View (Win32) V1.5.2 (Copyright Roderic D.M

UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

Identification and Characterization of Bacterial Communities in Warm

Groundwater Aquifers

Ian Laseke

B.S. University of Cincinnati, Cincinnati, OH 45268

THESIS

Submitted in partial fulfillment of the requirements for the

M.S. Degree in Environmental Engineering

In the College of Engineering in University of Cincinnati

Cincinnati, Ohio 2006

ii ABSTRACT

In Peoria, Arizona during the autumn season of 2002, two young children died from

Primary amoebic meningoencephalitis (PAM) infections. Naegleria fowleri, was detected in the residual water from household pipes and sinks and was linked to the cause of PAM infections that resulted in the death of both children. In this study, we tested the same groundwater in Peoria, Arizona that was suspected to be contaminated by N. fowleri, for the presence of N. fowleri. Results of the study indicated the presence of N.

fowleri in 12 samples. Five samples were analyzed for bacterial communities, of which

three were PCR positive for N. fowleri. Analysis of bacterial communities present in the

Peoria groundwater samples indicated the presence of β-proteobacteria such as

Caldimonas manganoxidans, Leptothrix species, Aquabacterium species and bacteria of

other genus such as Chloroflexus, Cytophaga, Flexibacter, Halochromatium,

Meiothermus, Nitrospira, Nocardia, Rasbo bacterium, and Thermus.

iii iv

ACKNOWLEDGEMENT

I feel grateful to the following people, without whom this thesis would not have been possible:

Dr. Daniel B. Oerther, advisor;

Dr. Sandhya Parshionikar, mentor and friend;

My lab-mates, especially Kai Zhang, Mau-Yi Wu, Ting Lu, Rob Smith, and Pascal E.

Saikaly, for their assistance in learning molecular biology.

TABLE OF CONTENTS

Abstract…………………………………………………………………………………...iii

Acknowledgement………………………………………………………………………...v

Table of Contents…………………………………………………………………………vi

Introduction……………………………………………………………………………….1

Methods and Materials…………………………………………………………………....5

Results……………………………………………………………………………………13

Validate Assay for N. fowleri…………………………………………………….13

Validate N. fowleri Assay in Environment……...…………………………….....17

Discussion………………………………………………………………………………..19

Validate Assay for E. coli O157:H7…………………………………………..…20

Validate E. coli O157:H7 in Environment…………………………………….....22

Identification of Predominant Bacterial Populations in Environment…………...22

Summary of Species Related to Sequences…………………………………...…28

Phylogenetic Analysis………………………………………………………...….40

Appendix…………………………………………………………………………...…….52

Tables………………………………………………………………………….…53

References……………………………………………………………………………..…73

INTRODUCTION

General water microbiology

More than 70 percent of the earth’s surface consists of water, both fresh and salt water.

Oceans, seas, and certain inland lakes make up the marine habitats that contain salt

concentrations of 3.5 percent and higher. Ponds, streams, lakes, rivers, and underground aquifers make up the freshwater habitats, which is only 2 percent of the total water on the planet (40).

Several chemical and physical factors influence the microbial populations present in

water environments. In fresh and salt waters there are various microorganisms found

naturally, while other microorganisms can infiltrate into these waters from soil, industrial

or domestic processes, and other contaminating events. Groundwater generally has good

overall quality with little color, turbidity, and low numbers of microorganisms. However,

groundwater may have high concentrations of dissolved minerals. On the other hand,

surface waters contain large numbers of microorganisms and originate from streams,

ponds, rivers, lakes, and shallow wells. Groundwater and surface water can be infiltrated and contaminated by potentially pathogenic bacteria, viruses, protozoa, and amoebae

originating from raw wastewater, which is one of the greatest threats to the quality of

water supplies (40).

A variety of bacteria, viruses, and protozoa cause waterborne infectious diseases and are

a large source of illness and death worldwide, especially in developing countries lacking

water treatment technology (40). There are very rigid water quality standards in

developed countries, which limit the spread of waterborne diseases. In the United States,

the Environmental Protection Agency (EPA) enforces the Clean Water Act and the Safe

Drinking Water Act (SDWA) to ensure high quality of drinking water.

Control of drinking water quality was given to the federal EPA in 1974, when congress

passed the SDWA. Over the years, namely 1986 and 1996, congress has passed

amendments to the SDWA. A federal regulation used to improve the microbiological

quality of drinking water is the Total Coliform Rule. This regulation sets a maximum

contaminant level for fecal coliform bacteria, E. coli, and total coliform bacteria in

treated drinking water. To protect the quality of surface water the Surface Water

Treatment Rule and the Enhanced Surface Water Treatment Rule focus on Legionella,

Giardia, enteric viruses, heterotrophic bacteria plate count, and Cryptosporidium spores.

Disinfection and other specific treatment technology are proposed by the EPA by this rule in order to achieve the reduction of these microorganisms (22).

For groundwater protection there are currently three programs run by the EPA: 1) the

Wellhead Protection Program (WPP) was designed to reduce the threat to the quality of

groundwater used for public drinking water by identifying and managing recharge areas

to specific wells or well fields, 2) the Source Water Assessment and Protection Program,

which was created by the 1996 Amendments to the SDWA of 1974, builds on the

Wellhead Protection Program approach to protecting groundwater, 3) the Comprehensive

State Groundwater Protection Programs establish a partnership between the EPA and the states, Native American tribes, and local governments to achieve a more efficient, coherent, and comprehensive approach to protecting the nation's groundwater resources.

Specific water microbiology

Microorganisms come from three domains of life: Bacteria, Archaea, and Eukarya.

While no human pathogens have been identified from the domain of Archaea, there are a number of Bacteria and Eukaryotes capable of harming humans. Fecal contamination can occasionally introduce bacterial and protozoan (belonging to Eukarya) pathogens in drinking water in the United States.

Infection of the free living amoeba Naegleria fowleri caused the death of two five-year old children in October 2002 from PAM (3). N. fowleri was discovered in the residual water from household pipes and sinks and linked to the cause of PAM infections that resulted in the death of both children (18). A total of 24 people in the United States have died by infection from N. fowleri during the period of 1989 to 2000 (6). Due to difficulties in detecting an uncommon infectious agent such as N. fowleri, many cases of infection are fatal. Amphotericin B, an anti-microbial agent, can be administered and is effective at eliminating the infection and allowing for a complete medical recovery of the infected individual if the identification of N. fowleri occurs quickly (3, 19).

The amoeboflagellate genus Naegleria contains pathogenic and nonpathogenic species,

and most species are morphologically indistinguishable requiring molecular methods to

define and identify members of the genus. The Naegleria isolates that cause PAM were

given species status and named N. fowleri, after Malcolm Fowler who first recognized the

disease in Australia (8, 19). N. fowleri is often isolated from warm soil and stagnant

water up to 45oC, with cysts surviving temperatures up to 46oC, and is found with large

quantities of fecal coliform bacteria as reported in the James River watershed, Virginia

(8). The three life stages of N. fowleri include cysts, trophozoites, and flagellates. By

entering the body through the mucous membranes of the nose, trophozoites infect

humans and animals by traveling to the central nervous system where they cause PAM

(8).

Problem Statement

The purpose of this study was to determine the presence of N. fowleri in the potable water

supply from warm water aquifers in Peoria, Arizona where two children died from PAM.

In a previous investigation a free living amoeba, N. fowleri, was discovered in the

residual water from household pipes and sinks and linked to the cause of PAM infections

that resulted in the death of both children. It was suspected that the probable vector of

infection was the potable water supply from warm water aquifers in the area. Another

goal of this study was to characterize the bacterial communities that were associated with

N. fowleri. PCR assays and sequencing were used to detect N. fowleri, bacterial pathogens, and predominant bacterial populations from groundwater samples.

METHODS AND MATERIALS

Samples

Eschericha coli O157:H7 cultivation

For the cultivation of Eschericha coli (E. coli) strains K12 and O157:H7, the pure

cultures were grown in Luria Bertani (LB) medium (see Table 1 for a list of media and

components). The medium was sterilized by autoclaving at 121oC for an amount of time

dependent on the volume. The cultures were grown overnight (12-16 hours) in a thermal incubator at 30oC. In addition, a sterile wire loop was used to streak the culture onto a

Petri dish of LB medium with agar. This plate was allowed to grow overnight at 30oC.

Single colonies were then picked by a sterile wire loop, streaked separately onto another

LB Petri dish, and stored at 4oC for future analysis. Once the cultures were fully grown,

cell counts were performed with the use of a hemocytometer. The information acquired

from the cell count was used to determine the volume of inoculated medium needed to

achieve a total of 103, 104, and 105 cells in three sets of separate 100 liter runs for the limit of detection (LOD) experiment.

Naegleria fowleri cultivation

For the cultivation of N. fowleri a modified Oxoid medium was prepared (Table 1).

Using a sterile pipet, a volume of 25 mL of the modified Oxoid medium was placed into

a 75 cm2 tissue culture flasks (Corning cell culture flasks, Corning Inc.) with a monolayer

of N. fowleri. The cultures were kept incubated at 37oC in a thermal incubator. The cell

monolayer was observed daily, after three or four days the original medium was poured

off and fresh medium was placed in the tissue culture flasks with a sterile pipet. With the

cultures at stationary growth phase, cell counts were performed with the use of a

hemocytometer. The information acquired from the cell count was used to determine the

volume of inoculated medium needed to achieve a total of 103, 104, and 105 cells in three

sets of separate100 liter runs for the LOD experiment.

Mason Raw Groundwater

With the cooperation of Greater Cincinnati Water Works, raw groundwater was collected

from the Mason Drinking Water Treatment Plant. Characteristics of the water are given

in Table 2. The water was stored in containers capable of holding approximately 121

liters of water. The raw groundwater was used to determine the LOD of the PCR assays

chosen for E. coli O157:H7 and N. fowleri. Two types of samples were made from the raw groundwater: spiked and unspiked. The unspiked sample consisted of 100 liters of the raw groundwater, completely unaltered with the addition of no microorganisms. The spiked samples consisted of three sets of 100 liters of raw groundwater with increasing

amounts of E. coli O157:H7 and N. fowleri cells in each set. The first spiked set

contained a total of 103 cells of E. coli O157:H7 and N. fowleri in the 100 liters of water,

the second set contained a total of 104 cells of E. coli O157:H7 and N. fowleri in the 100

liters of water, and the final set contained a total of 105 cells of E. coli O157:H7 and N.

fowleri in the 100 liters of water. The samples are listed in Table 3.

Environmental Filter and Water Samples

In the field, water from a specific groundwater aquifer was run through duplicate filters

(Cuno Micro-Wynd II cartilage filters); the 1.0 µm pore size first and 0.5 µm last, in

volumes ranging 62.5 gal to 1,162.9 gal from selected groundwater aquifers in the area

by representatives from US EPA Region 9. These samples (henceforth called ‘filter’

samples) were stored and analyzed by PCR. The filters were received in coolers

containing ice and were stored for later analyses. Warm groundwater samples not run

through filters are henceforth called ‘water’ samples. The water and filter samples are

listed in Table 3.

Processing Samples

Pure Cultures

E. coli strains K12 and O157:H7 were originally grown in LB media and stored as

isolated colonies on LB Petri dishes at 4oC. In addition to short term storage of 4oC, 0.5 mL of the liquid cultures were preserved in tubes containing 0.5 mL of 10 % glycerol solution and placed in -80oC for long term storage.

