis was carried out using GraphPad Prism® software, version 6.01. Data

was analysed using contingency tables with Fischer’s exact test. A probability value (p

value) of <0.05 was considered statistically significant.

2.8.1. Calculation of qPCR assay efficiency

The efficiency of each qPCR reaction was calculated using Equation 1 and the slope of

the appropriate Bacillus species standard curves prepared for limit of detection (LOD)

experiments.

Equation 1

2.8.2. Calculation of copy number

DNA concentration was converted into copy number using Equations 2-4.

Equation 2

28

Where, the average mass of each base pair = 1 x 10-21

g and genome lengths are as

given in Table 2.5.

Table 2.5: Genome sizes of bacterial species used in this study.

Species Genome length (bp) Source

B. thuringiensis 5,400,000 (Carlson et al., 1994)

B. cereus 5,400,000 (Rasko et al., 2005)

B. subtilis 4,214,814 (Sadeghi et al., 2012)

B. anthracis str. Sterne 5,228,700 (Kolstø et al., 2009)

Bp = base pairs

Equation 3

⁄

⁄

Equation 4

⁄

29

Chapter 3: Results

Development of singleplex qPCR assays for the detection and

quantitation of B. cereus, B. subtilis and B. thuringiensis subsp.

Israelensis.

The aim of this study was to develop and validate three singleplex qPCR assays capable

of both detecting and quantitating B. cereus, B. subtilis and B. thuringiensis subsp.

Israelensis.

3.1. In silico analysis of oligonucleotides

None of the primer sets tested were shown to be specific to B. cereus (Table 3.1). Two

primer sets were shown to be specific for B. subtilis, with the results of the Primer-

BLAST suggesting that the target sequence is not present in any other Bacillus species.

Table 3.1: In silico analysis of the cross-reactivity of primer sets using the Basic

Local Alignment Search Tool (BLAST) database

Primer sequence (5’ – 3’) Target Target sequence present in

F: AAT GGT CAT CGG AAC

TCT AT

R: CTC GCT GTT CTG CTG

TTA AT

B. cereus hblD

102 strains of B. cereus

32 strains of B. thuringiensis

7 strains of B. anthracis

F: GTG CAG ATG TTG ATG

CCG AT

R: ATG CCA CTG CGT GGA

CAT AT

B. cereus hblA

121 strains of B. cereus

34 strains of B. thuringiensis

6 strains of B. anthracis

F: CGG TAG TGA TTG CTG GG

R: CAG CAT TCG TAC TTG

CCA A

B. cereus nheC

88 strains of B. cereus

27 strains of B. thuringiensis

6 strains of B. anthracis

F: AAG TCG AGC GGA CAG

ATG G

R: CCA GTT TCC AAT GAC

CCT CCC C

B. subtilis 16S

rRNA

3 strains of B. cereus

Two strains of B. thuringiensis

36 strains of members within

the B. subtilis group

F: GCA TAT GAT GTA GCG

AAA CAA GCC

R: GCG TGA CAT ACC CAT

TTC CAG GTC C

B. thuringiensis

subsp.

Israelensis

Cry4

40 strains of B. thuringiensis

F: TTT ACG ATG GCG TTC

AGC AAC

R: GGA AGT GCC TTC ATT

TCC GGC T

B. subtilis

subtilisin E

30 strains of species within the

B. subtilis group

F: GCG GCG TGC CTA ATA

CAT GC

B. subtilis

Target spans 16S

39 strains within the B. subtilis

group

30

R: CAC CTT CCG ATA CGG

CTA CC

rRNA and the

rrnE gene

81 strains of B. cereus

25 strains of B. thuringiensis

5 strains of B. megatarium

3 strains of B. mycoides

5 strains of B. anthracis

F: TTT CTG GTG GTT TAC

ATG G

R: TTT TGA GCG ATT TAA

ATG C

B. cereus

gyrB gene

107 strains of B. cereus

27 strains of B. thuringiensis

6 strains of B. anthracis

3 strains of B. mycoides

F: CTT CTT GGC CTT CTT CTA

A

R: GAG ATT TAA ATG AGC

TGT AA

B. cereus

Cold shock

protein

131 strains of B. cereus

28 strains of B. thuringiensis

6 strains of B. anthracis

3 strains of B. mycoides

1 strain of B. megatarium

F = Forward primer; R = Reverse primer

3.2. Development of a singleplex qPCR assay for the detection and

quantification of B. subtilis

3.2.1. Determining the specificity of the 16S rRNA and aprE primers

To determine whether the 16S rRNA and aprE primer sets could amplify B. subtilis,

primers were combined with B. subtilis DNA and subjected to the qPCR protocol

outlined in Section 2.6.3. All reactions, excluding the NTC, were performed in triplicate

(n = 3). The results of the experiment are presented as CT values ± standard deviation.

To confirm that amplicons were the correct size, qPCR products were subjected to the

agarose gel electrophoresis protocol Section 2.7.

Amplification was observed for the aprE assay, with a CT value of 15.1 ± 0.5, and there

was no amplification for the NTC. The gel revealed a single large band which aligned

roughly with the 750 bp marker (data not shown).

Amplification was also observed for the 16S rRNA assay, with exponential growth

recorded early in the assay and a CT value of 10.3 ± 0.02. A large band with streaking

on either side was visible in the gel at approximately 600 bp. Amplification was also

observed in the NTC at a CT of 35.2 with a faint band visible on the gel at

approximately 50 bp.

31

3.2.2. Determining the cross-reactivity of the 16S rRNA and aprE

primers against other Bacillus species

To determine the cross-reactivity of the 16S rRNA and aprE primer sets, each primer set

was combined with the template DNA of B. anthracis str. Sterne, B. cereus, B.

thuringiensis subsp. Kurstaki, B. thuringiensis subsp. Israelensis, B. mycoides and B.

megatarium and subjected to the qPCR protocol. A concentration of 0.2 µM was used

for both primer sets. The CT values were recorded for each Bacillus species that

amplified in the qPCR protocol and are summarised in Table 3.2.

Table 3.2: Cross-reactivity of B. subtilis aprE and 16S rRNA primers

Bacterial species aprE CT 16S rRNA CT

B. subtilis 13 ± 0.6 8.8 ± 0.2

B. cereus 33.1 ± 0.6 27 ± 0.5

B. thuringiensis subsp. Kurstaki 36.6 ± 1.7 27.9 ± 0.3

B. thuringiensis subsp. Israelensis -- 27.6 ± 0.4

B. anthracis str. Sterne 34.6 ± 0.8 28.3 ± 0.5

B. mycoides -- --

B. megatarium -- --

NTC -- 31.3

Cycles at which fluorescence threshold was achieved (CT) are presented as an average ±

standard deviation. All reactions were performed in triplicate, with the exception of the

no template control (NTC) which was performed as a single reaction.

Amplification was observed in reactions containing aprE primers and B. subtilis, B.

cereus, B. thuringiensis subsp. Kurstaki and B. anthracis str. Sterne DNA.

Amplification of B. subtilis occurred much earlier in the assay than the other cross-

reacting Bacillus species, with an average CT value of 13 ± 0.6. CT values for the cross-

reacting species ranged from averages of 33.1 ± 0.6 to 36.6 ± 1.7. There was no

amplification observed for B. megatarium and B. mycoides. On the agarose gel there

was a single large band at approximately 750 bp for B. subtilis and a band at

approximately 800 bp for B. anthracis str. Sterne and B. cereus. No bands were visible

for B. thuringiensis subsp. Kurstaki (data not shown).

32

Cross-reactivity was also observed in the 16S rRNA assay, with B. anthracis str. Sterne,

B. cereus, B. thuringiensis subsp. Kurstaki and B. thuringiensis subsp. Israelensis all

amplifying at around CT 27. There was no amplification observed for B. megatarium

and B. mycoides. There was a band at approximately 600 bp on the agarose gel for B.

subtilis, B. anthracis and B. cereus and a faint band at approximately 50 bp for B.

anthracis, B. cereus, B. thuringiensis subsp. Kurstaki and B. thuringiensis subsp.

Israelensis (data not shown).

To determine whether the cross-reactivity of the aprE and 16S rRNA primers could be

alleviated through reducing the primer concentration, the concentrations of both primer

sets were lowered to 0.1 µM and 0.05 µM and re-tested. At primer concentrations of

0.1 µM and 0.05 µM, cross reactivity was still observed for the 16S rRNA assay, with

amplification occurring for B. anthracis str. Sterne, B. cereus, and B. thuringiensis

subsp. Kurstaki and B. thuringiensis subsp. Israelensis (Table 2.3).

Table 3.3: Cross-reactivity of the 16S rRNA B. subtilis assay at primer

concentrations of 0.1 µM and 0.05 µM

Primer concentration

Bacterial species 0.1 µM 0.05 µM

B. subtilis 12.2 ± 0.3 9.2 ± 0.5

B. thuringiensis subsp. Kurstaki 31.7 ± 0.2 32.5 ± 0.2

B. anthracis str. Sterne 35 ± 2.8 35.4 ± 0.6

B. cereus 31.2 ± 0.4 33.7 ± 0.3

B. thuringiensis subsp. Israelensis 31.6 ± 0.3

B. megatarium -- --

B. mycoides -- --

NTC 33.2 39.6

Cycles at which fluorescence threshold was achieved (CT) are presented as an average ±

standard deviation. All reactions were performed in triplicate, with the exception of the

no template control (NTC) which was performed as a single reaction.

At a primer concentration of 0.1 µM for the aprE assay, amplification still occurred for

B. thuringiensis subsp. Kurstaki, B. anthracis str. Sterne and B. cereus. At this primer

concentration, the average CT value for B. subtilis was 11.3 ± 0.6. At a primer

33

concentration of 0.05 µM only B. subtilis amplified. However, the average CT of B.

subtilis nearly doubled from an average of 11.3 ± 0.6 for 0.1 µM to 20.6 ± 1.5 µM.

