Developing Methodologies for the Investigation of Free-living Amoeba as a Tool for Pathogen ...

130 Pages·2014·3.18 MB·English

Developing Methodologies for the Investigation of Free-living Amoeba as a Tool for
Pathogen Surveillance on Dairy Farms and Aquaculture

by
John McLean

A Thesis
presented to
The University of Guelph

In partial fulfillment of requirements
for the degree of
Master of Science
in
Molecular and Cellular Biology

Guelph, Ontario, Canada
© John McLean, April, 2014
ABSTRACT

DEVELOPING METHODOLOGIES FOR THE INVESTIGATION OF FREE-LIVING
AMOEBA AS A TOOL FOR PATHOGEN SURVEILLANCE ON DAIRY FARMS AND
FISHERIES

John M. McLean Advisor:
University of Guelph, 2014 Dr. Lucy Mutharia

Free-living amoeba are phagocytic protozoans that act as environmental reservoirs,
a protective niche, and a vehicle for transmission for amoeba-resistant bacterial pathogens.
Many amoeba-resistant bacteria have been identified using only laboratory-adapted
Acanthamoeba. We isolated resident amoeba from target environments of dairy farms and
aquaculture settings to evaluate their use as a pathogen detection tool. Amoeba were only
isolated from 3 of 23 (13%) environmental samples using established methods. A two-step
sample decontamination protocol was developed and led to the isolation of 14 additional
amoeba. An amoeba co-culture method was developed to assess the survival of 12
mycobacterial species within environmental and laboratory-adapted amoeba. Major strain
differences were observed at the amoeba level which had drastic effects on the survival of
different bacterial species within individual amoeba. Targeted isolation of resident bacteria
from soils and feces using amoebal enrichment protocols were unsuccessful. However, the
methodologies developed in this study provide a valid technical starting point for future
studies.
Acknowledgments
First and foremost I would like to thank my advisor, Dr. Lucy Mutharia for her guidance
and continued support throughout the course of this work. I am ever grateful for her willingness
to provide direction and assistance at both the undergraduate and graduate level. I would also
like to thank my committee members, Dr. R.M.W. Stevenson and Dr. Cezar Khursigara for their
assistance and expertise during the course of my project, it would not have been possible without
you. Many thanks must also be given to the members of the Mutharia and Stevenson labs,
especially Tony Facciuolo and Melinda Raymond for being an ever present source of advice and
direction. I must also thank Steve Lord and members of the lab of Dr. Dave Kelton for obtaining
many of the environmental samples that made this work possible. Recognition must also go to
my office mates Daniel Jeffery, Kate Murphy, Alison Berezuk and Michelle Daigneault as a
constant source of both entertainment and advice. Finally I'd like to thank my family for their
unwavering support throughout this process.

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Table of Contents
List of Figures………………………………………………………………………………......vii
List of Tables………………………………………………………………………………….....ix
Chapter 1.0: Introduction….........……….....……………………………………………….......1
1.1 Free-living amoeba……………………………………………………………………........2
1.2 Cyst Resistance………………………………………………………………………........10
1.3 Amoeba-resistant Microbes………………………………………………………….........11
1.3.1 Pathogen Persistence…………………………………………………………….....17
1.3.2 Amoeba as a Bacterial Training Ground for Intracellular Pathogens.......................19
1.3.3 A Protective Environment.........................................................................................21
1.3.4 Pathogen Transmission.............................................................................................23
1.4 Research Rationale and Objectives......................................................................................25
Chapter 2.0: Materials and Methods.........................................................................................28
2.1. Bacterial Strains and Growth Conditions...........................................................................28
2.1.1 Preparation of NNA-E. coli Media...........................................................................30
2.1.2 Culture of Amoeba....................................................................................................30
2.1.3 Long-term Storage of Amoeba.................................................................................31
2.2 Development of Methodologies for Amoeba Isolation.......................................................32
2.2.1 Determining Cyst Resistance....................................................................................32
2.2.2 Primary Amoeba Isolation from Dairy Farms and Aquaculture...............................33
2.2.3 Methods Developed for Amoeba Isolation from Dairy Farms and Aquaculture.....34
2.2.4 Conversion of Xenically Culturable Amoeba to Axenically Culturable Forms.......36
2.3 Amoebal Enrichment and Co-culture..................................................................................37
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2.3.1 Preparation of Amoebal Microplates........................................................................37
2.3.2 Screening of Amoeba-resistant Bacteria by the Co-culture Protocol.......................38
2.3.3 Isolation of Environmental Amoeba-resistant Bacteria............................................39
2.3.4 MAP isolation from Cow Feces................................................................................40
2.3.5 MAP Isolation from Cow Feces by Amoebal Enrichment......................................41
2.4 DNA methods......................................................................................................................42
2.4.1 PCR conditions.........................................................................................................44
2.5 Microscopy..........................................................................................................................45
2.5.1 Staining of Amoeba Trophozoites and Amoeba-resistant Bacteria..........................45
2.5.2 Transmission Electron Microscopy..........................................................................46
Chapter 3.0: Results.....................................................................................................................48
3.1 Developing Methodologies for the Isolation of Free-living Amoeba from
Environmental Samples.......................................................................................................48
3.2 Molecular Identification of Free-living Amoeba.................................................................54
3.2.1 Diversity of Free-living Amoeba..............................................................................63
3.3 Axenic Culture of Free-living Amoeba...............................................................................67
3.4 Developing Methodologies for Identification of Amoeba-resistant Bacteria......................70
3.4.1 Identifying Amoeba-resistant Bacteria Resident in Free-living Amoeba.................75
3.4.2 Enriching for MAP from Feces................................................................................78
3.4.3 Persistence of MAP within Amoebal Cysts..............................................................82
Chapter 4.0: Discussion...............................................................................................................86
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4.1 Conclusions..........................................................................................................................96
4.2 Future Direction....................................................................................................................97
5.0 REFERENCES.......................................................................................................................98
6.0 APPENDICES......................................................................................................................108
6.1 Appendix 1: Media and Buffers..........................................................................................109
6.2 Appendix 2: 18S rRNA Sequences....................................................................................114