Environmental Sample Storage

Cuno Micro-Wynd II cartilage filters (0.5 and 1.0 µm pore size) were received from

Peoria, Arizona in coolers cooled by ice, removed, and stored at -80oC. In the field, water was run through duplicate filters; the 1.0 µm pore size first and 0.5 µm last, in volumes ranging from 62.5 gal to 1,162.9 gal from selected groundwater aquifers in the area by representatives from the US EPA Region 9. The aquifers sampled varied on temperature conditions, Heterotrophic plate counts (HPCs), and the type of pump used to elevate the water for drinking water treatment (Table 3).

Mason Raw Goundwater Samples and LOD Samples

The unspiked sample, unaltered Mason raw groundwater, and the spiked samples were collected in the form of duplicate Cuno Micro-Wynd II cartilage 0.5 µm filters and 50 mL water samples. After running the LOD experiment, all samples were stored at -80oC.

Molecular Methods

DNA Extraction

To identify the prokaryotic and eukaryotic communities present on the filters a sterile cutting device (e.g., sterile razor blade) was used to remove a 0.25 in2 area of the filter material, and the filter material was subsequently placed in a 2 mL tube of bead beating

solution from a MoBio Soil DNA Extraction Kit. The procedure followed for DNA extraction was that suggested by the manufacturer’s instructions. The final product was

50 µL of genomic DNA (gDNA), stored at -20oC, and used in PCR.

Polymerase Chain Reaction

For the pure culture of E. coli O157:H7 primer assays 1, 2, and 3 as reported in Table 4

were performed on dilutions of 1:1, 1:10, 1:100, and 1:1000 of its extracted gDNA to

verify the sensitivity of the assay. This was repeated for the pure culture of N. fowleri,

except primer assays 4 and 5 as reported in Table 4 were performed.

Environmental sample numbers 3 to 23, 26 to 32, 35 to 41, 43 to 47, and 49 to 53 for both filter and water samples (Table 3) were analyzed using primer assays 1, 2, 3, 4, and 5

(Table 4). Select environmental samples 3, 4, 5, 28, and 43 were further analyzed by primer assay 6 for use in the full cycle 16S rRNA approach (Figure 6).

For LOD sample numbers 54 to 67 for both filter and water samples (Table 3) were

analyzed using primer assays 1, 2, 3, 4, and 5 (Table 4).

Gel Electrophoresis

To verify the presence of PCR products, samples analyzed by primer assays 1, 2, 3, 6, and 7 (Table 4) were electrophoresed in a 1 % agarose gel stained with 0.008 % ethidium

bromide (EtBr) in 1x Tris-borate (TBE) buffer. Samples analyzed by primer assays 4 and

5 (Table 4) were electrophoresed in a 2 % agarose gel stained with 0.008 % EtBr in 1x

TBE buffer. Each gel was photo-documented using a Kodak EDAS 290 digital camera

and Spectroline ultraviolet transilluminator.

PCR Purification

Prior to cloning and sequencing, 16S rDNA PCR products were purified using a

QIAquick PCR Purification Kit (Qiagen Inc.) following the manufacturer’s instructions.

PCR products were eluted in 30 µL of elution buffer solution and stored at -20oC for future analyzes.

Cloning

Universal 16s rDNA PCR products were cloned into chemically competent E. coli DH5- alpha cells using the TOPO TA Cloning Kit (TOPO10 Electrocomp Cells, Invitrogen

Corp.). Briefly, fresh PCR products (right after gel verification of the PCR reaction) were ligated with the pCR® 2.1-TOPO vector by incubating at room temperature for 5 minutes. The ligation product (4 µL) was added into the microcentrifuge tube containing chemically competent E. coli DH5-alpha. The E. coli cells were transformed by heating to 42ºC for 30 seconds and immediately placed on ice. SOC medium, 250 µL, was added to the sample, followed by incubation at 37ºC for 1 hour. Kanamycin and 5-bromo-4- chloro-3-indolyl-alpha-D-galactopyranoside (X-Gal) were used to screen for transformed

E. coli cells containing recombinant plasmids. After the incubation, the transformed E. coli cells were transferred onto LB agar containing kanamycin (50 µg/mL) and X-Gal (10 mg/µL). The LB plates were incubated at 37ºC overnight. Single white colonies, 100 for each sample, (colonies containing plasmids) were randomly selected and transferred to a library plate containing LB medium and kanamycin only. After incubation at 37°C overnight, the plates were stored at 4°C.

Sequencing

Clone libraries on plates containing LB medium and kanamycin were analyzed by primer assay 7 to screen for colonies containing plasmids. Colonies containing plasmids were submitted to Cincinnati Children's Hospital Medical Center (CCHMC) DNA Sequencing

Facility for sequencing.

Sequence Analysis

Nucleic acid sequences received from the CCHMC DNA Sequencing Facility were transferred into FASTA format by using BioEdit v7.0 (13). Sequences of low quality were discarded. Sequences were aligned by using ARB software. A data base of well aligned sequences (SSUJAN03.ARB) was downloaded from the ARB home page

(www.arb-home.de) as reference used by the PT_SERVER. The closest relatives for each sequence of the environmental samples were identified in the data base. The sequences of the environmental samples were aligned by using the FastAligner integrated

in ARB followed by manual inspection. The aligned sequences of the environmental

samples and their relatives were used to reconstruct phylogenetic trees by using ARB (for

Neighbor Joining and FITCH) and PHYLIP (for Parsimony) (11) package integrated in

ARB. Neighbor Joining trees were constructed using ARB with Felsenstein and Jukes-

Cantor evolutionary models. FITCH was used to construct distance phylogenetic trees.

DNAPARS was used to construct parsimony phylogenetic trees. The aligned sequences

were exported into PAUP format. The Maximum Likelihood phylogenetic tree was

constructed in PAUP 4.0 (beta) using the default setting with 2 Jackknife replicates. The

Neighbor Joining and FITCH phylogenetic trees were manipulated with ARB. The

Maximum Likelihood phylogenetic tree was manipulated using TreeView (Win32) v1.5.2 (Copyright Roderic D.M. Page, 1998). Based on environmental sample results, well temperature, HPC, and pump type, primer assay number 6 was run on environmental sample numbers 3, 4, 5, 28, and 35.

RESULTS

Validate Assay for N. fowleri

To find the appropriate assay to detect N. fowleri by PCR, a literature review was performed. Table 5 provides a list of papers reviewed, application of the assay, the type

of assay used, and the limit of detection of the assay.

The assay selected was a nested PCR assay that targets the mip gene of N. fowleri (28).

This assay has a limit of detection of 5 cells per 50 mL of water. Nested PCR uses two

separate primer pairs; the first targets the mip gene, which is unique to the Naegleria genus. The second primer pair targets a conserved region within this gene and is capable of distinguishing pathogenic N. fowleri from other non-pathogenic members of the genus.

This nested PCR assay can be performed quickly and accurately by any laboratory that contains a basic thermocycler, and doesn’t require the advanced machinery of a real-time

PCR, multiplex PCR, or ELISA assay. Dr. Marciano-Cabral’s work focuses on pathogenic amoebae, specifically N. fowleri, and one of her published papers investigates the presence of N. fowleri in Peoria, Arizona, which is the site of investigation for this project (18). Previously, she was able to capture and isolate N. fowleri from bathtub water in homes of victims of PAM using Micro-wynd II filters (1.0 µm pore size) (18).

The bathtub water samples were tested for N. fowleri by nested PCR (28) and the assay was able to detect the presence of N. fowleri (18). Due to Dr. Marciano-Cabral’s past project investigating N. fowleri in the same area of interest as this project and her

extensive background and work related to N. fowleri (18, 19, 20, 28), her published assay

was chosen to detect the presence of N. fowleri.

To verify the specificity and sensitivity of the nested-PCR assay, a pure culture of N. fowleri was obtained from ATCC (strain 30984) and grown in 75 cm2 tissue culture

flasks with modified Oxoid medium. Genomic DNA was extracted from the pure culture

with a MoBio Soil DNA Extraction Kit and dilutions of the gDNA were made: 1:1

(362,500 cells), 1:10 (36,250 cells), 1:100 (3,625 cells), and 1:1000 (362.5 cells) in sterile

water. Figure 1 presents the results of the nested PCR assay on dilutions of N. fowleri

gDNA.

From the gel image presented in Figure 1, it can be seen for the first round of nested

PCR, no products are amplified. The first round of PCR uses MP2Cl5 primer pair designed to amplify a 166 bp product of all Naegleria species and increases the

sensitivity of the assay. The second round of nested PCR, amplification is seen around

110 bp for the MP2Cl5-in primer pair. This second round of PCR is designed to amplify

an inner region of the first round product that is specific for N. fowleri. A high intensity band is seen for 1:1 and 1:10 dilution of N. fowleri gDNA, a slightly lower intensity band is seen for 1:100 dilution of N. fowleri gDNA, and an even lower intensity band is seen

for 1:1000 dilution of N. fowleri gDNA. Therefore the assay chosen is specific for N.

fowleri gDNA and extremely sensitive as seen by the low intensity band even at 1:1000

dilution of gDNA.

1 2 3 4 5 6 7 8 9 10 11 12 13

200 bp 100 bp

Figure 1. Sensitivity of nested PCR assay on N. fowleri gDNA dilutions on 2% agarose gel stained with ethidium

bromide. Lane 1-100 bp ladder; lane 2-negative control; lane 3-1:1 gDNA dilution with Mp2Cl5 primer pair; lane 4-

1:10 gDNA dilution with Mp2Cl5 primer pair; lane 5-1:100 gDNA dilution with Mp2Cl5 primer pair; lane 6-1:1000 gDNA dilution with Mp2Cl5 primer pair; lane 7-100 bp ladder; lane 8-negative control; lane 9-1:1 gDNA dilution with

Mp2Cl5-in primer pair; lane 10-1:10 gDNA dilution with Mp2Cl5-in primer pair; lane 11-1:100 gDNA dilution with

Mp2Cl5-in primer pair; lane 12-1:1000 gDNA dilution with Mp2Cl5-in primer pair; and lane 13-100 bp ladder.

To find the limit of detection of the assay on environmental samples, raw groundwater

was collected from the Mason Drinking Water Treatment Plant in Mason, OH. Four sets

of 100 L volumes of the raw groundwater were run through four sets of two sterile 0.5

µm Micro-wynd filters in series. Two new filters were used for every 100 L of

groundwater filtered, sterilely removed after each run and stored. The first set of 100 L

was unaltered and used as a control to confirm the absence of N. fowleri in the Mason

raw groundwater. The second set of 100 L had a total of 103 cells of N. fowleri in the 100

L of water, the third had a total of 104 cells of N. fowleri in the 100 L of water, and the fourth set had 105 cells of N. fowleri total in 100 L of water. Prior to filtration and after

the addition of N. fowleri, a 50 mL sample of water was taken from the 100 L volume of

Mason raw groundwater. The four water samples were named and labeled according to

the number of N. fowleri cells that should have been captured in the 50 mL volume

according to the total number of cells added to the 100 L volume. Whereas the filter

samples for each 100 L experimental run were labeled according to the number of cells

per mL.

The two filters and the 50 mL water sample taken for each of the four 100 L groundwater

runs were analyzed using the nested PCR assay selected above (28), and the results are

reported in Table 6. All samples analyzed by PCR were run in triplicate and analyzed by

a gel stained with EtBr. For water samples 55 and 57, one of the triplicate runs detected

the presence of N. fowleri, sample 56 had two of the triplicate runs positive for N. fowleri,

and sample 54 tested negative for N. fowleri (Table 7). As a control, gDNA was

extracted from two sterile, unused, filters (samples 58 and 59) and tested for the presence

of N. fowleri with both filters negative. Out of four sets of filter pairs only one set,

samples 66 and 67, tested positive for N. fowleri and had amplification for all triplicate

runs. Figure 2 shows the presence or absence of N. fowleri amplification for LOD experiment samples (Table 7).