Table 3.4: Cross-reactivity of the aprE B. subtilis assay at primer concentrations of

0.1 µM and 0.05 µM

Primer concentration

Bacterial species 0.1 µM 0.05 µM

B. subtilis 11.3 ± 0.6 20.6 ± 1.5

B. thuringiensis subsp. Kurstaki 37.4 ± 0.3 --

B. anthracis str. Sterne 37.2 ± 0.3 --

B. cereus 35.2 ± 0.6 --

B. thuringiensis subsp. Israelensis -- --

B. megatarium -- --

B. mycoides -- --

NTC -- --

Cycles at which fluorescence threshold was achieved (CT) are presented as an average ±

standard deviation. All reactions were performed in triplicate, with the exception of the

no template control (NTC) which was performed as a single reaction.

As the cross-reactivity could not be eliminated from the 16S rRNA assay at primer

concentrations of either 0.05 µM or 0.1 µM the aprE assay was chosen for use in

detecting B. subtilis in all further assays.

3.2.3. Determining the limit of detection of the aprE primer assay

To determine the limit of detection (LOD) at a primer concentration of 0.05 µM, an

eight point serial dilution of B. subtilis DNA was constructed with an initial DNA input

of 13.5 ng/µL. The serial dilution consisted of the following DNA concentrations: 13.5

ng, 1.35 ng, 0.135 ng, 0.0135 ng, 1.35 x 10-3

ng, 1.35 x 10-4

ng, 1.35 x 10-5

ng and 1.35

x 10-6

ng. Reactions were performed in triplicate (n = 3). The standard curve is shown in

Figure 3.1.

34

Figure 3.1: Standard curve for the aprE assay.

Amplification was observed in the first four dilutions, with CT values ranging from 16.7

± 1.3 for 13.5 ng to 36.6 ± 0.7 for 0.0135 ng. No amplification was observed in the

dilutions containing 1.35 x 10-3

ng, 1.35 x 10-4

ng, 1.35 x 10-5

ng and 1.35 x 10-6

ng of

B. subtilis DNA.

Using the information from Figure 3.1 and the genome size and Equations 1-4 from

Section 2.8.2, the LOD was calculated to be 3144 copies. Additionally, using the slope

equation in Figure 3.1 and Equation 1 from Section 2.8.1, the efficiency of the reaction

was calculated to be 42%.

3.3. Development of a singleplex qPCR assay for the detection and

quantification of B. thuringiensis subsp. Israelensis

3.3.1. Determining the specificity of the Cry4 primers

To determine whether the primer set could amplify B. thuringiensis subsp. Israelensis,

the Cry4 primers were combined with B. thuringiensis subsp. Israelensis DNA and

subjected to the qPCR protocol outlined in Section 2.6.3. The reactions, excluding the

NTC, were performed in triplicate (n = 3). The primers were able to amplify B.

thuringiensis subsp. Israelensis with an average CT value of 18.2 ± 0.3 A band of

y = -6.573x + 24.123 R² = 0.9985

15

20

25

30

35

40

-2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5

CT

log10 [DNA concentration (ng)]

35

approximately 450 bp was observed in the agarose gel, which matched the expected size

of 434 bp.

3.3.2. Determining the cross-reactivity of the Cry4 primers against

other Bacillus species

To determine the specificity of the Cry4 primers, the primer set was combined with the

template DNA of B. anthracis str. Sterne, B. cereus, B. thuringiensis subsp. Kurstaki,

B. subtilis, B. thuringiensis subsp. Israelensis, B. mycoides and B. megatarium, at a

concentration of 0.2 µM and subjected to the qPCR protocol.

Table 3.5: Cross-reactivity of the Cry4 primers

Bacterial species Cry4 CT

B. subtilis 31.1 ± 0.5

B. cereus 37.6 ± 2.1

B. thuringiensis subsp. Kurstaki 37.6 ± 2.5

B. thuringiensis subsp. Israelensis 18 ± 0.6

B. anthracis str. Sterne 34.6 ± 0.8

B. mycoides --

B. megatarium 39.5 ± 0.4

NTC --

Cycles at which fluorescence threshold was achieved (CT) are presented as an average ±

standard deviation. All reactions were performed in triplicate, with the exception of the

no template control (NTC) which was performed as a single reaction.

Cross-reactivity was observed in the B. thuringiensis subsp. Israelensis Cry4 assay,

with amplification evident in all species tested except for B. mycoides. All species

amplified late in reaction after 30 cycles, with the exception of the target, B.

thuringiensis subsp. Israelensis, which amplified at an average CT value of 18 ± 0.6.

To determine whether the cross-reactivity observed in the previous experiment could be

abated through the lowering of the primer concentration, the assay was re-tested with

primer concentrations of 0.1 µM and 0.05 µM.

36

At 0.1 µM, only B. thuringiensis subsp. Israelensis and B. subtilis amplified, with

average CT values of 20.5 ± 1.1 and 35.7 ± 0.2, respectively. At 0.05 µM, the assay

became specific to the target, with amplification observed only in reactions containing

B. thuringiensis subsp. Israelensis (Table 3.6)

Table 3.6: Cross-reactivity of the Cry4 B. thuringiensis subsp. Israelensis assay at

primer concentrations of 0.1 µM and 0.05 µM

Primer concentrations

Bacterial species 0.1 µM 0.05 µM

B. thuringiensis subsp. Israelensis 20.5 ± 1.1 21.4 ± 1.3

B. thuringiensis subsp. Kurstaki -- --

B. anthracis -- --

B. cereus -- --

B. subtilis 35.7 ± 0.2 --

B. megatarium -- --

B. mycoides -- --

NTC -- --

Cycles at which fluorescence threshold was achieved (CT) are presented as an average

± standard deviation. All reactions were performed in triplicate, with the exception of

the no template control (NTC) which was performed as a single reaction.

3.3.3. Determining the limit of detection of the cry4 primers

To determine the LOD for the Cry4 primers at a concentration of 0.05 µM, an eight

point serial dilution of a known concentration of standard B. thuringiensis subsp.

Israelensis was set up, as described in section (#). The final concentrations of the

dilutions assayed were 0.902 ng, 0.0902 ng, 9.02 x 10-3

ng, 9.02 x 10-4

ng, 9.02 x 10-5

ng, 9.02 x 10-6

ng, 9.02 x 10-7

ng and 9.02 x 10-8

ng. The standard curve is shown in

Figure 3.2.

37

Figure 3.2: Standard curve for the cry4 assay.

The CT values for the assay ranged from averages of 19 ± 0.7 for 0.902 ng to 33.9 ± 0.4

for 9.02 x 10-4

ng of DNA. The last four dilutions in the serial dilution, 9.02 x 10-5

ng,

9.02 x 10-6

ng, 9.02 x 10-7

ng and 9.02 x 10-8

ng, did not amplify.

Using the information from Figure 3.2 and the genome size and Equations 2-4 from

Section 2.8.2, the LOD was calculated to be approximately 155 copies. Using the slope

equation in Figure 3.2 and Equation 1 from Section 2.8.1, the efficiency of the reaction

was calculated to be 59%.

3.4. Development of a singleplex assay for the detection and

quantitation of B. cereus

3.4.1. Determining the specificity of hblC and gyrB primers

To determine whether the primer sets could amplify B. cereus, both the hblC and gyrB

primer sets were combined with B. cereus DNA and subjected to the qPCR protocol.

All reactions, excluding the NTC, were performed in triplicate (n = 3). The results of

the experiment are presented as CT values ± standard deviation. Following the qPCR,

products were electrophoresed (see Section 2.7) to confirm that correct amplicons were

being obtained.

y = -4.978x + 18.977 R² = 0.9978

15

20

25

30

35

40

-3.5 -3 -2.5 -2 -1.5 -1 -0.5 0

CT

Log10 [DNA Concentration (ng)]

38

At a primer concentration of 0.2 µM there was no amplification observed in the hblC

assay. Similarly, no bands were visible in the gel. Similarly, no amplification was

observed at primer concentrations of 0.1 µM or 0.05 µM, or with diluted template DNA.

As the primers were not able to amplify B. cereus DNA, their use was not carried

forward in future testing.

Amplification was observed in the gyrB assay, with an average CT value of 21 ± 0.3.

The gel revealed a single band which aligned roughly with the 400 bp marker (data not

shown).

3.4.2. Determining the cross-reactivity of the gyrB primers against

other Bacillus species

To determine the specificity of the gyrB primers, the primer set was combined with the

template DNA of B. anthracis str. Sterne, B. cereus, B. thuringiensis subsp. Kurstaki,

B. subtilis, B. thuringiensis subsp. Israelensis, B. mycoides and B. megatarium, at a

concentration of 0.2 µM.

Amplification was observed in all Bacillus species examined, with the exception of B.

mycoides and B. megatarium (Table 3.7). B. cereus, B. thuringiensis subsp. Kurstaki

and B. thuringiensis subsp. Israelensis all amplified at similar times, mid-way through

the reaction at approximately a CT value of 20. B. anthracis str. Sterne amplified shortly

after at an average CT value of 23.7 ± 0.9. B. subtilis amplified very late in the reaction

at 37.8 ± 0.3. The gel image revealed identical banding at approximately the 400 bp

marker in lanes containing B. cereus, B. anthracis and B. thuringiensis subsp. Kurstaki

and B. thuringiensis subsp. Israelensis. No banding was visible in the lane containing B.

subtilis.

39

Table 3.7: Cross-reactivity of the gyrB primers

Bacterial species gyrB CT

B. subtilis 37.8 ± 0.3

B. cereus 20.2 ± 0.8

B. thuringiensis subsp. Kurstaki 19.1 ± 1.1

B. thuringiensis subsp. Israelensis 19.8 ± 0.6

B. anthracis str. Sterne 23.7 ± 0.9

B. mycoides --

B. megatarium --

NTC --

Cycles at which fluorescence threshold was achieved (CT) are presented as an average ±

standard deviation. All reactions were performed in triplicate, with the exception of the

no template control (NTC) which was performed as a single reaction.