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List of Figures
Figure 1.1 Locomotive form of an amoebal trophozoite................................................................3
Figure 1.2 Overview of phagocytosis of bacteria by free-living amoeba.......................................6
Figure 1.3 Cross-section of an Aca. polyphaga cyst showing cyst-wall structure..........................9
Figure 1.4 Mechanisms used by amoeba-resistant bacteria to resist phagocytic killing..............14
Figure 3.1 NNA-E. coli plate during primary amoeba isolation showing
fungal overgrowth..........................................................................................................................50
Figure 3.2 Amoebal walk-out.......................................................................................................51
Figure 3.3 Schematic overview of the developed methods for primary amoeba
isolation..........................................................................................................................................56
Figure 3.4 Morphology of different environmental amoeba isolated...........................................58
Figure 3.5 Representation of the amoebal 18S rRNA gene showing variable regions.................59
Figure 3.6 Agarose gel of amplified amoebal DNA using the JDP1/JDP2 primer set.................60
Figure 3.7 Agarose gel of amplified amoebal DNA using the P-FLA primer set........................61
Figure 3.8 Agarose gel of products from a nested (CRN5/1137R) PCR......................................62
Figure 3.9 Agarose gel of Ami-primer amplified DNA from the amoebal isolates.....................64
Figure 3.10 A Phylogenetic tree showing evolutionary relationship of amoebal
isolates............................................................................................................................................68
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Figure 3.11 A co-culture of Aca. polyphaga ATCC 50372 with mycobacteria...........................71
Figure 3.12 Co-culture of 2 Aca. polyphaga isolates with 3 mycobacterial species before and
after encystment.............................................................................................................................73
Figure 3.13 Medium (24)-well plates used for cyst recovery during amoebal enrichment..........80
Figure 3.14 Results of a comparative study for amoeba-based isolation of MAP from feces......81
Figure 3.15 Repeat attempt at amoebal enrichment for MAP from feces....................................83
Figure 3.16 MAP persistence through multiple rounds of Aca. polyphaga encystment..............84
Figure 3.17 Transmission electron micrograph of amoeba cyst after co-culture with MAP........85

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List of Tables
Table 1.1 Predicted biosynthetic capacities of different amoeba....................................................5
Table 1.2 List of amoeba-resistant bacteria identified to date......................................................13
Table 2.1 Bacterial strains and growth conditions........................................................................29
Table 2.2 Oligonucleotide primers used in this study...................................................................43
Table 3.1 Sample locations for amoeba isolation.........................................................................49
Table 3.2 Aca. polyphaga cyst resistance to HClO and moist heat..............................................52
Table 3.3 Cyst resistance of environmental amoeba to 0.1N HCl and 2% (v/v)
glutaraldehyde................................................................................................................................55
Table 3.4 Identities of isolated free-living amoeba.......................................................................65
Table 3.5 Axenic cultures of environmental amoeba ...................................................................69
Table 3.6 Results of co-cultures with environmental amoeba and mycobacterial species...........76
Table 3.7 Results of co-cultures between Aca. polyphaga and bacterial pathogens.....................77
Table 3.8 Potential amoeba-resistant bacteria isolated using amoeba enrichment.......................79
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Chapter 1.0: Introduction
Amoeba are a diverse group of unicellular protozoans that differ from other protozoans
by their characteristic amoeboid movement, facilitated by cytoplasmic extensions known as
pseudopodia. Amoeba co-evolved with environmental microorganisms resulting in many
bacterial species becoming resistant to amoebal killing, allowing them to survive and even
multiply within amoebal hosts (Adiba et al., 2010). Many virulence factors required for intra-
amoebal survival of pathogens mirror those required for macrophage resistance in mammalian
hosts (Segal and Shuman, 1999, Swanson and Hammer, 2000). In some cases, pathogenic
bacteria utilize free-living amoeba as a “Trojan horse”, allowing them to enter the host protected
and undetected (Barker and Brown, 1994). In the case of the best studied amoeba-resistant
bacterium, Legionella pneumophila, amoeba act as a vector for transmission and a training
ground for the selection and up-regulation of virulence factors (Molmeret et al., 2005, Swanson
and Hammer, 2000). The study of amoeba-resistant bacteria has garnered attention in the
scientific community and led to the identification of an increasing number of bacteria and other
microorganisms as amoeba resistant (reviewed by Greub and Raoult, 2004).
Our current understanding of the diversity of free-living amoeba in the environment is
limited. This is in part due to a lack of established methodologies for isolation and identification
of free-living amoeba (Szenasi et al., 1998). The ability of most amoeba-resistant bacteria to
survive within environmental amoeba has not been tested experimentally. Additionally, there is
no consensus on the criteria by which a microorganism is deemed to be amoeba-resistant. This
review highlights characteristics of free-living amoeba with specific attention to their
interactions with bacteria, highlighting the role of free-living amoeba in the persistence,
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