1 2 3 4 5 6

200 bp 100 bp

Figure 2. Gel image (2% agarose gel stained with EtBr) showing the intensity of the amplification product of the

second set of nested PCR primers (27). Lane 1-100 bp ladder; lane 2-negative control; lane 3-positive control with

gDNA from N. fowleri; lane 4-10 cells / mL series 1; lane 5-10 cells / mL series 2; and lane 6-100 bp ladder.

Validate N. fowleri Assay in Environment

Environmental samples were received in the form of filters (Micro-wynd II 0.5 µm and

1.0 µm pore size) and small volumes of water drawn from warm groundwater aquifers.

The groundwater sites sampled varied by temperature, pump type, HPC numbers, pH,

dissolved oxygen, and conductivity (Table 3).

One of the triplicate runs for samples 28, 29, 43, 44, and 50 tested positive for N. fowleri.

Sample 5 had two of the triplicate runs positive and sample 6 had all triplicate runs positive for N. fowleri. Water samples 18, 19, 21, and 22 had one of the triplicate runs positive and sample 9 had all triplicate runs positive for N. fowleri.

When comparing the collected field data (Table 3 or 8) of all filter samples positive for

N. fowleri, three pieces of information could indicate the presence of N. fowleri; HPC,

well temperature, and pump type. N. fowleri has been known to grow in warm water

environments, tolerate a wide range of pH, and found in association with high coliform

counts (19). While no coliform count was performed, HPC can indicate the presence of

heterotrophic bacteria that can serve as one of many food sources for amoebae. The

presence of N. fowleri for these groundwater samples could be correlated to the high

temperatures of the wells and the use of oil lube pumps. Oil from these pumps leaks into

17 the groundwater, providing a carbon source for bacteria and other microorganisms.

These microorganisms in turn become the food source for N. fowleri, a pathogenic amoeba that is also found in the soil (19).

DISCUSSION

In Peoria, Arizona during the autumn season of 2002, two young children died from

PAM. A free living amoeba, Naegleria fowleri, was discovered in the residual water from household pipes and sinks and was linked to the cause of PAM infections that

resulted in the death of both children (18). It was suspected that the probable vector of

infection was the potable water supply from warm water aquifers in the area. Since N.

fowleri was detected in pipes and sinks, it was suspected that the warm groundwater was

contaminated with N. fowleri. The goal of this study was to test the warm groundwater in

Peoria, Arizona for the presence of N. fowleri. According to the scope and aim of the

project, a nested PCR assay was chosen which is capable of detecting N. fowleri at concentrations of 5 cells per 50 mL (28).

Before testing the nested PCR assay on environmental samples from Peoria, Arizona, the

assay was first run on pure cultures and spiked raw groundwater from Mason, Ohio to

determine the limit of detection for this project. From 50 mL samples collected from the

spiked raw groundwater prior to filtration, N. fowleri was detected for 0.1, 1, and 10 cells

(Table 7), but the results were inconsistent suggesting that high variability in outcome

would be present in samples without concentration. Out of four filter pairs, samples 66

and 67, both 0.5 µm pore size and run in series, tested positive for N. fowleri. From

mock environmental samples, the nested PCR assay was able to detect a minimum of 10

cells per mL of N. fowleri from a Micro-wynd II 0.5 µm filter with consistent results

among replicate PCR runs. When the nested PCR assay was applied to environmental

filter and water samples from Peoria, Arizona, several samples tested positive for N.

fowleri, but the results were not consistent across replicates. Filter samples 5, 6, 28, 29,

43, 44, 50 and water samples 9, 18, 19, 21, 22 tested positive for N. fowleri (Table 6).

Validate assay for E. coli O157:H7

In addition to looking for N. fowleri, a secondary objective was to look for the presence

of pathogenic E. coli O157:H7, which is known to be associated with N. fowleri (19). A

literature review was conducted and PCR primers were selected that were capable of

detecting general E. coli, E. coli O157:-- serotype, and E. coli --:H7 serotype (24). To

verify the specificity and sensitivity of this PCR assay (containing the above three primer pairs), a pure culture of E. coli O157:H7 was obtained from ATCC (strain 43895) and grown in a 250 mL Erlenmeyer flask with LB medium. Genomic DNA was extracted from the pure culture with a MoBio Soil DNA Extraction Kit and dilutions of the gDNA were made: 1:1 (107 cells), 1:10 (106 cells), 1:100 (105 cells), and 1:1000 (104 cells) in

sterile water. Figures 3, 4, and 5 present the results of the PCR assay on dilutions of E.

coli O157:H7 gDNA. The best amplification is seen for the primer pair targeting general

E. coli 16S rRNA, which is able to detect gDNA at a dilution of 1:1000. The other two

primer pairs were not as sensitive as the first and the primer pair targeting E. coli O157:--

and E .coli --:H7 serotypes were able to detect gDNA at a dilution of 1:100.

To find the limit of detection of the E. coli O157:H7 PCR assay on environmental samples, groundwater from the Mason Drinking Water Treatment Plant was spiked with

20 a pure culture of E. coli O157:H7. Four sets of 100 L volumes of the raw groundwater were run through four sets of two sterile 0.5 µm Micro-wynd filters in series. On top of the addition of N. fowleri to three of the 100 L volumes of raw groundwater, E. coli

O157:H7 was also added during the experiment. The first set of 100 L was unaltered and used as a control to confirm the absence of E. coli O157:H7 in the Mason raw groundwater. The second set of 100 L had a total of 103 cells of E. coli O157:H7, the third had 104 cells of E. coli O157:H7, and the fourth set had 105 cells of E. coli

O157:H7. Prior to filtration and after the addition of E. coli O157:H7 and N. fowleri, a

50 mL sample of water was taken from the 100 L volume of Mason raw groundwater.

Two new filters were used for every 100 L of groundwater filtered, sterilely removed after each run, and stored.

The two filters and the 50 mL water sample taken for each of the four 100 L groundwater runs were analyzed using the E. coli O157:H7 PCR assay. All samples analyzed by PCR were run in triplicate and analyzed by an agarose gel stained with EtBr. For water and filter samples, all triplicate runs showed the presence of E. coli, but not E. coli O157:-- serotype or E. coli --:H7 serotype. As a control, gDNA was extracted from two sterile, unused, filters and tested for the presence of E. coli O157:H7 and both filters tested negative (Table 7). Together, these results suggest the presence of E. coli in the raw groundwater from Mason, OH but not present on virgin filter material.

Validate E. coli O157:H7 Assay in Environment

The environmental samples received from Peoria, Arizona were tested for the presence of

E. coli O157:H7 using primer assays 1, 2, and 3. Sixteen samples: 3, 4, 7, 8, 13, 14, 30,

39, 40, 43, 44, 45, 46, 49, 50 and 51 tested positive for E. coli, four samples: 3, 4, 27, and 35 tested positive for E. coli O157:-- serotype, and no samples tested positive for E. coli --:H7 serotype (Table 6). Two samples, 27 and 35, tested positive for E. coli O157:-- serotype but not for general E. coli. Figures 3 and 4 show the ability of primer assays 1

and 2 (Table 4) to detect dilutions of E. coli and E. coli O157:-- gDNA in sterile H2O.

Although primer assay 1 (targeting E. coli 16S rRNA) is capable of detecting E. coli gDNA to a dilution of 1:1000 and primer assay 2 (targeting rfb O157 gene) is capable of detecting E. coli O157:-- gDNA to a dilution of 1:100, the latter is known to show less sensitivity to inhibition (21). This could explain why E. coli O157:-- serotype was able to be detected by primer assay 2 in the two samples where E. coli was not detected. In addition to feces, surface water and groundwater contain organic and inorganic compounds which act as PCR inhibitors (1, 15). It is possible that primer assay 2 is designed better and less impacted by the presence of PCR inhibitors on the environmental filters as compared to primer assay 1.

Identification of Predominant Bacterial Populations in Environment

The full cycle 16S rRNA approach (Figure 6) was used to identify and characterize bacterial communities from select warm groundwater filter samples. The purpose of

22 identifying the predominant bacterial communities in the warm groundwater was to develop a molecular fingerprint to indicate the presence of N. fowleri, which uses the microorganisms as a food source. The oligonucleotide primers for this were specific for conserved bacterial 16S rRNA sequences (31). Environmental samples 3, 4, 5, 28, and

43 were selected because they represented variability in temperatures, pump types, and

PCR detection of N. fowleri (Table 9). Samples 3 and 4 had high groundwater temperature, used a submersible pump, and the lacked PCR detection of N. fowleri.

Sample 5 had high groundwater temperature, used a submersible pump, and had a positive PCR detection of N. fowleri. Sample 28 had a lower groundwater temperature than samples 3, 4, and 5, used a submersible pump, and had a positive PCR detection of

N. fowleri. Finally, sample 43 had the lowest groundwater temperature as compared to the other samples, used an oil lube pump, and had a positive PCR detection of N. fowleri.

Sequences of filter samples 3, 4, 5, 28, and 43 were initially analyzed by BLASTn

(http://www.ncbi.nlm.nih.gov/BLAST) before being aligned into a phylogenetic tree.

BLASTn results of samples 3 and 4 indicated a close homology of the sequences to

Caldimonas species and Leptothrix species (4).

BLASTn results for sample 5 produced a diverse community closely homologous to

Caldimonas taiwanesis, uncultured Nitrospira species, Meiothermus cerebereus, uncultured β-proteobacteria species, and a variety of uncultured bacterium clones (4).

BLASTn results of sample 43 yielded sequences closely homologous to Aquabacterium parvum, Nocardia asteroides, and a few uncultured bacterium clones (4).

The BLASTn result for sample 28 revealed a close homology to Aquabacterium hongkongesis strain (4). Unlike filter samples 3, 4, 5, and 43 that were analyzed by the full cycle 16S rRNA approach, PCR products from sample 28 were unable to be cloned and transformed into the competent E. coli. Blue and white screening was used to distinguish transformed colonies from non-transformed colonies. Even though white colonies did form, these cells lacked the PCR insert and only contained the vector. This is probably due to the 16S rDNA region amplified from sample 28 being toxic to the competent cells, and therefore being expelled from the cells during the incubation period.

To overcome this difficulty, several courses of action were tried: 1) drop the incubation temperature from 37oC to 30oC, slowing down cellular growth and cellular expulsion of the toxic PCR insert, 2) use a special cell line (D10 competent cells) capable of cloning and transforming a toxic PCR insert, 3) use M9 minimal medium plates to slow the growth of competent cells and expulsion of the toxic PCR insert, and 4) use a combination of M9 plates with D10 cells and 30oC incubation. After following the listed courses of action, sample 28 was still unable to be cloned. This result was confirmed by another study that was unable to clone Aquabacterium hongkongensis from biofilms with the use of TA Cloning Kit (Dr. T. Zhang, Environmental Biotechnology Lab, Department of Civil Engineering in Hong Kong, personal communication). Therefore a direct sequence of the PCR product had to be performed, and the resulting sequence identified

Aquabacterium hongkongensis. Thus, it appears that the bacterial community in sample

28 was predominated by a difficult to clone strain of Aquabacterium.

The phylogenetic trees for samples 3 and 4 (Figures 7 and 8) show that most of the

sequences are closely related to β-proteobacteria Caldimonas manganoidans, and

weakly related to Acidovorax temperans, Leptothrix species, Rubrivivax gelatinosus,

Rhodocyclus purpureus, and Aquabacterium species.

The sequence of sample 28, obtained from a purified PCR product, was added to all

phylogenetic trees and closely related to β-proteobacteria Aquabacterium species.