To determine whether the cross-reactivity in the gyrB assay could be abated through

lowering the primer concentration, the assay was re-tested at primer concentrations of

0.1 µM and 0.05 µM. Lowering the primer concentration to 0.1 µM abates the cross-

reactivity of the gyrB primers against B. subtlis (Table 3.8).

Table 3.8: Cross-reactivity of the gyrB assay at primer concentrations of 0.1 µM

and 0.05 µM.

Primer concentrations

Bacterial species 0.1 µM 0.05 µM

B. thuringiensis subsp. Israelensis 22.1 ± 0.4 22.5 ± 1.2

B. thuringiensis subsp. Kurstaki 21.8 ± 0.5 21 ± 1.3

B. anthracis 29.4 ± 0.2 35 ± 1.9

B. cereus 23.7 ± 0.8 23.5 ± 0.4

B. subtilis -- --

B. megatarium -- --

B. mycoides -- --

Cycles at which fluorescence threshold was achieved (CT) are presented as an

average ± standard deviation. All reactions were performed in triplicate, with the

exception of the no template control (NTC) which was performed as a single

reaction.

40

3.4.3. Determining the limit of detection of the gyrB assay

To determine the LOD for the gyrB assay, an eight point serial dilution of a known

concentration of standard B. cereus was set up, as described in section (#). The final

concentrations of the dilutions assayed were 7.82 ng, 0.782 ng, 0.0782 ng, 7.82 x 10-3

ng, 7.82 x 10-4

ng, 7.82 x 10-5

ng, 7.82 x 10-6

ng and 7.82 x 10-7

ng. The standard curve

is shown in Figure 3.3.

Figure 3.3: standard curve for the gyrB assay.

Amplification was observed in the first three dilutions, with CT values ranging from

averages of 19.8 ± 2.4 for 7.82 ng to 33.2 ± 0.9 for 0.0782 ng. No amplification was

observed in the dilutions between 7.82 x 10-3

ng and 7.82 x 10-7

ng.

Using the information from Figure 3.3 and the genome size and Equations 2-4 from

Section 2.8.2, the LOD was calculated to be 14,481 copies. Additionally, using the

slope equation in Figure 3.3 and the Equation 1 from 2.8.1, the efficiency of the reaction

was calculated to be 41%.

3.5. Determining the LOD of the B. anthracis str. Sterne assays

The B. anthracis str. Sterne assays, including the singleplex qPCR’s for the detection of

the genome (PL3) and pXO1 (cya), were developed and optimised by Rossi (2011).

y = -6.695x + 25.834 R² = 1

0

5

10

15

20

25

30

35

-1.5 -1 -0.5 0 0.5 1

CT

log10 [DNA Concentration (ng)]

41

However, the LOD for these assays was not determined. The aim of this section was to

determine the LOD for the B. anthracis str. Sterne PL3 and cya assays.

To determine the LOD of the B. anthracis str. Sterne assay, an eight point serial dilution

of B. anthracis str. Sterne DNA was prepared. The final concentrations of the dilutions

assayed were 4.69 ng, 0.469 ng, 0.0469 ng, 4.69 x 10-3

ng, 4.69 x 10-4

ng,

4.69 x 10-5

ng, 4.69 x 10-6

ng and 4.69 x 10-7

ng. Reactions were performed in triplicate

(n = 3). The standard curve is shown in Figure 3.4.

Figure 3.4: Standard curve for the B. anthracis str. Sterne PL3 assay.

The CT values for the assay ranged from averages of 16.92 ± 0.3 for 4.69 ng to 35.5 ±

1.0 for 4.69 x 10-5

ng/µL of DNA. The last two dilutions in the serial dilution,

4.69 x 10-6

and 4.69 x 10-7

ng/µL, did not amplify.

Using the information from Figure 3.4 and the genome size and Equation 2-4 from

Section 2.8.2, the LOD was calculated to be approximately 9 copies. Using the slope

equation in Figure 3.4 and Equation 1 from Section 2.8.1, the efficiency of the reaction

was calculated to be 87.5%.

y = -3.7042x + 19.207 R² = 0.9992

0

10

20

30

40

-5 -4 -3 -2 -1 0 1

CT

Log10 [DNA Concentration (ng)]

42

3.6. Determining the LOD of the of B. thuringiensis subsp. Kurstaki

assay

The B. thuringiensis subsp. Kurstaki assay, which targets the Cry1 gene, was developed

and optimised by Rossi (2011). However, the LOD was not determined. To determine

the LOD of the B. thuringiensis subsp. Kurstaki assay, an eight point serial dilution of

B. thuringiensis subsp. Kurstaki DNA was prepared. The final concentrations of the

dilutions assayed were 19.9 ng, 1.99 ng, 0.199 ng, 0.0199 ng, 1.99 x 10-3

ng, 1.99 x 10-4

ng, 1.99 x 10-5

ng and 1.99 x 10-6

ng. Reactions were performed in triplicate (n = 3).

The standard curve is shown in Figure 3.5.

Figure 3.5: Standard curve for the B. thuringiensis subsp. Kurstaki Cry1 assay.

The CT values for the assay ranged from averages of 13.1 ± 0.3 for 19.9 ng to 38.2 ± 1.0

for 1.99 x 10-5

ng of B. anthracis str. Sterne DNA. The last dilution in the serial

dilution, 1.99 x 10-6

ng, did not amplify.

Using the information from Figure 3.5 and the genome size and Equations 2-4 from

Section 2.8.2, the LOD was calculated to be approximately 4 copies. Additionally, using

the slope equation in Figure 3.5 and Equation 1 from Section 2.8.1, the efficiency of the

reaction was calculated to be 65.2%.

y = -4.439x + 18.005 R² = 0.9933

0

10

20

30

40

-6 -5 -4 -3 -2 -1 0 1 2

CT

Val

ue

Log10 of DNA Concentration (ng)

43

Chapter 4: Results

4.1. Determining the presence of PCR inhibition in samples

collected from the Canberra Airport

All samples collected from the Canberra Airport were assessed for PCR inhibition to

ensure that negative results were true negatives and were not false negatives caused by

the presence of PCR inhibitors. To determine whether PCR inhibition was present, each

sample was spiked with a known concentration of B. thuringiensis subsp. Kurstaki

DNA and subjected to the Cry1 qPCR protocol (see Section 2.6.2). Alongside these

spiked samples, a positive using the same known concentration of B. thuringiensis

subsp. Kurstaki was also subjected to the qPCR protocol. The CT values of the spiked

Canberra Airport samples and the positive control were compared and if no difference

was observed, the sample was considered not to be inhibited.

There were no differences between any of the CT values for spiked Canberra Airport

samples and positive controls with all spiked samples being within 1 CT of the positive

controls (data not shown). As there were no differences, all 331 samples analysed were

determined not to be inhibited.

4.2. Determining the presence and concentration of B. anthracis at

the Canberra Airport

To determine the presence of B. anthracis at the Canberra Airport and at Air Freight

Australia, 331 samples December 2011 and July 2012 were analysed using the

B. anthracis PL3, cya and capB assays (see Section 2.6.2). All samples were analysed in

duplicate (n = 2), with the exception of the no template control (NTC), which was

performed as a single reaction.

Of the 331 samples analysed, 17 samples returned a positive result for the B. anthracis

PL3 genomic marker (Table 4.1). Amplification occurred late in these samples, with CT

values ranging from 34.4 to 38.1 Out of the 17 samples that, only the replicates of four

samples amplified uniformly; only one replicate amplified for the other 13 samples.

February 2012 had a significantly higher of proportion of positive samples than any

other month (p < 0.05). April 2012 also had a significantly higher proportion of positive

samples than any other month, except February 2012 (p < 0.05).

44

Table 4.1: Samples positive for the B. anthracis PL3 target

Sample Replicate 1 CT Replicate 2 CT

CA9 PO 20/02 35.9 --

CA9 LO 20/02 36.7 35.9

CA9 LI 20/02 37.4 --

CA9 POV 20/02 36.7 35.6

CA9 LC 20/02 35.6 --

CA9 PO 21/02 35.3 36.4

CA9 LO 21/02 35.3 --

CA9 POV 22/02 34.4 --

CA9 LO 24/02 36.8 36.5

CA9 LC 24/02 36.3 --

CA9 LO 25/02 35.3 --

CA9 LC 25/02 37.7 --

CA11 LO 23/04 37.3 --

CA11 PO 29/04 38.1 --

CA11 LI 29/04 38.1 --

CA11 POV 29/04 37.4 --

CA13 LO 21/06 35.9 --

Sample notation = [sampling month, sampling location, date sample was taken]; PO =

People OUT; LO = Luggage OUT; LI = Luggage IN; POV = People OUT vacuum; LC

= Low control. Cycles at which fluorescence threshold was achieved (CT) are presented.

The greatest incidence occurred in samples collected from the carousel on which the

luggage leaving Canberra was transported (Luggage OUT; LO), with six of the 17

positive samples collected from this site. The LO sampling site a significantly higher

proportion of positive samples than any other sampling site (p < 0.05). Three samples

collected from the site near the security checkpoint, which captured people departing

Canberra (People OUT; PO) also tested positive. The vacuum sample collected near this

point also returned three positive results, two of which overlapped with samples

collected from PO on the same days. Three samples from the “Low Control” (LC) site

tested positive as did two samples collected from the QANTAS baggage claim carousel

(Luggage IN; LI). No samples collected from the base of the arrival escalator (People

In; PI) during the study period tested positive. Apart from PI and LO, no sampling site

was significantly different any other (p > 0.05)

The concentration of the samples which returned a positive result for the B. anthracis

PL3 genomic marker in the presence/absence screening were determined using

quantitative PCR (qPCR) as described in Section #. Of the 17 samples analysed, only

five samples amplified during the quantification assay (Table 4.2), however all CT

45

values fell outside of the standard curve and were thus reported as below limit of

detection (BLOD).

Table 4.2: Concentration of samples positive for the B. anthracis PL3 genomic

marker

Sample Concentration of DNA (ng) Copy number

CA9 PO 20/02

CA9 LO 20/02 BLOD BLOD

CA9 LI 20/02 BLOD BLOD

CA9 POV 20/02

CA9 LC 20/02

CA9 PO 21/02

CA9 LO 21/02 BLOD BLOD

CA9 POV 22/02 BLOD BLOD

CA9 LO 24/02 BLOD BLOD

CA9 LC 24/02

CA9 LO 25/02

CA9 LC 25/02

CA11 LO 23/04

CA11 PO 29/04

CA11 LI 29/04

CA11 POV 29/04

CA13 LO 21/06

PO = People OUT; LO = Luggage OUT; LI = Luggage IN; POV = People OUT

vacuum sample; LC = Low control; BLOD = Below limit of detection; NQ = Not able

to be quantitated. All samples, including the no template control (NTC), were

performed in duplicate. Cycles at which fluorescence threshold was achieved (CT) are

presented as an average ± standard deviation. Samples in which only one of replicates

amplified are presented as a CT value.

Three samples out of the 331 samples analysed were positive for the B. anthracis capB

target found on pXO2 (Table 4.3). Amplification occurred late in all three samples, with

CT values ranging from 32.5 ± 0.07 to 39.9 ± 0.07. All three positive samples came

from different sampling sites as well as from different sampling months. Due to

restrictions involving the use of pOX1 and pXO2 containing strains in the same

laboratory the concentration of these samples could not be determined.

Table 4.3: Samples positive for the B. anthracis capB target

Sample Replicate 1 CT Replicate 2 CT

CA7 FI 19/12 32.5 32.6

CA9 POV 21/02 39.9 40

CA13 LO 20/06 36.6

46

FI = Freight IN; POV = People OUT vacuum; LO = Luggage OUT. Cycles at which

fluorescence threshold was achieved (CT) are presented as an average ± standard

deviation. Samples in which the duplicate did not amplify are presented as a CT value.

Of the 331 samples 29 tested positive for the cya gene found on PXO1 (Table 4.4).

Amplification occurred late in all positive samples, with CT values ranging from 35.5 to

39.9. Of the 29 samples that were positive for the B. anthracis cya assay, 24 were from

samples collected in February 2012. The remaining five samples were collected in

December 2011, April 2012 and June 2012. The sampling location with greatest

incidence of positive samples across all months was PO.

Table 4.4: Samples positive for the B. anthracis cya target

Sample CT1 CT2

CA7 PO 15/12 39.7

CA7 FI 18/12 38.6

CA9 POV 20/02 37.7 37.5

CA9 LC 20/02 38.2

CA9 PO 21/02 38.5 36.9

CA9 LO 21/02 37.9

CA9 LI 21/02 37.4

CA9 POV 21/02 39.1

CA9 LC 21/02 38.9

CA9 PO 22/02 38.2

CA9 PI 22/02 37.3

CA9 LO 22/02 38.2

CA9 LI 22/02 38.2

CA9 PO 23/02 38.2 38.9

CA9 PI 23/02 39.7 40

CA9 LO 23/02 37.6

CA9 LI 23/02 38.2

CA9 POV 23/02 37.9

CA9 LC 23/02 39.9 40

CA9 PO 24/02 35.5

CA9 LI 24/02 38

CA9 LC 24/02 37.7

CA9 PO 25/02 36.0 37.2

CA9 PI 25/02 38.7 38.0

CA9 LO 25/02 38.9

CA9 LI 25/02 38.1 40

CA11 POV 23/04 39.9

CA11 POV 26/04 39.9

CA13 PI 18/06 39.4

PO = People OUT; PI = People IN; LO = Luggage OUT; LI = Luggage IN; POV =

People OUT vacuum sample; LC = Low control; FI = Freight IN. All samples,

including the no template control (NTC), were performed in duplicate. Cycles at which

47

fluorescence threshold was achieved (CT) are presented as an average ± standard

deviation. Samples in which the duplicate did not amplify are presented as a CT value.

Of the 17 samples that tested positive for the B. anthracis PL3 marker, six of them also

tested positive for the cya gene (Table 4.5). All six samples were taken in February

2012. One sample tested positive for the cya and capB genes.

Table 4.5: Summary of the overlap between samples testing positive for the PL3,

cya and capB genes

Sample PL3 Positive Cya Positive capB Positive

CA9 POV 20/02 Yes Yes No

CA9 LC 20/02 Yes Yes No

CA9 PO 21/02 Yes Yes No

CA9 LO 21/02 Yes Yes No

CA9 LC 24/02 Yes Yes No

CA9 LC 25/02 Yes Yes No

CA9 POV 21/02 No Yes Yes

POV = People OUT vacuum; LC = Low control; PO = People OUT; LO = Luggage

OUT; LC = Low control.

4.3. Determining the presence and concentration of B. thuringiensis

subsp. Kurstaki at the Canberra Airport

To determine the presence of B. thuringiensis subsp. Kurstaki at the Canberra Airport

and Air Freight Australia, samples were analysed using the Cry1 assay (see Section #).

All 331 samples were analysed in duplicate (n = 2), with the exception of the NTC,

which was performed as a single reaction.

Of the 331 samples analysed with the B. thuringiensis subsp. Kurstaki Cry1 assay, 136

returned a positive a result (data not shown). Amplification occurred late in the all of the

positive samples, with CT values ranging from 30.9 ± 0.2 to 39.8 ± 0.3. April 2012 had a

significantly higher proportion of positive samples than all other months (p < 0.05) with

35 out of 36 positive samples (Figure 4.1). May 2012 also had a significantly higher

proportion of positive than all other months, except for April 2013, with 31 out of 36

positive samples (p < 0.05).

48

Figure 4.1: Positive Cry1 samples by month. 1, 2, 3,4 5 = Pair-wise comparisons (p <

0.05); # = significantly different from all other months (p < 0.05); * = significantly

different from all other months, except April.

Samples taken from the LO sampling site returned the most positive results, with 26

positive results (Figure 4.2) The POV had the next greatest incidence at 24 positive

results, followed by PO (22), PI (21), LI (20) and LC (18). No sampling location had a

significantly higher proportion of positive samples than any other sampling location (p

> 0.05).

Figure 4.2: Positive Cry1 samples by sampling location

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Pe

rce

nta

ge P

osi

tive

Sam

ple

s

Month

1, 3

1, 2, 4

2

2, 5

#

*

4, 5

0

5

10

15

20

25

30

PO PI LO LI POV LC

Po

siti

ve S

amp

les

Sampling Location

49

4.4. Determining the presence and concentration of B. thuringiensis

subsp. Israelensis at the Canberra Airport

To determine the presence of B. thuringiensis subsp. Israelensis at the Canberra Airport

and Air Freight Australia, all 331 samples collected were analysed in duplicate using

the Cry4 assay (see Section #). Amplification was not observed in any of the 331

samples analysed (data not shown).

4.5. Determining the presence and concentration of B. subtilis at the

Canberra Airport

To determine the presence of B. subtilis at the Canberra Airport and Air Freight

Australia, all 331 samples collected were analysed in duplicate using the aprE assay

(see Section #). Amplification was not observed in any of the 331 samples analysed

(data not shown).

4.6. Determining the presence and concentration of B. cereus at the

Canberra Airport

To determine the presence of B. cereus at the Canberra Airport and Air Freight

Australia, all 331 samples collected were analysed in duplicate using the gyrB assay

(see Section 2.6.2). Amplification was not observed in any of the 331 samples analysed

(data not shown).

51

Chapter 5: Discussion

5.1. Selection of primers

5.1.1. Selection of primers for B. cereus

B. cereus is both a species within the B. cereus group and an opportunistic food-borne

pathogen. In that respect, it is often examined in the literature from two different

perspectives: a forensic perspective and a biomedical perspective. From a biomedical

point of view, the syndrome-causing enterotoxins are largely the target of investigation.

From a forensic perspective, it is the close-relatedness of B. cereus to B. anthracis and

B. thuringiensis which is discussed, with methods of differentiation being the primary

target for investigation.

As stated above, the different enterotoxins that B. cereus produces are of biomedical

importance. These enterotoxins, while large in number, have been identified in a

number of closely related bacterial species within the B. cereus group, in particular, B.

thuringiensis (Hansen and Hendriksen, 2000, Rosenquist et al., 2006, Ngamwongsatit et

al., 2008). This is also shown in the Primer-BLAST results (see Section 3.1), where

every primer set targeting an enterotoxic factor returned results confirming that the

targeted sequences were present in B. cereus, B. thuringiensis and B. anthracis. As

these enterotoxins appear frequently in B. thuringiensis, all of the reviewed biomedical

literature concerning B. cereus tended toward identifying the enterotoxic factors present

in a sample, rather than singularly identifying B. cereus.

On the forensic side, B. anthracis is the primary topic of investigation, with B. cereus

and B. thuringiensis discussed secondarily as potential false-positive-causing and hoax

agents. Given the high degree of genetic similarity between B. anthracis, B. cereus and

B. thuringiensis, and the overwhelming support in the literature to re-classify them as

the same species (Helgason et al., 2000, Rasko et al., 2005, Vilas-Bôas et al., 2007,

Stenfors Arnesen et al., 2008, Kolstø et al., 2009), most studies no longer attempt to

genetically differentiate all three from each other. Instead, most studies aimed to

differentiate B. anthracis from B. cereus and B thuringiensis. In that respect, forensic

studies also tended not to identify B. cereus individually in a sample.

52

In summary, recent studies seeking to identify B. cereus genetically and singularly in a

sample were scarce. The literature review alongside the Primer-BLAST results reinforce

the suggestion many authors posit in their studies: B. thuringiensis and B. cereus are not

able to be genetically differentiated from one another and should be re-classified as a

single species (Helgason et al., 2000, Rasko et al., 2005, Vilas-Bôas et al., 2007,

Stenfors Arnesen et al., 2008, Kolstø et al., 2009).