The phylogenetic tree for sample 5 (Figure 9) shows a wide distribution of the sequences

among different groups of bacteria. A minority of the sequences are related to

Rubrivivax gelatinosus, Caldimonas manganoxidans, Cytophaga marinoflava,

Meiothermus ruber, Bergella zoohelcum, Nitrospira moscoviensis, and Flexibacter

flexilis. A majority of sample 5 sequences are strongly related to β-proteobacteria

Aquabacterium species and weakly related to Leptothrix species, Rhodocyclus purpureus,

Acidovorax temperans and Rubrivivax gelatinosus.

The phylogenetic tree for sample 43 (Figure 10) shows a few sequences related to

Nocardia asteroides, Flexibacter flexilis, Myxobacterium, Rasbo bacterium, and

Aquabacterium species. A majority of the sequences from sample 43 are strongly related

to the β-proteobacterium Caldimonas manganoxidans and have a weak relation to

Acidovorax temperans, Leptothrix species, Rhodocyclus purpureus, Rubrivivax

gelatinosus, and Aquabacterium species.

1 2 3 4 5 6

200 bp

100 bp

Figure 3. Gel image (1% agarose gel stained with EtBr) showing the intensity of the amplification product of 16SF and

16SR primers targeting 16S rRNA specific to E. coli. Lane 1-100 bp ladder; lane 2-negative control; lane 3-1:1 gDNA

dilution; lane 4-1:10 gDNA dilution; lane 5-1:100 gDNA dilution; and lane 6-1:1000 gDNA dilution.

1 2 3 4 5 6

200 bp

100 bp

Figure 4. Gel image (1% agarose gel stained with EtBr) showing the intensity of the amplification product of PF and

PR primers targeting rfb gene specific to E. coli O157:-- serotype. Lane 1-100 bp ladder; lane 2-negative control; lane

3-1:1 gDNA dilution; lane 4-1:10 gDNA dilution; lane 5-1:100 gDNA dilution; and lane 6-1:1000 gDNA dilution.

1 2 3 4 5 6

200 bp

100 bp

Figure 5. Gel image (1% agarose gel stained with EtBr) showing the intensity of the amplification product of 1806 and

1809 primers targeting fliC gene specific to E. coli --:H7 serotype. Lane 1-100 bp ladder; lane 2-negative control; lane

3-1:1 gDNA dilution; lane 4-1:10 gDNA dilution; lane 5-1:100 gDNA dilution; and lane 6-1:1000 gDNA dilution.

Environmental Sample sample Sample storage at –20oC storage at –80oC

Extract Phylogenetic analysis genomic DNA

Automated PCR amplify sequencing 16S rRNA genes

Clone 16S rRNA genes

Figure 6. Diagram of the full cycle 16S rRNA approach.

Summary of species related to sequences from samples 3, 4, 5, 28, and 43:

Genus Acidovorax consists of Gram-negative cells that are straight to slightly curved rods

ranging 0.2-1.2 x 0.8-5.0 µm in size. Cells of this genus can occur singly, in pairs, or

short chains and move by means of one, two, or three polar flagella. Species are

chemoorganotrophic and aerobic, using oxygen as the terminal electron acceptor and two

species, A. delafieldii and A. temperans, are capable of heterotrophic denitrification of

nitrate. A. facilis and A. delafieldii can grow lithoautotrophically by means of the

oxidation of hydrogen as an energy source. Organic acids, amino acids, and peptone

provide cells with good growth characteristics. Habitats include soil, water, clinical samples, activated sludge, and infected plants. Acidovorax temperans characteristics are as given for the genus and found in various samples from clinical environments and one strain was isolated from activated sludge (12).

Genus Aquabacterium consists of Gram-negative cells that are rod-shaped ranging 0.5 x

1-4 µm in size. They are motile by means of monotrichous polar flagella. Storage materials of polyalkanoate and polyphosphate inclusion bodies have been observed.

Under oligotrophic conditions, extracellular polymeric substances are formed. The genus is microaerophilic, having a respiratory type of metabolism with oxygen as the terminal electron acceptor. During anaerobic respiration, nitrate can be used as an alternate electron acceptor, but nitrite, chlorate sulfate, and ferrous iron are not used as electron acceptors. No growth occurs during fermentation. A wide variety of carbon sources can be used, such as: Tweens 20, 40, 60, and 80, acetate, butyrate, valerate, caproate,

caprylate, succinate, adipate, pimelate, azelate, sebacate, fumarate, β-hydroxybutyrate,

malate, and butanol. Optimum conditions for growth include a pH near 7; however

growth can occur between pH 5.5 to 10 depending on the species. The optimal

temperature for growth is approximately 20oC with a temperature growth range of 6 to

36oC. Genus can grow in the presence of 1.8 percent sodium chloride. Species are

usually found in drinking water biofilms originating from various raw water sources

including: groundwater, surface water, artificially recharged groundwater used for

drinking water production. Aquabacterium commune characteristics are as given for the

genus. It is the only species capable of utilizing benzoate, Casamino acids, and glutamate. Out of all species, A. commune shows the lowest temperature growth.

Aquabacterium citratiphilum characteristics are as given for the genus, however its preferred growth on citrate and its utilization of lactate, γ-hydroxybutyrate, and glycerol distinguish the species from other Aquabacterium. Aquabacterium parvum

characteristics are as given for the genus (12).

Genus Bergeyella consists of Gram-negative cells that are non-spore forming rods ranging 0.6 x 2-3 µm in size. Gliding motility is not present, and cells contain

sphingophospholipids, show urease activity. Grow in nutrient broth at room temperature

or 37oC, or on β-hydroxybutyrate. The source of type species Bergeylla Zoohelcum is

from human wounds caused by dog bites, the organism is apart of the normal oral flora of certain animals and dogs. However, the pathogenicity of the species is unknown (39).

Genus Burkholderia consists of Gram-negative cells are straight or curved rods,

occurring in single or pairs with dimensions generally 0.5-1 x 1.5-4 µm. Several or one

polar flagella are used for movement, and no resting stages have been observed. Genus

does not produce sheaths or prosthecae. Poly-β-hydroxybutyrate (PHB) is accumulated

as a carbon reserve by most species. The Burkholderia genus is characterized as

chemoorganotrophs with a strict respiratory type of metabolism using oxygen as the

terminal electron acceptor; however some species can exhibit anaerobic respiration with

nitrate. B. cepacia and B. vietnamiensis are able to fix diatomic nitrogen. Carbon and

energy sources for growth include a wide variety of organic compounds. A majority of

the species are pathogenic for plants, animals, and humans. B. mallei and B.

pseudomallei are the pathogenic species capable of harming humans and animals.

Burkholderia multivorans, a reference species used in the phylogenetic trees, cells are

rods ranging 0.6-0.9 x 1.0-2.0 µm in size. Species can grow at a temperature of 42oC

with some strains growing at room temperature, but none of the species grow at 5oC.

Production of acids from some compounds is known, but in general no information given

on utilization of carbon compounds for growth. Nitrate is reduced, but not nitrite. PHB

is accumulated and utilized by this species. Certain strains can tolerance cyanide (12).

Genus Caldimonas consists of Gram-negative, aerobic rods ranging 0.5-0.7 x 2.2-3.5 µm in size. Cells are motile by means of a single polar flagellum. Sheath formation and filamentous growth is not observed for any species of the genus. Characterized as chemoorganotrophs and utilizes a large number of organic acids and carbohydrates. PHB granules are accumulated as storage material. Optimum growth conditions for

temperature and pH are 50°C and 8 to 9, respectively. Caldimonas manganoxidans cells are rods with an average size range 0.6 x 2.6 µm. On agar plates with 0.5 to 5.0 mM manganese, cells slowly and weakly oxidize the metal. In the presence of 3 percent (w/v) sodium chloride, growth is not observed. D-sorbitol, maltose, sucrose, D-glucose,

mannitol, D-galactose, starch, glycerol, L-malate, lactate, citrate, tartrate, succinate,

pyruvate, gluconate, malonate, 3-hydroxybutyrate, beef extract, yeast extract, malt

extract, tryptone, Casamino acids and peptone are compounds utilized as energy and

carbon sources. PHB is rapidly degraded and utilized in addition to the substrates listed

above (12).

Genus Chloroflexus cells stain Gram-negative and appear as filaments of indefinite

length, individual cells range 0.5-1.5 x 2-6 µm in size. Cellular division is by fission, and

not by branching. No flagella present on cells, motile by gliding. Thin sheath may be

present. Some species are anaerobic and facultatively aerobic. Genus is primarily

photoheterotrophic and secondarily photoautotrophic and chemoheterotrophic. Acetate,

glycerol, glucose, pyruvate, and glutamate utilized as carbon sources. Ammonium and

some amino acids will serve as nitrogen sources; nitrate will not. The optimum growth temperature is known for strains C. aurantiacus and C. aggregans, 52 to 60oC. Habitats

include hot spring habitats and mesophilic Chloroflexus populations are common in the

anaerobic portions of freshwater lakes. Chloroflexus aurantiacus have the same

characteristics as those described by the genus. Filament diameter of the type strain is

less than 1.0 µm, whereas filaments of most strains are less than 1.5 µm in diameter. The

type species, thermophilic, grows in the temperature range of 40 to 66oC with an

optimum between 52 to 60oC. The optimum growth pH is 7.6 to 8.4. Mesophilic strains of C. aurantiacus grow in the temperature range of 10 to 40oC with an optimum range

between 20 to 25oC and optimum pH 7.0 to 7.2 (14).

Genus Cytophaga consists of Gram-negative cells that are very short to moderately long rods with rounded or slightly tapered ends. Cells range 0.3-0.8 x 1.5-15 µm in size and occasionally the rods can be longer and flexible. No flagellum present, motile by gliding.

Cells are strict aerobes or facultative anaerobes with some using nitrate as a terminal electron acceptor. Chemoorganotrophs with a fermentative or respiratory metabolism.

Optimum temperature and pH are 20 to 35oC and 7, respectively. Habitats include soil, decomposing organic matter, salt and freshwater. Cytophaga marinoflava cells are short rods that may become coccoid, frequently found in pairs, ranging 0.5-0.6 x 1-3 µm in size. Gliding motility is very fast in liquid, but nonmotile when fixed on an agar surface.

Sole nitrogen source are peptones, which can serve as energy and carbon sources. C. marinoflava is a strict aerobe that cannot grow under low oxygen conditions, but capable of growing in a marine environment with high salt concentrations. Growth occurs within a wide temperature range of 4 to 30o C, with optimum growth at 30oC (14).

Genus Flexibacter consists of Gram-negative rod-shaped cells of variable length, ranging

10-50 µm long and 0.2-0.6 µm wide. Cells are flexible and have rounded or tapered ends. Cell has both stages of gliding motility and nonmotility. Contain carotenoids or flexirubin type pigments within the cell. The genus consists of chemoorganotrophs that are facultatively anaerobic or strictly aerobic. A wide range of sugars are utilized by the

cells, and amino acids and peptones are supplied as nitrogen sources. Habitats include

soil and freshwater. Flexibacter flexilis cells are long and thread-like ranging 0.5 x 10-60

µm in size. Cells are capable of limited gliding motility. Develops slime in the presence

of many substrates. Cells are strictly aerobic using peptones and Casamino acids as

nitrogen sources. Optimum temperature and pH at 25oC and 7, respectively. Originally

isolated from various freshwater environments (14).

Genus Halochromatium consists of Gram-negative cells that are straight to slightly

curved rods that occur singly or in pairs. Cells multiply by binary fission and are motile

by polar flagella. Contain bacteriochlorophyll-a and carotenoids photosynthetic

pigments. Photolithoautotrophic growth is capable with light under anoxic conditions with sulfide or elemental sulfur as electron donor. Stored in the form of highly refractile globules inside the cells, elemental sulfur is formed as an intermediate oxidation product and sulfate is the ultimate oxidation product. Capable of chemolithotrophic and chemoorganotrophic growth under micro-oxic conditions in the dark. Genus is mesophilic with optimal growth temperatures of 20 to 35°C with optimum pH 7.2 to 7.6.

For growth high salt concentrations are required and habitat includes reduced sediments in Salinas, microbial mats in hypersaline environments, and coastal lagoons with elevated salt concentrations. Halochromatium salexigens has an extensive salt dependence and salt tolerance, which is the highest within the family Chromatiaceae (12).

Genus Leptothrix consists of Gram-negative straight rods ranging 0.6-1.5 x 2.5-15 µm in

size. Cells are naturally occurring within a sheath or free-swimming as single cells, in

pairs, or as motile short chains containing up to 8 cells in some species. Free cells are

motile by means of one polar flagellum, only one species has a subpolar tuft that contains

several flagella. Globules of PHB are stored by most species as reserve material. Iron

and manganese oxides have a tendency to cover the sheaths. Characterized as

chemoorganotrophs with a respiratory metabolism and never fermentative. For growth,

the temperature range extends from 10 to 35oC, with an optimum temperature for most

strains around 25oC and the optimum pH ranges between 6.5 and 7.5. Glucose, fructose

and sucrose, organic acids, including lactic, malic and β-hydroxybutyric acids, and

glycerol are utilized by most Leptothrix species as carbon and energy sources. Species are widely distributed in a variety of freshwater habitats, varying from sediments, sewage, unpolluted springs and slowly running water rich in soluble iron and manganese

compounds. Leptothrix cholodnii, a reference species used in the phylogenetic trees,

cells are usually found in long chains inside the sheaths ranging 0.7-1.3 x 2-7 µm in size.

However, outside the sheaths single motile cells can be found. The cells become covered

with granular manganese dioxide when manganese is present. Slowly running iron- and

manganese-containing unpolluted or polluted waters, particularly in activated sludge are

natural habitats for L. cholodnii. Leptothrix discophora, a reference species used in the

phylogenetic trees, cells are smaller in comparison to other Leptothrix species described.

Cells may be free swimming or occur in narrow sheaths, and the free cells are motile by

thin polar flagella at one or both poles. The species is well known for its well defined

manganese-oxidizing and ferric oxide-storing capacities. For growth, the temperature

range extends between 15 and 33oC and pH from 6.0 to 8.0. PHB granules are formed by

this species. The normal habitat for L. discophora is slow running, unpolluted, iron- and

manganese-containing water of ditches, rivers, and ponds. Leptothrix mobilis, a

reference species used in the phylogenetic trees, cells are similar in width to L.

discophora, but are typically shorter and motile by single polar flagella. Sheaths are not

formed under laboratory conditions for this species, but PHB granules are formed. For

growth, the temperature range extends between 10 and 37oC with the pH from 6.5 to 8.5.

This species was originally isolated from the sediment of a freshwater lake (12).

Genus Meiothermus cells stain Gram-negative and appear as straight rods 0.5-0.8 µm in

diameter with variable cell length, with the formation of filaments under some culture

conditions. Cells do not possess flagella and are nonmotile. Species are aerobic with a

strictly respiratory type of metabolism with some capable of using nitrate as terminal

electron acceptor. Slightly thermophilic with an optimal growing temperature range

between 50 to 65oC and are not capable of growing at 70oC with optimum pH ranges

from 7.5 to 8.0. Carbon and energy sources include: hexoses, a few pentoses and a few

polyols, disaccharides, amino acids, and organic acids. Found in hydrothermal areas with

neutral to alkaline pH and also isolated from fermentors. Meiothermus ruber cells are

similar to all other species of this genus. High optimum growth temperature and the fatty acid composition distinguish the strains of this species from the other species of this

genus. Succinate, malate, and myo-inositol are utilized by the type strain. Habitats

include hot springs and strains similar to the type strain have been isolated from hot

springs and runoffs in other geographical areas as well as from fermentors (14).

Order Myxococcales have three types of cells: cube-shaped cylindrical cells with rounded or truncated ends, cylindrical cells with rounded ends, and long slender cells with tapering ends. Myxobacteria are motile by gliding along surfaces. Multicellular fruiting bodies that contain spores are formed during the developmental cycle of

Mycoccus. Cells are Gram-negative, chemoorganotrophic, obligate aerobes.

Myxobacteria form colonies on agar that appear film-like and can be cultivated on a liquid medium containing a nitrogen source. Myxobacteria most commonly inhabit topsoil in the pH range from 5 to 8, although they can also be found at extreme pH.

Other habitats include decaying organic material and fresh water (14).

Genus Nitrospira consists of Gram-negative cells and appear as vibrio-like to spiral- shaped rods ranging 0.2-0.4 x 0.9-2.2 µm in size. Cells lack intracytoplasmic membranes and reproduce by binary fission. Cells are predominantly nonmotile and aerobic. The oxidation of nitrite to nitrate provides the major source of energy. Mainly lithoautotrophs, however can grow mixotrophically. Habitats include ocean environments, heating systems, soil samples, freshwater, and activated sludge.

Nitrospira moscoviensis cells are curved rods with 1-3 turns, with growing cells having irregular shapes. Strains of the species are mesophilic, growth was not supported by organic matter, and the doubling time was 12 hours in a mineral medium with nitrate.

Growth occurs between 33 to 40oC with optimum growth at 39oC. Extracellular polymeric substances cause the formation of flocs 1-2 mm in diameter after the consumption of nitrite. Originally isolated from an iron pipe of municipal heating system

(14).

Genus Nocardia are Gram-positive to Gram-variable cells rudimentary to extensively branched vegetative hyphae ranging 0.5-1.2 µm in diameter. The hyphae grow on the surface of agar media and penetrate turning into nonmotile rod-shaped and coccoid forms with the ability to form aerial hyphae. The genus is described as chemoorganotrophic with and oxidative type of metabolism, aerobic, and mesophilic. The genus can be found in a wide variety of environments and abundant in the soil with some strains as opportunistic pathogens of humans and animals. Nocardia asteroides has characteristics similar to the genus description. Cellular morphology varies greatly in terms of colony color and form, numbers of aerial hyphae, and degree of fragmentation. Strains decompose urea and produce nitrate reductase. A majority of the strains are capable of growth between 10 to 45oC and can survive up to 50oC. Most strains are soil saprophytes and some are pathogenic for man and animals (14).

Unclassified α-proteobacteria Rasbo bacterium cells are rod shaped and coccoid ranging

4-7 x 1 µm and 1-3 x 0.7-1 µm in size, respectively. In liquid medium, motility can be seen with a polar flagellum. The organism showed electron-dense granules within the cytoplasm and a three-layer laminar cell wall. Human pathogen isolated from the blood and the pericardial fluid and displays a clinical presentation of sepsis and multi-organ disease. Also has the ability to harbor in tissues or compartments within the human body to avoid anti-biotics. Cells are Gram-negative and optimal growing temperature is 25 to

30oC. Oxidase, urease, alkaline phosphatase, esterase, lipase, leucine arylamidase,

trypsin, acid phosphatase, and naphthol phosphohydrolase are produced. No reduction of

nitrate or fermentation of carbohydrates recorded (5).

Genus Rhodocyclus consists of Gram-negative cells that are slender, curved, or straight

thin rods both nonmotile and motile by means of polar flagella. Cells multiply by binary

fission and contain photosynthetic pigments bacteriochlorophyll-a and carotenoids.

Under anoxic conditions in the light with different organic substrates as carbon and

electron sources, species of Rhodocyclus prefer to grow photoheterotrophically.

Photoautotrophic and chemotrophic growth are also possible. While assimilatory sulfate

reduction is possible, reduced sulfur compounds are not used as photosynthetic electron

donors. The genus is comprised of both mesophilic and neutrophilic freshwater bacteria.

Habitat includes freshwater ponds, sewage, ditches, and swine waste lagoon.

Rhodocyclus purpureus cells are 0.6-0.7 µm wide with the diameter 2-3 µm, and

described as being half-ring-shaped to ring-shaped before cellular division. Cells are

nonmotile under all growth conditions. Bacteriochlorophyll-a and carotenoids of

rhodopinal series serve as photosynthetic pigments. Under anoxic conditions in the light

with relatively small number of organic substrates as carbon and electron sources

photoheterotrophic growth occurs. Cells also grow photoautotrophically with hydrogen

as the electron donor, chemotrophiclly under microoxic to oxic conditions in the dark, and photoheterotrophically using only ammonia and glutamine as nitrogen sources; diatomic nitrogen is not assimilated. The sole sulfur source can be sulfate. R. purpureus

is a mesophilic freshwater bacterium with optimal growth at 30oC and pH 7.2 and found

in swine waste lagoons (12).

Genus Rubrivivax consists of Gram-negative cells that are straight or curved rods, with

polar flagella, and multiply by binary fission. Bacteriochlorophyll-a and carotenoids of

the spheroidene series serve as photosynthetic pigments. Hydrogen and a variety of

carbon compounds serve as electron donors necessary for photoautotrophic and

photoheterotrophic growth. Under microoxic to oxic conditions chemotrophic growth is possible by respiration in the dark or by fermentation. Simple organic compounds used

as electron donors and carbon sources support good growth for cells. The genus is comprised of mesophilic and neutrophilic freshwater bacteria. Habitats include freshwater ponds, sewage ditches, and activated sludge. Rubrivivax gelatinosus cells are

rod shaped, straight, or slightly curved ranging 0.4-0.7 x 1-3 µm in size with older

cultures capable of reaching 15 µm in length. The cells clump together and appear immotile due to abundant mucous production in all media. Cells are highly motile by means of polar flagella in young cultures. In the dark with carbon monoxide as sole carbon and energy sources, some strains can also adapt to grow anaerobically. At low concentrations, fatty acids are utilized. Suitable nitrogen sources include: ammonia, diatomic nitrogen, and a number of amino acids. The sole sulfur source can be sulfate.

Optimal pH 6.0 to 8.5 and temperature at 30oC. Habitat includes freshwater ponds,

sewage ditches, and activated sludge (12).

Genus Thermus cells stain Gram-negative are straight rods ranging 0.5-0.8 µm in

diameter with variable length. Under some culture conditions short filaments are also

formed. Cells do not possess flagella and are nonmotile. Cells are aerobic with a strictly

39 respiratory type of metabolism, and some grow anaerobically with nitrate and nitrite as terminal electron acceptors. Species are thermophilic with an optimum growth temperature below 80oC, with some strains capable of growing at higher temperatures.

Optimum pH for growth is approximately 7.8. Carbon and energy sources include monosaccharides, disaccharides, amino acids, and organic acids. Habitat includes hydrothermal areas with neutral to alkaline pH and man-made thermal environments.

Thermus thermophilus cells are rod shaped and short filaments capable of growing as high as 80 to 82oC. Habitat includes inland and marine hot springs (14).

Phylogenetic Analysis

All phylogenetic trees had a majority of sequences closely related to three genus of β- proteobacteria: Leptothrix, Caldimonas, and Aquabacterium (Figures 7, 8, 9, 10, 11).