Given that no specific primer sets targeted singularly at B. cereus could be identified in

the literature, an alternative approach was taken to instead target B. anthracis, B.

thuringiensis and B. cereus with a single primer set. A positive sample using such a

primer set would mean that either B. anthracis, B. thuringiensis or B. cereus had been

detected. B. anthracis would be differentiated through the PL3 assay that had been

developed by Rossi (2011). If the PL3 assay did not amplify alongside the proposed

primer set, it would be assumed that either B. thuringiensis or B. cereus was present in

the sample. B. thuringiensis by definition produces crystalliferous inclusions and so

must therefore contain Cry genes (Vilas-Bôas et al., 2007, Rasko et al., 2005). If the

Cry1 and Cry4 assays did not amplify alongside the proposed primer set, then it would

be assumed that B. cereus had been obtained.

To that end, two primer sets were chosen to detect B. anthracis, B. thuringiensis and B.

cereus in a sample. As one of the early aims of this study was to design qPCR assays so

that they could be multiplexed downstream, primer sets with similar optimal annealing

temperatures to those designed by Rossi (2011) (54°C) were chosen. These included the

gyrB primer set, developed by Manzano et al. (2003), which had a reported optimal

annealing temperature of 57°C. The hblC primer set, developed by Hansen and

Hendrikson (2000) was also chosen for testing. This primer set had a reported optimal

annealing temperature of 55°C.

5.1.2. Selection of primers for B. subtilis

As the model organism for sporulated bacteria, B. subtilis is a well-studied bacterium.

However, studies focusing on the detection of B. subtilis in a sample are scarce. Most

studies are focused on elucidating new pathways within the bacterium, rather than

53

detecting it in a sample. This was not unexpected, as B. subtilis is neither a medically

important bacterium nor a bioterrorism agent.

Three studies were identified in the literature where the focus was on detecting B.

subtilis or members of the B. subtilis group in a sample (Sadeghi et al., 2012, Green et

al., 1999, Wattiau et al., 2001). Of these three, primer sets from two of the studies were

chosen for testing based on the Primer-BLAST result.

The primer sets designed in the study by Sadeghi et al. 2012 target the aprE gene.

Results of the Primer-BLAST show that the target sequence was present only in 30

strains of species, all belonging to the B. subtilis group (Table 3.1). The 16S rRNA

primers developed in the study by Wattiau et al. (2001) were shown to only cross-react

with three strains of B. cereus and two strains of B. thuringiensis. The aprE primer set

had a reported optimal annealing temperature of 57°C, whereas the 16S rRNA primer set

had an optimal annealing temperature of 65°C.

5.2. Development of a singleplex qPCR assay for the detection and

quantification of B. subtilis

The data presented in Section 3.2.1 shows that the aprE and 16S rRNA primers were

capable of amplifying B. subtilis DNA, with CT values of 15.1 ± 0.5 and 10.3 ± 0.02,

respectively. This was supported by the banding in the agarose gel, which showed that

amplicons of the correct size were being obtained. For the aprE assay, a single band of

roughly 750 bp was obtained, which corresponded to the 744 bp amplicon size

published in the study by Sadeghi et al. (2012). For the 16S rRNA assay, a band of

approximately 600 bp was obtained, which matched the expected size of 595 bp

(Wattiau et al., 2001).

Table 3.2 revealed that the aprE primers were cross-reactive, with B. cereus, B.

anthracis and B. thuringiensis subsp. Israelensis all amplifying in the assay alongside B.

subtilis. The agarose gel revealed different banding patterns between the target and the

cross-reacting species, with B. subtilis yielding a single band of 750 bp and B. anthracis

str. Sterne and B. cereus producing bands of approximately 800 bp. The Primer-BLAST

results for the aprE primers returned only species within the B. subtilis group; no

54

members of the B. cereus group were shown in these results, let alone any products of

800 bp (see Section 3.2). From this information it was concluded that the cross-

reactivity was most likely due to non-specific binding and could potentially be abated

through optimisation of the PCR conditions.

Table 3.2 also revealed that the 16S rRNA primers were cross-reactive, with B. cereus,

B. anthracis, B. thuringiensis subsp. Kurstaki and B. thuringiensis subsp. Israelensis all

amplifying. The agarose gel showed bands of identical size for B. subtilis, B. cereus and

B. anthracis at the expected 600 bp marker. Both B. thuringiensis subsp. Kurstaki and

B. thuringiensis subsp. Israelensis had faint bands of approximately 50 bp on the

agarose gel. Given the position of these bands, it was concluded that amplification

observed in the qPCR for these two species was the result of primer-dimer and not

because of cross-reactivity. Primer-BLAST results for the 16S rRNA primers (see

Section 3.1) suggest that the primers were only cross-reactive with two B. cereus strains

and just one B. thuringiensis strain. Given the late amplification of B. anthracis and B.

cereus, as well as their differences in banding intensity from B. subtilis and the results

of the Primer-BLAST, it was hypothesised that the cross-reactivity observed in this

experiment was caused from non-specific binding due to sub-optimal PCR conditions.

As with the cross-reactivity seen with the aprE assay, it was thought that the cross-

reactivity in the 16S rRNA could potentially be abated through the optimisation of PCR

conditions.

As one of the early aims of this project was to design assays so that they could be later

multiplexed alongside the B. thuringiensis subsp. Kurstaki and B. anthracis assays, a

number of PCR conditions were not able to be optimised or changed. The four assays

that were designed by Rossi (2011) all used the same FastStart SYBR Green Master

Mix, BSA concentration, and annealing temperature (54°C). Changing any of these

variables in the B. subtilis assay would have made multiplexing difficult if not

impossible, as only a single annealing temperature, FastStart SYBR Green Master Mix

concentration and BSA concentration can be used in a multiplex assay. The only

variable that could be changed without affecting a downstream multiplex assay was the

concentration of individual primer sets.

Table 3.3 demonstrates that decreasing the primer concentration to 0.1 µM and 0.05 µM

had no effect on the cross-reactivity observed in the 16S rRNA assay. Although the

55

sensitivity of the assay actually improved at the lower concentration 0.05 µM,

amplification was still observable in the cross-reacting species at only marginally higher

CT values to that seen in the initial cross-reactivity assay. The PCR cycling conditions

from the journal article that the 16S rRNA primers were selected from record an optimal

annealing temperature of 65°. It was hypothesised that the 11°C difference between the

optimal annealing temperature and the temperature used in this assay had most likely

added to the cross-reactivity exhibited in the assay. Another conclusion would be that

the target sequence was present in the templates of B. anthracis and B. cereus being

used in this study. As changing the annealing temperature would have interfered with

multiplexing, the experiment was not conducted. As the cross-reactivity exhibited by

these primer sets could not be abated through lowering the primer concentration, their

use was discontinued in favour of the aprE primer set, which did not display any cross-

reactivity at lower primer concentrations.

Table 3.4 demonstrates that lowering the primer concentration in the aprE assay to 0.05

µM mitigates the cross-reactivity observed in higher primer concentrations. At 0.1 µM,

the sensitivity of the assay actually improved, with a shift from 13 ± 0.6 for 0.2 µM to

11.3 ± 0.6 for 0.1 µM. As the sensitivity decreased again for 0.05 µM, it was

hypothesised that the optimal primer concentration for the assay was approximately at

0.1 µM. Despite being at or near the optimal primer concentration, cross-reactivity was

still observed late in the experiment at only marginally lower CT values. At 0.05 µM

only B. subtilis amplified. Lowering the primer concentration to 0.05 µM had a

significant effect on the sensitivity of the assay. The average CT of B. subtilis assay

shifted from 11.3 ± 0.6 for 0.1 µM to 20.6 ± 1.5 for 0.05 µM, which represents a

sizeable loss in sensitivity.

The LOD experiment was performed on the B. subtilis assay at 0.05 µM to determine its

sensitivity (Figure 3.1). The LOD for the assay, with an aprE primer concentration of

0.05 µM, was determined to be 3144 copies. This was a less than optimal amount, as it

was initially hypothesised that any bacteria present on surfaces at the Canberra Airport

would most likely exist in low copy numbers. Mitigating the cross-reactivity without

changing the annealing temperature, or any other PCR variable, presented a great

impediment to creating an assay which was both sensitive and specific. However, due to

56

time constrictions and the scarcity of studies geared toward detection B. subtilis in a

sample (see Section 5.1.2), another primer set was not sought.

5.3. Development of a singleplex assay for the detection and

quantitation of B. thuringiensis subsp. Israelensis

The data in Section 3.3.1 shows that the Cry4 primers were able to amplify B.

thuringiensis subsp. Israelensis DNA. A band of approximately 450 bp was observed in

the agarose gel, which matched the expected amplicon size of 439 bp (Ben-Dov et al.,

1997). This suggested that the correct sequence had been amplified.

Table 3.5 revealed that the Cry4 primers also amplified B. cereus, B. thuringiensis

subsp. Kurstaki, B. anthracis str. Sterne, B. subtilis and B. megatarium; the primers

were cross-reactive. In direct comparison to B. thuringiensis subsp. Israelensis, all

cross-reacting species amplified late in the in the PCR, with average CT values all

exceeding 30. Primer-BLAST results (see Section 3.1) revealed the primer set did not

amplify any other species other than the intended target of B. thuringiensis subsp.

Israelensis. Given the late amplification of the cross-reacting species and the Primer-

BLAST results, it was hypothesised that the cross-reactivity observed in the experiment

was due to non-specific binding and could be potentially abrogated through lowering

the primer concentration, in a similar way to that which was done for the B. subtilis

aprE assay.

Table 3.6 demonstrated that lowering the primer concentration from 0.2 µM to 0.05 µM

reduces the cross-reactivity to undetectable levels. While the specificity was increased

through the reduction in the primer concentration, the sensitivity was decreased.