While samples 3 and 4 did not test PCR positive for N. fowleri, the presence of sequences related to Leptothrix, Caldimonas, and Aquabacterium species were dominant. Samples

5 and 43, which were PCR positive for N. fowleri, showed more diversity among different groups of bacteria. The diversity of sequences for samples 5 and 43 included:

1) species capable of growing in saline environments: Halochromatium salexigens and

Cytophaga marinoflava (12, 14), 2) thermophilic species: Chloroflexus aurantiacus,

Thermus thermophilus, and Meiothermus ruber (14), 3) species from wastewater, activated sludge, sewage, or swine waste lagoons: Rhodocyclus purpureus, Rubrivivax gelatinosus, Acidovorax temperans, and Nitrospira moscoviensis (12, 14), 4) some human pathogens: Rasbo bacterium, Nocardia asteriodes, and Burkholderia species (5,

12, 14), and 5) filamentous-like species such as Flexibacter flexilis, Chloroflexus aurantiacus, and Nocardia asteroides (14).

The diversity of samples 5 and 43, which tested PCR positive for N. fowleri, may indicate that N. fowleri prefers these classes of bacteria for a source of food in addition to the dominant β-proteobacteria related sequences. Aquabacterium species have the ability to avoid protistan predation (35) and may be able to avoid N. fowleri as a predator. This may explain the abundant presence of sequences related to Aquabacterium species.

Pathogenic Naegleria strains have been isolated from water at 10°C (19), the pathogenic species N. fowleri has been isolated from environments at 45oC, with cysts surviving temperatures up to 46oC (8). In addition, N. fowleri has been isolated mainly from thermal effluents, hot springs, and waters with naturally or artificially elevated temperatures (19). Leptothrix and Aquabacterium species can grow at 35 and 36oC, respectively. Caldimonas has the highest growth temperature of 50oC. Therefore, the presence of these species along with high aquatic temperatures may indicate the presence of N. fowleri.

Lakes that contained high concentrations of iron and manganese yielded a large number of pathogenic Naegleria strains, including N. fowleri (19). It has been shown in laboratory experiments that the viability of Naegleria species is enhanced by the uptake of exogenous iron (19). Leptothrix species can oxidize manganese and iron, whereas C. manganoxidans can only oxidize manganese. However Leptothrix cellular sheaths

become covered with iron and manganese oxides. Therefore, the presence of Leptothrix

species and C. manganoxidans can indicate iron- and manganese-containing waters, which in turn can point to the potential presence of N. fowleri. Also, N. fowleri could use

Leptothrix species as a food and iron source since the species stores iron oxides on its sheaths.

Bacterial communities present in an aqueous environment can greatly influence the

presence of N. fowleri, and waters contaminated with coliforms have a high occurrence of

Naegleria species (19). Leptothrix species, Rhodocyclus purpureus, Rubrivivax gelatinosus, Acidovorax temperans, and Nitrospira moscoviensis can be found in or isolated from swine waste lagoons, activated sludge, and sewage ditches. While no

sequences were related to fecal coliforms, the presence of above listed species could be

an indicator of wastewater infiltration into drinking water supplies, which can indicate

the presence of N. fowleri.

A few sequences were related to known human pathogens such as Rasbo bacterium,

Nocardia asteroides, and Burkholderia species. In addition to Nocardia asteroides and

Burkholderia species as human pathogens, N. asteroides and some Burkholderia species

can also survive temperatures up to 50oC and 42oC, respectively, which are close to the

extreme temperature range for N. fowleri. In general, water containing human pathogens

could contain other pathogens, including N. fowleri, which originate from soil, runoff,

surface water or wastewater infiltration (7).

Chloroflexus aurantiacus, Thermus thermophilus, and Meiothermus ruber are

thermophilic to slightly thermophilic species capable of growing at high temperatures (up

to 80oC for T. thermophilus) similar to N. fowleri (8, 19, 34). With thermophilic species

and N. fowleri both having the ability to grow in warm water environments, the presence

of Chloroflexus aurantiacus, Thermus thermophilus, and Meiothermus ruber could

indicate the presence of the pathogenic amoeba.

Some sequences were related to Flexibacter flexilis, Chloroflexus aurantiacus, and

Nocardia asteroides which have cells that are long and thread-like or structured similar to

filaments. This is an interesting observation as a large number of amoebae, including N.

fowleri, have been isolated from waters containing filamentous cyanobacteria, which

serve as a food source (19). In addition, N. asteroides and C. aurantiacus can sustain

temperatures at 50oC and above, which means it can tolerate the warm water

temperatures N. fowleri is typically found (19).

While most of the species described above could indicate the presence of N. fowleri one

way or another, a couple of species could show the absence of N. fowleri in certain

environments. While no sequences were closely related to Halochromatium salexigens and Cytophaga marinoflava, both species are distinct for growth at high salt concentrations. In addition, Aquabacterium species can grow in the presence of 1.8 percent sodium chloride and Caldimonas species can not grow in the presence of 3 percent sodium chloride, but growth may be possible at lower concentrations. However, most N. fowleri strains can only tolerate 0.5 to 1.0 percent sodium chloride, whereas

some strains are inhibited by 0.2 percent sodium chloride (19). If a saline water

environment existed, it would be expected that species similar to Halochromatium

salexigens and Cytophaga marinoflava would exist, with a few sequences related to

Aquabacterium and Caldimonas species depending on the salt concentration. While it

may be possible to assume the presence of N. fowleri given the presence of

Aquabacterium and Caldimonas species from reasons discussed above, the presence of

high salt tolerant species would indicate that N. fowleri could not exist due to its low salt

tolerance.

As mentioned above, phylogenetic trees show that sequences for samples 3 and 4 are

related to β-proteobacteria which are capable of growing at moderate to high

temperatures (35 to 50oC), oxidizing iron and manganese, and forming sheaths covered in

iron and manganese oxides. Although these conditions are favorable for the growth of N.

fowleri (19), samples 3 and 4 did not test PCR positive for N. fowleri. This could

probably be due to the numbers of N. fowleri, being below the detection limit of the

nested PCR assay. Samples 5 and 43 have a wider distribution of sequences in their phylogenetic trees. While the trees have a majority of the sequences related to β- proteobacteria such as Leptothrix, Caldimonas, and Aquabacterium species, there are

other species capable of indicating the presence of N. fowleri.

In summary, N. fowleri was detected by nested PCR from groundwater in Peoria, Arizona that was suspected to be the cause of PAM in two children. E. coli 16S rRNA and E. coli

O157:-- were detected by PCR, and E. coli O157:H7 was not detected by PCR from

44 groundwater in Peoria, Arizona. Bacterial communities in the groundwater were identified using the full cycle 16S rRNA approach and β-proteobacteria were dominant in all samples analyzed and associated with N. fowleri. Other genus such as

Chloroflexus, Cytophaga, Flexibacter, Halochromatium, Meiothermus, Nitrospira,

Nocardia, Rasbo bacterium, and Thermus were associated with N. fowleri. In conclusion, N. fowleri was detected in the groundwater in Peoria, Arizona. Bacterial communities in samples positive for N. fowleri can be used as a molecular fingerprint to identify N. fowleri and other pathogens in samples N. fowleri was not detected.

Figure 7. Phylogenetic tree for sample 3.

Figure 8. Phylogenetic tree for sample 4.

Figure 9. Phylogenetic tree for sample 5.

Figure 10. Phylogenetic tree for sample 43.

Figure 11. Phylogenetic tree for all sequence

Figure 11. Phylogenetic tree for all sequence (continued)

APPENDIX

Table 1. Media and Components.

Modified Oxoid Medium Luria Bertani Medium Broth Luria Bertani Medium Agar

To 1 liter of Page's Amoeba Saline add: To 1 liter of deionized water add: To 1 liter of deionized water add:

5.5 g Oxoid Neutralized Liver Digest 10.0 g Tryptone 10.0 g Tryptone 3.0 g Dextrose 5.0 g Yeast Extract 5.0 g Yeast Extract 5.0 g Proteose Peptone 10.0 g NaCl 10.0 g NaCl 2.5 g Yeast Extract 15.0 g Agar Stir for 10 minutes and autoclave. Stir for an hour and autoclave. Stir for 10 minutes and autoclave. When medium is ready to be used add:

0.5 mL Hemin / 500 mL Oxoid medium

53 5.0 mL Donor Calf Serum / 500 mL Oxoid medium

Hemin solution:

Add 10.0 mg Bovine Hemin to 10 mL 1 N NaOH. Sterilze by filtering solution through 0.25 micron filter. Do not autoclave solution. Table 2. Mason, OH Raw Ground Water Characteristics.

Coliforms Heterotrophic Plate Count E. coli Total Total Plate number Plate number CFUs CFUs CFUs Plate 1 13 94 Plate 1 48 Plate 2 16 67 Plate 2 43 Plate 3 18 86 Plate 3 108 Average 15.6 82.3 Average 66.3

Total Organic Carbon Total Inorganic Total Organic Carbon Carbon Carbon (mg C/L) (mg C/L) (mg C/L) Mean 105.6 94.21 11.39 Standard Dev. 120 171 Coeff. of Var. 0.40% 0.57%

ICP Analysis Analyte mg/L Ammonium 6.3 Nitrate 0.7 Nitrite 0.081 Phosphorous 0.1 pH 7.33 As 0.004 Ba 0.559 Ca 160.039 Fe 0.452 Mg 39.812 Mn 0.177 Na 10.713 S 64.011 Si 6.950

SiO2 14.893

SO4 192.033 Zn 0.020

54 Table 3. List of Environmental and Mock Environmental Samples.

Groundwater Dissolved Final Meter Volume Sample Sample Identification Sample HPC Cond. Sample Initial Meter Filter Size Analyzed Temperature pH Oxygen Reading Filtered Pump Type Number Name Date/Time (CFU/ml) (µS) Date/Time Reading (gal) (µm) by PCR (oC) (mg/L) (gal) (gal)

Well Site 1 1 TON1 8/23/2004 270 44.9 8.1 3 938 N/A N/A N/A N/A N/A submersible No 12:55 2 TON1 Duplicate 8/23/2004 330 44.8 8.4 3.2 - N/A N/A N/A N/A N/A submersible No 13:00 3 TON1A 12/14/2004 2 43.9 8.6 4.1 840 12/14-15/04 20297.2 21460.1 1,162.90 1 submersible Yes 8:14 8:30-7:30 4 TON1B 12/14/2004 3 44.2 8.6 4.2 896 12/14-15/04 20297.2 21460.1 1,162.90 0.5 submersible Yes 8:15 8:30-7:30 5 TON1C 9/1/2005 <1.0 47 8.4 - 968 9/1/2005 21941.8 22260.1 318.3 1 submersible Yes 7:27 7:45-13:37 6 TON1D 9/1/2005 2 48.1 8.3 - 921 9/1/2005 21941.8 22260.1 318.3 0.5 submersible Yes 7:28 7:45-13:37 7 TON1F 9/28/2005 11 47.9 8.6 - 910 9/28/2005 22739.5 22920 180.5 1 submersible Yes 7:09 7:30-10:45 8 TON1E 9/28/2005 <1.0 46.8 8.6 - 912 9/28/2005 22739.5 22920 180.5 0.5 submersible Yes

55 7:08 7:30-10:45 9 TON1DD Water sample submersible Yes

10 TON1FF Water sample submersible Yes Table 3. List of Environmental and Mock Environmental Samples (continued).

Well Site 2 11 VAL1A 9/1/2005 3 35.4 8.1 - 2.31m 9/1/2005 22267 22366.8 99.8 1 submersible Yes 8:25 14:30-16:15 12 VAL1B 9/1/2005 2 35.1 8 - 2.31m 9/1/2005 22267 22366.8 99.8 0.5 submersible Yes 8:26 14:30-16:15 13 VAL1D 9/28/2005 3400 34.7 8.3 - 2.28m 9/28/2005 13748.5 13855.4 106.9 1 submersible Yes 8:10 8:30-11:40 14 VAL1C 9/28/2005 3200 34.5 8.3 - 2.23m 9/28/2005 13748.5 13855.4 106.9 0.5 submersible Yes 8:09 8:30-11:40 15 VAL1AA Water sample submersible Yes