However, as previously noted, the only variable able to be changed without affecting a

downstream multiplex was the concentration of individual primer sets.

The LOD experiment (see Section 3.3.3), revealed that the assay could detect a

minimum of 155 copies. The efficiency, which was calculated from the slope of the

standard curve at Figure 3.2, was calculated to be 59%.

57

5.4. Development of a singleplex assay for the detection and

quantitation of B. cereus

Amplification was observed in the gyrB assay, as described in Section 3.4.1. A band of

approximately 400 bp was observed in the agarose gel, which roughly matched the

published amplicon size of 374 bp (Manzano et al., 2003). This suggests that amplicons

of the correct size were being obtained, and is indicative that the assay worked.

As displayed in Table 3.7, cross-reactivity was observed with B. subtilis, B.

thuringiensis subsp. Kurstaki, B. thuringiensis subsp. Israelensis and B. anthracis str.

Sterne. Amplification of members of the B. cereus group was not unexpected, as the

Primer-BLAST results for the primer set (see Section 3.1) indicated that the target

sequence was present in 27 strains of B. thuringiensis and 3 strains of B. anthracis.

The amplification of B. subtilis in the assay was unexpected, as the Primer-BLAST

results suggested that the target sequence was not present in any members of the B.

subtilis group. No banding was seen in the agarose gel for B. subtilis. Given the late CT

of B. subtilis and the lack of observable banding in the agarose gel, it was hypothesised

that amplification occurred from non-specific binding or primer-dimer, which may have

been too faint to see on the agarose gel. As previously discussed, the only variable that

was able to be optimised was the primer concentration, as changing any other variable

would have impeded the development of a multiplex assay downstream.

Table 3.8 revealed that reducing the primer concentration of the gyrB primer set to 0.1

µM abrogated the cross-reactivity of B. subtilis observed at a primer concentration of

0.2 µM. The sensitivity of the assay decreased with the primer concentration, from

average CT of 20.2 ± 0.8 to 23.7 ± 0.8. To assess the assay’s sensitivity at a primer

concentration of 0.1 µM, a LOD experiment was undertaken. Only three of the eight 10-

fold serial dilutions amplified in the experiment and copy number was determined to be

approximately 14,481 copies. This was not ideal considering that any B. cereus group

species on surfaces at the Canberra Airport would most like be found in low copy

numbers.

58

Mitigating the cross-reactivity without changing the annealing temperature, or any other

PCR variable, presented a great impediment to creating an assay which was both

sensitive and specific. The sensitivity of the assay may have been improved if changing

the annealing temperature (54°C) to the reported optimal annealing temperature could

have been allowed.

5.5. Determining LOD for the B. anthracis and B. thuringiensis

subsp. Kurstaki assays

The LOD of the B. anthracis assay was determined to be nine copies, which was

considered a good result. Nine copies is below to infectious dose of inhalational B.

anthracis, which is estimated to be between 2500 and 5500 copies. This is also below

the infectious dose of cutaneous anthrax, which is estimated to be around 10 copies.

A standard curve for the B. anthracis pXO1 cya assay was not able to be generated.

Although different dilutions and fresh aliquots of B. anthracis stock DNA were used,

amplification appeared erratic and dilutions did not amplify such that a linear

correlation could be observed. A possible explanation is that the strain of pXO1+ B.

anthracis used in this study was accidently cured of the pXO1 plasmid sometime before

the project started. Due to time constrictions, a fresh B. anthracis str. Sterne stock was

not able to obtained. The LOD of the capB assay could not be determined due to

restrictions in keeping pXO1+ and pXO2

+ B. anthracis strains in the same laboratory.

The LOD B. thuringiensis subsp. Kurstaki Cry1 assay, developed and optimised by

Rossi (2011) was found to be four copies (see Figure 3.5).

5.6. Detection of B. anthracis at the Canberra Airport

Out of the 17 samples that returned a positive result for the B. anthracis PL3 genomic

marker, 12 of the samples were collected in February 2012. February 2012 had a

significantly higher of proportion of positive samples than any other month (p < 0.05).

April 2012 also had a significantly higher proportion of positive samples than any other

month, except February 2012 (p < 0.05).

59

Interestingly, LO had a significantly higher proportion of positive samples than any

other sampling location over all months (p < 0.05). This may indicate that B. anthracis

spores are primarily entering the airport locally on the luggage of people departing

Canberra. The PI sampling location did not return any positive results, which suggests

that no spores are being introduced to the airport on people arriving into Canberra.

However, it was noted that samples collected from the LO and LI conveyer belts were

always dirty and dark samples, as were the samples collected from PO. Apart from the

LC sampling site, samples taken from the PI sampling location were always the cleanest

samples, which is was unexpected given the high traffic of people through the area. This

may suggest that the PI sampling location was cleaned frequently, or more frequently

than any other sampling location, which may have affected the results.

Table 4.1 also shows that out of the 331 samples analysed, 17 tested positive in the

presence/absence screening for the B. anthracis PL3 genomic marker. Out of these 17

samples, just four produced a final amplification product when they were quantitated.

The concentrations of all four of these samples fell just outside of the standard curve

and were thus reported as being below the limit of detection (BLOD). The LOD for the

B. anthracis PL3 assay is nine copies (see Section 3.5).

At such small quantities, it was not unexpected that only a single replicate amplified for

13 of the 17 samples that tested positive in presence/absence screening. Likewise, it was

also not unexpected that 13 of the 17 samples were not able to quantitated, particularly

as each of these samples had been frozen and thawed three times between the

presence/absence screening and the quantitation testing.

Table 4.4 shows that out of the 331 samples analysed, just 29 tested positive for the cya

gene found on pXO1. Of these 29 samples, 24 were samples collected in February 2012.

February had a significantly higher proportion of positive samples than any other month

(p < 0.05). Interestingly, there was an overlap between the peak months of PL3 and cya

incidence, which was February 2012. One possible explanation for the peak in February

2012 was the flooding that occurred in Queensland and New South Wales during

December 2011 – January 2012. Durrheim (2009) concluded in his investigation of an

anthrax outbreak in livestock 350 km away from the anthrax belt in Hunter Valley, that

the emergence of anthrax in this area was most likely due to a 1-in-100 year rain event

60

that immediately preceded the outbreak. Another explanation is that there may be

increased traffic through the airport in February.

Of the 17 positive PL3 samples, six also tested positive for the cya gene (Table 4.5).

This suggests that a pXO1+ pXO2

- strain of B. anthracis was present in this six samples.

All six of these samples were unexpectedly in the month of February 2012, where the

majority of PL3- and cya-positive samples were collected.

Table 4.3 reveals that three samples tested positive for the capB pXO2 target. Each of

these samples was collected from a different month and a different sampling location.

The positive sample collected in February, CA9POV 21/02, also tested positive for the

cya gene, which indicates that a pXO1+ and pXO2

+ organism had been detected (Table

4.5). As the B. anthracis PL3 genomic marker didn’t test positive in this sample, it can

be concluded that B. anthracis was not present in the sample. B. thuringiensis subsp.

Kurstaki tested positive in this sample (data not shown). Given the high mobility of

plasmids between members of B. cereus group (Hoffmaster et al., 2004), it is possible

that the organism detected was a pXO1+ and pXO2

+ strain of B. thuringiensis.

5.7. Detection of B. thuringiensis subsp. Kurstaki at the Canberra

Airport

Of the 331 field samples that were analysed, 136 returned a positive result for B.

thuringiensis subsp. Kurstaki . April 2012 had a significantly higher proportion of

positive samples than all other months (p < 0.05) with 35 out of 36 positive samples

(see Figure 4.1). May 2012 also had a significantly higher proportion of positive than all

other months, except for April 2013, with 31 out of 36 positive samples (p < 0.05).

Overall, no sampling location had a significantly higher proportion of positive incidents

than any other sampling location (p > 0.05). Likewise, no sampling location had a

significantly higher proportion of positive incidents within each month. This could

suggest that the spores equalise across the airport as they enter. Modelling performed by

Sextro et al. (2002) demonstrated that a complex number of interactions between

airflow, ventilation and tracking can disburse spores widely across buildings from a

single point of origin.

61

With no site sampling site presenting a significant difference from another sampling

site, it is difficult to predict whether the spores are being introduced into the airport

locally or from regions outside of the ACT. Likewise, it is difficult to understand the

peaks in April and May without knowing where the spores are being introduced into the

airport. Personal communication with the owners of apple orchards in the ACT revealed

that no biopesticides, including B. thuringiensis-based products, are in use in orchards

in the ACT. Similarly, the owners of plant nurseries confirmed that no biopesticides

were being used to control pests in their stocks. In future, it may be useful to analyse

soil samples around the ACT to check whether B. thuringiensis naturally dwells in the

area.

5.8. Detection of other B. cereus, B. subtilis and B. thuringiensis

subsp. Israelensis at the Canberra Airport

B. cereus, B. subtilis and B. thuringiensis subsp. Israelensis were not detected at the

Canberra Airport using the qPCR assays designed in this study. This was not

unexpected, as the LOD for each of these assays were high, particularly given that the

LOD for the B. anthracis PL3 assay was nine copies and the positive field samples were

not able to be quantitated.

In the design of each of these three assays, sensitivity had to be sacrificed for specificity

to meet the aim of developing assays for downstream multiplexing. In future, SYBR

Green Master Mixes should be avoided, to allow more freedom to optimise PCR

components such as dNTPs, taq polymerase and magnesium concentrations.

5.9. Conclusions

This study broadly aimed at expanding on the work done by Rossi (2011) and establish

background levels for B. anthracis, B. thuringiensis subsp. Kurstaki, B. thuringiensis

subsp. Israelensis, B. cereus and B. subtilis at the Canberra Airport over a 12 month

period.

62

Three assays were developed to detect and quantitate B. cereus, B. subtilis and B.

thuringiensis subsp. Israelensis. Each of these assays was designed in such a way that

they could they could be streamlined into a multiplex assay. The LOD of each of these

assays was too high to be useful in detecting trace amounts of bacterial DNA on

surfaces at the Canberra Airport.

In this study, the creation of a baseline of B. anthracis and B. thuringiensis subsp.

Kurstaki over 12 month period was achieved. The key findings are that non-virulent B.

anthracis may be present at the Canberra Airport in minute quantities below

quantifiable levels. A high incidence in positive B. anthracis and pXO1 samples was

found in the month of February. B. thuringiensis subsp. Kurstaki showed a peak

incidence in samples collected in April and May 2012.

5.10. Future directions

There is room for further work to optimise the B. cereus group, B. thuringiensis subsp.

Israelensis and B. subtilis assays. Results indicated that the LOD for each of these

assays was too high to be useful in detecting trace amounts of bacterial DNA on

surfaces at the Canberra Airport.

In addition, there is room for further work in the interpretation of the positive B.

anthracis and B. thuringiensis subsp. Kurstaki results. Canberra Airport data, in

particular, the volume of flights arriving and departing Canberra each day or each

month, would help determine whether peaks of positive samples are tethered to traffic

through the airport. In future, it would also be prudent to sample the ventilation systems

of the airport to confirm whether ventilation is equalising spores across sampling areas.

Likewise, a selection of soil samples from across the ACT should be analysed to check

whether B. thuringiensis and B. anthracis are natural dwellers in the soils of the region.

63

Chapter 6: References

ALAEDDINI, R. 2012. Forensic implications of PCR inhibition—A review. Forensic Science International: Genetics, 6, 297-305.

AUSTRALIAN PESTICIDES AND VETERINARY MEDICINES AUTHORITY. 2012. Public Chemical Registration Information System (PUBCRIS) [Online]. Available: http://services.apvma.gov.au/PubcrisWebClient/search.do;jsessionid=2v1qQHfBGLv9cnHfxJNv7JCVd3Jrn8G1lqnBKWvFGhyzzZXwcYhb!-3295173.

BAAR, C., D’ABBADIE, M., VAISMAN, A., ARANA, M. E., HOFREITER, M., WOODGATE, R., KUNKEL, T. A. & HOLLIGER, P. 2011. Molecular breeding of polymerases for resistance to environmental inhibitors. Nucleic Acids Research, 39, e51.

BAVYKIN, S. G., MIKHAILOVICH, V. M., ZAKHARYEV, V. M., LYSOV, Y. P., KELLY, J. J., ALFEROV, O. S., GAVIN, I. M., KUKHTIN, A. V., JACKMAN, J., STAHL, D. A., CHANDLER, D. & MIRZABEKOV, A. D. 2008. Discrimination of Bacillus anthracis and Closely Related Microorganisms by Analysis of 16S and 23S rRNA with Oligonucleotide Microarray. Chemico-biological interactions, 171, 212-235.

BEN-DOV, E., ZARITSKY, A., DAHAN, E., BARAK, Z. E., SINAI, R., MANASHEROB, R., KHAMRAEV, A., TROITSKAYA, E., DUBITSKY, A., BEREZINA, N. & MARGALITH, Y. 1997. Extended Screening by PCR for Seven cry-Group Genes from Field-Collected Strains of Bacillus thuringiensis. Applied and Environmental Microbiology, 63, 4883–4890.

BLACKWOOD, K. S., TURENNE, C. Y., HARMSEN, D. & KABANI, A. M. 2004. Reassessment of Sequence-Based Targets for Identification of Bacillus Species. Journal of Clinical Microbiology, 42, 1626-1630.

BLAUSTEIN, R. O., KOEHLER, T. M., COLLIER, R. J. & FINKELSTEIN, A. 1989. Anthrax toxin: Channel-forming activity of protective antigen in planar phospholipid bilayers. Proceedings of the National Academy of Science of the United States of America, 86, 2209-2213.

BODE, E., HURTLE, W. & NORWOOD, D. 2004. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis. Journal of Clinical Microbiology, 42, 5825-5831.

BOTTONE, E. J. 2010. Bacillus cereus, a Volatile Human Pathogen. Clinical Microbiology Reviews, 23, 382-398.

CARLSON, C. R., CAUGANT, D. A. & KOLSTØ, A.-B. 1994. Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains. Applied and Environmental Microbiology, 60, 1719-1725.

CHRISTOPHER, G. W., CIESLAK, T. J., PAVLIN, J. A. & EITZEN, E. M. 1997. Biological Warfare: A Historical Perspective. Journal of American Medical Association, 278, 417-417.

CIESLAK, T. & EITZEN, E. M. 1999. Clinical and Epidemiologic Principles of Anthrax. Emerging Infectious Diseases, 5, 552-555.

CRIGHTON, T., HOILE, R. & COLEMANN, N. 2012. Comparison of quantitative PCR and culture-based methods for evaluating dispersal of Bacillus thuringiensis endospores at a bioterrorism hoax crime scene. Forensic Science International.

DE BOER, A. S. & DIDERICHSEN, B. 1991. On the safety of Bacillus subtilis and B. amyloliquefaciens: a review. Applied Microbiology and Biotechnology, 36, 1-4.

DEPARTMENT OF HEALTH. 2013. Security Sensitive Biological Agents [Online]. Available: http://www.health.gov.au/SSBA.

DURRHEIM, D. N., FREEMAN, P., ROTH, I. & HORNITZKY, M. 2009. Epidemiologic Questions from Anthrax Outbreak, Hunter Valley, Australia. Emerging Infectious Diseases, 15, 840-842.

ELLERBROK, H., NATTERMANN, H., ÖZEL, M., BEUTIN, L., APPEL, B. & PAULI, G. 2002. Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiology Letters, 214, 51–59.

64

GOTO, K., OMURA, T., HARA, Y. & SADAIE, Y. 2000. Application of the partial 16S rDNA sequence as an index for rapid identification of species in the genus Bacillus. The Journal of General and Applied Microbiology, 46, 1-8.

GREEN, D. H., WAKELEY, P. R., PAGE, A., BARNES, A., BACCIGALUPI, L., RICCA, E. & CUTTING, S. M. 1999. Characterization of Two Bacillus Probiotics. Applied and Environmental Microbiology, 65, 4288-4291.

HANSEN, B. M. & HENDRIKSEN, N. B. 2000. Detection of Enterotoxic Bacillus cereus and Bacillus thuringiensis Strains by PCR Analysis. Applied and Environmental Microbiology, 67, 185-189.

HELGASON, E., ØKSTAD, O. A., CAUGANT, D. A., JOHANSEN, H. A., FOUET, A., MOCK, M., HEGNA, I. & KOLSTØ, A.-B. 2000. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence. Applied and Environmental Microbiology, 66, 2627-2630.

HOFFMASTER, A. R., RAVEL, J., RASKO, D. A., CHAPMAN, G. D., CHUTE, M. D., MARSTON, C. K., DE, B. K., SACCHI, C. T., FITZGERALD, C., MAYER, L. W., MAIDEN, M. C. J., PRIEST, F. G., BARKER, M., JIANG, L., CER, R. Z., RILSTONE, J., PETERSON, S. N., WEYANT, R. S., GALLOWAY, D. R., READ, T. D., POPOVIC, T. & FRASER, C. M. 2004. Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proceedings of the National Academy of Sciences of the United States of America, 101, 8449-8454.

HURTLE, W., BODE, E., KULESH, D. A., KAPLAN, R. S., GARRISON, J., BRIDGE, D., HOUSE, M., FRYE, M. S., LOVELESS, B. & NORWOOD, D. 2004. Detection of the Bacillus anthracis gyrA Gene by Using a Minor Groove Binder Probe. Journal of Clinical Microbiology, 42, 179-185.

INGLESBY, T. V. 2002. Anthrax as a Biological Weapon, 2002: Updated Recommendations for Management. JAMA: The Journal of the American Medical Association, 287, 2236-2252.

JANSE, I., BOK, J. M., HAMIDJAJA, R. A., HODEMAEKERS, H. M. & VAN ROTTERDAM, B. J. 2012. Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays. PLoS ONE, 7, e31958.

JERNIGAN, D. B., RAGHUNATHAN, P. L., BELL, B. P., BRECHNER, R., BRESNITZ, E. A., BUTLER, J. C., CETRON, M., COHEN, M., DOYLE, T., FISCHER, M., GREENE, C., GRIFFITH, K. S., GUARNER, J., HADLER, J. L., HAYSLETT, J. A., MEYER, R., PETERSEN, L. R., PHILLIPS, M., PINNER, R., POPOVIC, T., QUINN, C. P., REEFHUIS, J., REISSMAN, D., ROSENSTEIN, N., SCHUCHAT, A., SHIEH, W.-J., SIEGAL, L., SWERDLOW, D. L., TENOVER, F. C., TRAEGER, M., WARD, J. W., WEISFUSE, I., WIERSMA, S., YESKEY, K., ZAKI, S., ASHFORD, D. A., PERKINS, B. A., OSTROFF, S., HUGHES, J., FLEMING, D., KOPLAN, J. P. & GERBERDING, J. L. 2002. Investigation of Bioterrorism-Related Anthrax, United States, 2001: Epidemiologic Findings. Emerging Infectious Diseases, 8, 1019-1028.

KERMEKCHIEV, M. B., KIRILOVA, L. I., VAIL, E. E. & BARNES, W. M. 2009. Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Research, 37, e40.

KIM, K., SEO, J., WHEELER, K., PARK, C., KIM, D., PARK, S., KIM, W., CHUNG, S.-I. & LEIGHTON, T. 2005. Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis. FEMS Immunology & Medical Microbiology, 43, 301–310.

KOLSTØ, A.-B., TOURASSE, N. J. & ØKSTAD, O. A. 2009. What Sets Bacillus anthracis Apart from Other Bacillus Species? Annual Review of Microbiology, 63, 451-476.