16 VAL1BB Water sample submersible Yes

17 VAL1DD Water sample submersible Yes

18 VAL1-X1 Water sample submersible Yes 56 19 VAL1-X2 Water sample submersible Yes

20 VAL1-X3 Water sample submersible Yes

21 VAL1-X4 Water sample submersible Yes

22 VAL1-X5 Water sample submersible Yes

23 VAL1-X6 Water sample submersible Yes Table 3. List of Environmental and Mock Environmental Samples (continued).

Well Site 3 24 SAF3.1 8/26/2004 100 32.1 8.3 6.3 344 N/A N/A N/A N/A N/A submersible No 11:35 25 SAF3.1 Duplicate 8/26/2004 130 31.8 8.5 6.5 344 N/A N/A N/A N/A N/A submersible No 11:38 26 SAF3.1A 12/14/2004 50 30.5 8.5 5.9 326 12/14/2005 13029.6 13246.3 216.7 1 submersible Yes 10:44 11:00-16:00 27 SAF3.1B 12/14/2004 23 30.5 8.5 6.1 321 12/14/2005 13029.6 13246.3 216.7 0.5 submersible Yes 10:45 11:00-16:00 28 SAF3.1C 9/1/2005 8 35.9 8.6 - 352 9/1/2005 13436 13588 152 1 submersible Yes 10:29 11:10-15:15 29 SAF3.1D 9/1/2005 1 35.6 8.6 - 343 9/1/2005 13436 13588 152 0.5 submersible Yes 10:30 11:10-15:15 30 SAF3.1F 9/26/2005 <1.0 34.7 8.8 4.9 340 9/26/2005 22376.9 22554.8 177.9 1 submersible Yes 8:41 9:30-12:45 31 SAF3.1E 9/26/2005 <1.0 34.5 8.8 5.1 340 9/26/2005 22376.9 22554.8 177.9 0.5 submersible Yes

57 8:40 9:30-12:45 32 SAF3.1FF Water sample submersible Yes Table 3. List of Environmental and Mock Environmental Samples (continued).

Well Site 4 33 PX244 8/24/2004 400 30.9 7.9 5.3 438 N/A N/A N/A N/A N/A water lube No 8:55 turbine 34 PX244 Duplicate 8/24/2004 350 30.9 7.9 5.3 439 N/A N/A N/A N/A N/A water lube No 8:57 turbine 35 PX244A 12/15/2004 2 34.1 8.1 6.3 444 12/15/2004 13253.2 13429.2 176 1 water lube Yes 13:03 13:30-16:30 turbine 36 PX244B 12/15/2004 13 34.3 8.1 6.1 437 12/15/04 13:30- 13253.2 13429.2 176 0.5 water lube Yes 13:04 turbine 37 PX244C 8/30/2005 <1 34.5 8.3 - 381 8/30/2005 21462.3 21668.7 206.4 1 water lube Yes 8:50 9:07-12:50 turbine 38 PX244D 8/30/2005 <1 34.8 8.3 - 378 8/30/2005 21462.3 21668.7 206.4 0.5 water lube Yes 8:51 9:07-12:50 turbine 39 PX244F 9/26/2005 <1.0 37.9 8 6.9 447 9/26/2005 13599 13677.8 78.8 1 water lube Yes 10:21 11:00-13:45 turbine 40 PX244E 9/26/2005 <1.0 37.7 8.2 6.9 457 9/26/2005 13599 13677.8 78.8 0.5 water lube Yes

58 10:20 11:00-13:45 turbine 41 PX244FF Water sample water lube Yes turbine Table 3. List of Environmental and Mock Environmental Samples (continued).

Well Site 5 42 SC85 8/25/2004 110 31.4 7.7 5.9-28.8 -5 N/A N/A N/A N/A N/A oil lube turbine No 10:45 43 SC85A 8/31/2005 7 31.2 8.4 - 365 8/31/2005 21665.4 21932.2 266.8 1 oil lube turbine Yes 6:49 7:07-11:40 44 SC85B 8/31/2005 15 31.4 8.2 - 470 8/31/2005 21665.4 21932.2 266.8 0.5 oil lube turbine Yes 6:50 7:07-11:40 45 SC85D 9/27/2005 83 31.2 8.2 7.2 478 9/27/2005 22562 22723.9 161.9 1 oil lube turbine Yes 6:35 6:55-9:45 46 SC85C 9/27/2005 32 31.1 8.4 7.4 487 9/27/2005 22562 22723.9 161.9 0.5 oil lube turbine Yes 6:34 6:55-9:45 47 SC85DD Water sample oil lube turbine Yes

Well Site 6 48 SC8 8/25/2004 110 28.4 7.3 9.4-27.8 496 N/A N/A N/A N/A N/A oil lube turbine No 10:30

59 49 SC86A 8/31/2005 10 29.2 7.8 - 611 8/31/2005 13423.3 13424 155 1 oil lube turbine Yes 7:28 7:42-12:00 50 SC86B 8/31/2005 21 29.2 7.7 - 624 8/31/2005 13423.3 13424 155 0.5 oil lube turbine Yes 7:27 7:42-12:00 51 SC86D 9/27/2005 52 29.1 7.6 10.3-11.9 616 9/27/2005 13672 13734.5 62.5 1 oil lube turbine Yes 7:19 7:35-10:00 52 SC86C 9/27/2005 19 29 7.7 8.8-10.0 617 9/27/2005 13672 13734.5 62.5 0.5 oil lube turbine Yes 7:19 7:35-10:00 53 SC86DD Water sample oil lube turbine Yes Table 3. List of Environmental and Mock Environmental Samples (continued).

Limit of Detection Samples 54 0 cells Water Sample N/A Yes

55 5 cells Water Sample N/A Yes

56 50 cells Water Sample N/A Yes

57 500 cells Water Sample N/A Yes

58 filter material rep 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

59 filter material rep 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

60 0 cells series 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

61 0 cells series 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes 60

62 0.1 cell/ml series 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

63 0.1 cell/ml series 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

64 1 cells/ml series 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

65 1 cells/ml series 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

66 10 cells/ml series 1 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes

67 10 cells/ml series 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.5 N/A Yes Table 4. Polymerase Chain Reaction Assays.

PCR Target Target Size of PCR Forward Forward Primer Reverse Reverse Primer PCR Mixture per Reaction Thermocycler Conditions Reference Number Microorganism Gene amplicon Primer Sequence Primer Sequence (bp) Name (5' → 3') Name (5' → 3')

1 General E. coli 798 16S-F AGAGTTTGAT 16S-R GGACTACCAG 5 µL of 10X Taq buffer, 5 min at 94oC for 1 cycle. 24 E. coli 16S rRNA CATGGCTCAG GGTATCTAAT 2 Units Taq DNA polymerase, 1 min at 94oC, 1 min at 53oC, o 200 µM dNTPs, 5 mM of MgCl2, and 1 min at 72 C at 30 cylces. 0.5 µM of each primer, 53oC for 5 min. and water to a final volume of 50 µL.

2 E. coli rfb O157 420 PF8 CGTGATGAT PR8 AGATTGGTTG 5 µL of 10X Taq buffer, 5 min at 94oC for 1 cycle. 24 O157:-- GTTGAGTTG GCATTACTG 2 Units of Takara rTaq DNA polymerase, 1 min at 94oC, 1 min at 53oC, o serotype 200 µM dNTPs, 5 mM of MgCl2, and 1 min at 72 C at 30 cylces. 0.5 µM of each primer, 53oC for 5 min. and water to a final volume of 50 µL.

3 E. coli fli C H7 948 1806 GCTGCAACG 1809 GGACTACCAG 5 µL of 10X Taq buffer, 5 min at 94oC for 1 cycle. 24 --:H7 GTAAGTGAT GGTATCTAAT 2 Units of Takara rTaq DNA polymerase, 1 min at 94oC, 1 min at 53oC, o serotype 200 µM dNTPs, 5 mM of MgCl2, and 1 min at 72 C at 30 cylces. 0.5 µM of each primer, 53oC for 5 min. and water to a final volume of 50 µL. 61

4 General Mp2Cl5 166 Mp2Cl5.for TCTAGAGATC Mp2Cl5.rev ATTCTATTCAC 5 µL 10X Taq DNA polymerase buffer, 5 min at 95oC for 1 cycle. 18 Naegleria CAACCAATGG TCCACAATCC 0.2 mM dNTP, 0.6 µM primer, 1 min at 95oC, 1 min at 65oC, o 2.5 mM MgCl2, and 2 min at 72 C at 35 cycles. and 2.5 U Taq DNA polymerase to a final volume of 50 µL.

5 Naegleria Mp2Cl5 110 Mp2Cl5.for-in GTACATTGTTTT Mp2Cl5.rev-in GTCTTTGTGA 5 µL 10X Taq DNA polymerase buffer, 1 min at 95oC, 1 min at 55oC, 18 fowleri TATTAATTTCC AAACATCACC 0.2 mM dNTP, 0.5 µM primer, and 1 min at 72oC at 35 cycles.

2.5 mM MgCl2, and 2.5 U Taq DNA polymerase to a final volume of 50 µL.

6 Universal 16S rDNA 918 S-D-Bact- AGAGTTTGAT S-D-Bact- CCGTCAATTC 5 µL 10X Taq buffer, 200 µM dNTP, 3 min at 94oC for 1 cycle. 31 rDNA 0008-a-S-20 CCTGGCTCAG 0926-a-S-20 CTTTRAGTTT 0.025U of Taq DNA polymerase/ µL, 45 sec at 94oC, 1 min at 55oC, o 2 mM MgCl2, and 1.5 min at 72 C. and 0.3 mM of each primer 15 min at 72oC. to a final volume of 50 µL.

7 Chemically PCR Insert ~100 bp M13F TGTAAAACG M13R CAGGAAACA 5 µL 10X Taq buffer, 0.6 mM dNTP, 5 min at 94ºC. 41 Compotent larger than ACGGCCAGT GCTATGACC 0.025U of Taq DNA polymerase/ µl, 0.5 min at 94ºC, 0.5 min at 55ºC,

E. Coli PCR Insert 0.5 mM MgCl2, and 0.5 min at 72ºC for 35 cycles. and 0.8 mM of each primer 72ºC for 7 min. to a final volume of 50 µL. Table 5. Summary of Literature Review. .

Reference Reference Name Sample H2O source Assay Type Limit of Detection Number Locaton

18 Marciano-Cabral, F. et al. (2003) Arizona Groundwater PCRA 5 intact N. fowleri in 50 mL of water

29 Reveiller, F. L. et al. (2003) France Cooling ponds and rivers ELISAB Sensitivity = 97.4% = 100 X [true positive] / [true positive + false negative]

32 Sheehan, K. B. et al. Yellowstone and Grand Hot springs PCR No limit of detection mentioned Teton National Parks

25 Pelandakis, M. and Pernin, P. France Cooling ponds and rivers Multiplex PCR DNA from as few as five trophozoites or cysts

36 Smits, H. L. and Hartskeerl, R. A. Review of PCR assays Cerebrospinal fluid or PCR No limit of detection mentioned nasal swabs

2 Behets, J. et al. Belgium Cooling water circuits FTC, ELISA, PCR 2 Naegleria fowleri per liter from power plants 62

26 Pelandakis, M., Serre, S. and Pernin, P. None Pure cultures PCR No limit of detection mentioned

37 Sparagano, O. et al. None Pure cultures PCR Equivalent DNA of 1 cell in 100 mL

23 McLaughlin, G. L. et al. Mice specimens Brain tissue PCR 3 ng DNA per 0.1 g of tissue

28 Reveiller, F. L. et al. (2002) USA-Virgina and Florida Mock environmental PCR 5 pg of N. fowleri DNA or 5 intact N. fowleri amoebae samples

30 Robinson, B. S. et al. None Pure cultures Real-Time PCR A product was amplified from extracted DNA equivalent to 0.1 to and Melting-Curve 0.2 cells. The reaction efficiency calculated from the standard Analysis curve was 0.92 for N. fowleri .