KUNST, F., OGASAWARA, N., MOSZER, I., ALBERTINI, A. M., ALLONI, G., AZEVEDO, V., BERTERO, M. G., BESSIÈRES, P., BOLOTIN, A., BORCHERT, S., BORRISS, R., BOURSIER, L., BRANS, A., BRAUN, M., BRIGNELL, S. C., BRON, S., BROUILLET, S., BRUSCHI, C. V., CALDWELL, B., CAPUANO, V., CARTER, N. M., CHOI, S.-K., CODANI, J.-J., CONNERTON, I. F., CUMMINGS, N. J., DANIEL, R. A., DENIZOT, F., DEVINE, K. M., DÜSTERHÖFT, A.,

65

EHRLICH, S. D., EMMERSON, P. T., ENTIAN, K. D., ERRINGTON, J., FABRET, C., FERRARI, E., FOULGER, D., FRITZ, C., FUJITA, M., FUJITA, Y., FUMA, S., GALIZZI, A., GALLERON, N., GHIM, S.-Y., GLASER, P., GOFFEAU, A., GOLIGHTLY, E. J., GRANDI, G., GUISEPPI, G., GUY, B. J., HAGA, K., HAIECH, J., HARWOOD, C. R., HÉNAUT, A., HILBERT, H., HOLSAPPEL, S., HOSONO, S., HULLO, M.-F., ITAYA, M., JONES, L., JORIS, B., KARAMATA, D., KASAHARA, Y., KLAERR-BLANCHARD, M., KLEIN, C., KOBAYASHI, Y., KOETTER, P., KONINGSTEIN, G., KROGH, S., KUMANO, M., KURITA, K., LAPIDUS, A., LARDINOIS, S., LAUBER, J., LAZAREVIC, V., LEE, S.-M., LEVINE, A., LIU, H., MASUDA, S., MAUËL, C., MÉDIGUE, C., MEDINA, N., MELLADO, R. P., MIZUNO, M., MOESTL, D., NAKAI, S., NOBACK, M., NOONE, D., O'REILLY, M., OGAWA, K., OGIWARA, A., OUDEGA, B., PARK, S.-H., PARRO, V., POHL, T. M., PORTETELLE, D., PORWOLLIK, S., PRESCOTT, A. M., PRESECAN, E., PUJIC, P., PURNELLE, B., et al. 1997. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature, 390, 249-256.

LEASK, A., DELPECH, V. & MCANULTY, J. 2003. Anthrax and other suspect powders: initial responses to an outbreak of hoaxes and scares. NSW Public Health Bulletin, 14, 218-221.

LIM, D. V., SIMPSON, J. M., KEARNS, E. A. & KRAMER, M. F. 2005. Current and Developing Technologies for Monitoring Agents of Bioterrorism and Biowarfare. Clinical Microbiology Reviews, 18, 583-607.

MACINTYRE, A. G. 2000. Weapons of Mass Destruction Events With Contaminated Casualties: Effective Planning for Health Care Facilities. JAMA: The Journal of the American Medical Association, 283, 242-249.

MANZANO, M., GIUSTO, C., IACUMIN, L., CANTONI, C. & COMI, G. 2003. A molecular method to detect Bacillus cereus from a coffee concentrate sample used in industrial preparations. Journal of Applied Microbiology, 95, 1361–1366.

MILLETT, P. D. 2006. The Biological and Toxin Weapons Convention in context: from monolith to keystone. United Nations: Department for Disarmament Affairs.

MOCK, M. & FOUET, A. 2001. Anthrax. Annual Review of Microbiology, 24. NGAMWONGSATIT, P., BUASRI, W., PIANARIYANON, P., PULSRIKARN, C., OHBA, M.,

ASSAVANIG, A. & PANBANGRED, W. 2008. Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel primers. International Journal of Food Microbiology, 121, 352-356.

NICHOLSON, W. L., MUNAKATA, N., HORNECK, G., MELOSH, H. J. & SETLOW, P. 2000. Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments. Microbiology and Molecular Biology Reviews, 64, 548-572.

OGGIONI, M. R., POZZI, G., VALENSIN, P. E., GALIENI, P. & BIGAZZI, C. 1998. Recurrent Septicemia in an Immunocompromised Patient Due to Probiotic Strains of Bacillus subtilis. Journal of Clinical Microbiology, 36, 325-326.

OPEL, K. L., CHUNG, D. & MCCORD, B. R. 2009. A Study of PCR Inhibition Mechanisms Using Real Time PCR. Journal of Forensic Sciences.

QI, Y., PATRA, G., LIANG, X., WILLIAMS, L. E., ROSE, S., REDKAR, R. J. & DELVECCHIO, V. G. 2001. Utilization of the rpoB Gene as a Specific Chromosomal Marker for Real-Time PCR Detection of Bacillus anthracis. Applied and Environmental Microbiology, 67, 3720-3727.

RAO, S. S., MOHAN, K. V. K. & ATREYA, C. D. 2010. Detection technologies for Bacillus anthracis: Prospects and challenges. Journal of Microbiological Methods, 82, 1-10.

RASKO, D. A., ALTHERR, M. R., HAN, C. S. & RAVEL, J. 2005. Genomics of the Bacillus cereus group of organisms. FEMS Microbiology Reviews, 29, 303–329.

ROSENQUIST, H., SMIDT, L., ANDERSON, S. R., JENSEN, G. B. & WILCKS, A. 2006. Occurence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food. FEMS Microbiology Letters, 250, 129-136.

ROSSI, R. 2011. An Environmental Survey of Forensically Significant Microorganisms Located at the Canberra Airport. Bachelor of Applied Science (Honours), University of Canberra.

66

SADEGHI, A., MORTAZAVI, S. A., BAHRAMI, A. R. & SADEGHI, B. 2012. Design of Multiplex PCR for simultaneous detection of rope-forming Bacillus strains in Iranian bread dough. Journal of the Science of Food and Agriculture, 92, 2652 - 2656.

SCOBIE, H. M., WIGELSWORTH, D. J., MARLETT, J. M., THOMAS, D., RAINEY, G. J. A., LACY, D. B., MANCHESTER, M., COLLIER, R. J. & YOUNG, J. A. T. 2006. Anthrax Toxin Receptor 2–Dependent Lethal Toxin Killing In Vivo. PLoS Pathog, 2, e111.

SEXTRO, R. G., LORENZETTI, D. M., SOHN, M. D. & THATCHER, T. L. Modeling the Spread of Anthrax in Buildings. Proceedings of the Indoor Air 2002 Conference, 2002 Monterey, CA. Berkley, CA: Lawrence Berkley National Lab.

SKOTTMAN, T., PIIPARINEN, H., HYYTIÄINEN, H., MYLLYS, V., SKURNIK, M. & NIKKARI, S. 2007. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. European Journal of Clinical Microbiology & Infectious Diseases, 26, 207-211.

SPENCER, R. C. 2003. Bacillus anthracis. Journal of Clinical Pathology, 56, 182-187. STENFORS ARNESEN, L. P., FAGERLUND, A. & GRANUM, P. E. 2008. From soil to gut: Bacillus

cereus and its food poisoning toxins. FEMS Microbiology Reviews, 32, 579–606. TOMIOKA, K., PEREDELCHUK, M., ZHU, X., ARENA, R., VOLOKHOV, D., SELVAPANDIYAN, A.,

STABLER, K., MELLQUIST-RIEMENSCHNEIDER, J., CHIZHIKOV, V., KAPLAN, G., NAKHASI, H. & DUNCAN, R. 2005. A Multiplex Polymerase Chain Reaction Microarray Assay to Detect Bioterror Pathogens in Blood. The Journal of molecular diagnostics : JMD, 7, 486-494.

VALASEK, M. A. & REPA, J. J. 2005. The power of real-time PCR. Advances in Physiology Education, 29, 151-159.

VILAS-BÔAS, G. T., PERUCA, A. P. S. & ARANTES, O. M. N. 2007. Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Canadian Journal of Microbiology, 53, 673-687.

VOLKMANN, H., SCHWARTZ, T., KIRCHEN, S., STOFER, C. & OBST, U. 2007. Evaluation of inhibition and cross-reaction effects on real-time PCR applied to the total DNA of wastewater samples for the quantification of bacterial antibiotic resistance genes and taxon-specific targets. Molecular and Cellular Probes, 21, 125-133.

WALLIN, A., LUKSIENE, Z., ZAGMINAS, K. & SURKIENE, G. 2007. Public health and bioterrorism: renewed threat of anthrax and smallpox. Medicina (Kaunas), 43, 278-284.

WANG, L.-T., LEE, F.-L., TAI, C.-J. & KASAI, H. 2007. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA–DNA hybridization in the Bacillus subtilis group. International Journal of Systematic and Evolutionary Microbiology, 57, 1846-1850.

WANG, S.-H., WEN, J.-K., ZHOU, Y.-F., ZHANG, Z.-P., YANG, R.-F., ZHANG, J.-B., CHEN, J. & ZHANG, X.-E. 2004. Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip. Biosensors and Bioelectronics, 20, 807-813.

WATTIAU, P., RENARD, M.-E., LEDENT, P., DEBOIS, V., BLACKMAN, G. & AGATHOS, S. N. 2001. A PCR test to identify Bacillus subtilis and closely related species and its application to the monitoring of wastewater biotreatment. Applied Microbiology and Biotechnology, 56, 816-819.

WESSELY, S., HYAMS, K. C. & BARTHOLOMEW, R. 2001. Psychological implications of chemical and biological weapons. BMJ : British Medical Journal, 323, 878-879.

ZASADA, A. A., GIERCZYŃSKI, R., RADDADI, N., DAFFONCHIO, D. & JAGIELSKI, M. 2006. Some Bacillus thuringiensis Strains Share rpoB Nucleotide Polymorphisms Also Present in Bacillus anthracis. Journal of Clinical Microbiology, 44, 1606-1607.