APCR = Polymerase Chain Reaction BELISA = Enzyme-Linked ImmunoSorbent Assay CFT = Flagellation Test Table 6. PCR Results for Environmental Samples.

Naegleria fowleri Sample Number Sample Name Results Negative Control Positive control Date abcabcabc 3 TON1A 11/7/2005 ------+++ 4 TON1B 11/7/2005 ------+++ 5 TON1C 10/31/2005 - + + - - - + + + 6 TON1D 10/31/2005 + + + - - - + + + 9 TON1DD 10/31/2005 + + + - - - + + + 8 TON1E 10/28/2005 ------+++ 7 TON1F 10/28/2005 ------+++ 10 TON1FF 10/28/2005 ------+++ 11 VAL1A 10/31/2005 ------+++ 15 VAL1AA 10/31/2005 ------+++ 12 VAL1B 10/31/2005 ------+++ 16 VAL1BB 10/31/2005 ------+++ 14 VAL1C 10/28/2005 ------+++ 13 VAL1D 10/28/2005 ------+++ 17 VAL1DD 10/28/2005 ------+++ 18 VAL1-X1 10/31/2005 - + ----+++ 19 VAL1-X2 10/31/2005 + -----+++ 20 VAL1-X3 10/31/2005 ------+++ 21 VAL1-X4 10/31/2005 - + ----+++ 22 VAL1-X5 10/31/2005 - - + - - - + + + 23 VAL1-X6 10/31/2005 ------+++ 26 SAF3.1A 11/7/2005 ------+++ 27 SAF3.1B 11/7/2005 ------+++ 28 SAF3.1C 10/31/2005 - - + - - - + + + 29 SAF3.1D 10/31/2005 + -----+++ 31 SAF3.1E 10/28/2005 ------+++ 30 SAF3.1F 10/28/2005 ------+++ 32 SAF3.1FF 10/28/2005 ------+++ 35 PX244A 11/7/2005 ------+++ 36 PX244B 11/7/2005 ------+++ 37 PX244C 9/7/2005 ------+++ 38 PX244D 9/11/2005 ------+++ 40 PX244E 10/28/2005 ------+++ 39 PX244F 10/28/2005 ------+++ 41 PX244FF 10/28/2005 ------+++ 43 SC85A 10/31/2005 + -----+++ 44 SC85B 10/31/2005 + -----+++ 46 SC85C 10/28/2005 ------+++ 45 SC85D 10/28/2005 ------+++ 47 SC85DD 10/28/2005 ------+++ 49 SC86A 10/31/2005 ------+++ 50 SC86B 10/31/2005 - - + - - - + + + 52 SC86C 10/28/2005 ------+++ 51 SC86D 10/28/2005 ------+++ 53 SC86DD 10/28/2005 ------+++

63 Table 6. PCR Results for Environmental Samples (continued).

E. coli 16s Sample Number Sample Name Results Negative control Positive control Date abcabcabc 3 TON1A 7/29/2005 + + + - - - + + + 4 TON1B 7/29/2005 + + + - - - + + + 5 TON1C 9/18/2005 ------+++ 6 TON1D 9/18/2005 ------+++ 9 TON1DD 9/19/2005 ------+++ 8 TON1E 9/29/2005 + + + - - - + + + 7 TON1F 9/29/2005 + + + - - - + + + 10 TON1FF 9/29/2005 ------+++ 11 VAL1A 9/18/2005 ------+++ 15 VAL1AA 9/19/2005 ------+++ 12 VAL1B 9/18/2005 ------+++ 16 VAL1BB 9/19/2005 ------+++ 14 VAL1C 9/29/2005 + + ----+++ 13 VAL1D 9/29/2005 + + + - - - + + + 17 VAL1DD 9/29/2005 ------+++ 18 VAL1-X1 9/29/2005 ------+++ 19 VAL1-X2 9/29/2005 ------+++ 20 VAL1-X3 9/29/2005 ------+++ 21 VAL1-X4 9/29/2005 ------+++ 22 VAL1-X5 9/29/2005 ------+++ 23 VAL1-X6 9/29/2005 ------+++ 26 SAF3.1A 7/29/2005 ------+++ 27 SAF3.1B 7/29/2005 ------+++ 28 SAF3.1C 9/19/2005 ------+++ 29 SAF3.1D 9/19/2005 ------+++ 31 SAF3.1E 9/27/2005 ------+++ 30 SAF3.1F 9/27/2005 + + + - - - + + + 32 SAF3.1FF 9/27/2005 ------+++ 35 PX244A 7/29/2005 ------+++ 36 PX244B 7/29/2005 ------+++ 37 PX244C 8/31/2005 ------+++ 38 PX244D 8/31/2005 ------+++ 40 PX244E 9/27/2005 + -----+++ 39 PX244F 9/27/2005 + + + - - - + + + 41 PX244FF 9/27/2005 ------+++ 43 SC85A 9/7/2005 + + + - - - + + + 44 SC85B 9/7/2005 + + + - - - + + + 46 SC85C 9/28/2005 + + + - - - + + + 45 SC85D 9/28/2005 + + + - - - + + + 47 SC85DD 9/28/2005 ------+++ 49 SC86A 9/7/2005 + + + - - - + + + 50 SC86B 9/7/2005 + + + - - - + + + 52 SC86C 9/28/2005 ------+++ 51 SC86D 9/28/2005 + + + - - - + + + 53 SC86DD 9/28/2005 ------+++

64 Table 6. PCR Results for Environmental Samples (continued).

E. coli O157:-- Sample Number Sample Name Results Negative control Positive control Date abcabcabc 3 TON1A 7/27/2005 - - + - - - + + + 4 TON1B 7/27/2005 + + + - - - + + + 5 TON1C 9/9/2005 ------+++ 6 TON1D 9/9/2005 ------+++ 9 TON1DD 9/9/2005 ------+++ 8 TON1E 9/29/2005 ------+++ 7 TON1F 9/29/2005 ------+++ 10 TON1FF 9/29/2005 ------+++ 11 VAL1A 9/9/2005 ------+++ 15 VAL1AA 9/9/2005 ------+++ 12 VAL1B 9/9/2005 ------+++ 16 VAL1BB 9/9/2005 ------+++ 14 VAL1C 9/29/2005 ------+++ 13 VAL1D 9/29/2005 ------+++ 17 VAL1DD 9/29/2005 ------+++ 18 VAL1-X1 9/29/2005 ------+++ 19 VAL1-X2 9/29/2005 ------+++ 20 VAL1-X3 9/29/2005 ------+++ 21 VAL1-X4 9/29/2005 ------+++ 22 VAL1-X5 9/29/2005 ------+++ 23 VAL1-X6 9/29/2005 ------+++ 26 SAF3.1A 7/27/2005 ------+++ 27 SAF3.1B 7/27/2005 + -----+++ 28 SAF3.1C 9/9/2005 ------+++ 29 SAF3.1D 9/9/2005 ------+++ 31 SAF3.1E 9/27/2005 ------+++ 30 SAF3.1F 9/27/2005 ------+++ 32 SAF3.1FF 9/27/2005 ------+++ 35 PX244A 7/27/2005 - - + - - - + + + 36 PX244B 7/27/2005 ------+++ 37 PX244C 8/31/2005 ------+++ 38 PX244D 8/31/2005 ------+++ 40 PX244E 9/27/2005 ------+++ 39 PX244F 9/27/2005 ------+++ 41 PX244FF 9/27/2005 ------+++ 43 SC85A 9/7/2005 ------+++ 44 SC85B 9/7/2005 ------+++ 46 SC85C 9/28/2005 ------+++ 45 SC85D 9/28/2005 ------+++ 47 SC85DD 9/28/2005 ------+++ 49 SC86A 9/7/2005 ------+++ 50 SC86B 9/7/2005 ------+++ 52 SC86C 9/28/2005 ------+++ 51 SC86D 9/28/2005 ------+++ 53 SC86DD 9/28/2005 ------+++

65 Table 6. PCR Results for Environmental Samples (continued).

E. coli --:H7 Sample Number Sample Name Results Negative control Positive control Date abcabcabc 3 TON1A 7/27/2005 ------+++ 4 TON1B 7/27/2005 ------+++ 5 TON1C 9/9/2005 ------+++ 6 TON1D 9/9/2005 ------+++ 9 TON1DD 9/9/2005 ------+++ 8 TON1E 9/29/2005 ------+++ 7 TON1F 9/29/2005 ------+++ 10 TON1FF 9/29/2005 ------+++ 11 VAL1A 9/9/2005 ------+++ 15 VAL1AA 9/9/2005 ------+++ 12 VAL1B 9/9/2005 ------+++ 16 VAL1BB 9/9/2005 ------+++ 14 VAL1C 9/29/2005 ------+++ 13 VAL1D 9/29/2005 ------+++ 17 VAL1DD 9/29/2005 ------+++ 18 VAL1-X1 9/29/2005 ------+++ 19 VAL1-X2 9/29/2005 ------+++ 20 VAL1-X3 9/29/2005 ------+++ 21 VAL1-X4 9/29/2005 ------+++ 22 VAL1-X5 9/29/2005 ------+++ 23 VAL1-X6 9/29/2005 ------+++ 26 SAF3.1A 7/27/2005 ------+++ 27 SAF3.1B 7/27/2005 ------+++ 28 SAF3.1C 9/9/2005 ------+++ 29 SAF3.1D 9/9/2005 ------+++ 31 SAF3.1E 9/27/2005 ------+++ 30 SAF3.1F 9/27/2005 ------+++ 32 SAF3.1FF 9/27/2005 ------+++ 35 PX244A 7/27/2005 ------+++ 36 PX244B 7/27/2005 ------+++ 37 PX244C 8/31/2005 ------+++ 38 PX244D 8/31/2005 ------+++ 40 PX244E 9/27/2005 ------+++ 39 PX244F 9/27/2005 ------+++ 41 PX244FF 9/27/2005 ------+++ 43 SC85A 9/7/2005 ------+++ 44 SC85B 9/7/2005 ------+++ 46 SC85C 9/28/2005 ------+++ 45 SC85D 9/28/2005 ------+++ 47 SC85DD 9/28/2005 ------+++ 49 SC86A 9/7/2005 ------+++ 50 SC86B 9/7/2005 ------+++ 52 SC86C 9/28/2005 ------+++ 51 SC86D 9/28/2005 ------+++ 53 SC86DD 9/28/2005 ------+++

66 Table 7. PCR Results for Limit of Detection Experiment.

Naegleria fowleri Sample Number Sample Name Results PCR Negative Control PCR Positive Control Date abcabcabc 54 0 cells 11/15/2005 ------+++ 55 0.1 cells 11/15/2005 - - + - - - + + + 56 1 cells 11/15/2005 + + ----+++ 57 10 cells 11/15/2005 - - + - - - + + + 58 filter material rep 1 11/16/2005 ------+++ 59 filter material rep 2 11/16/2005 ------+++ 60 0 cells series 1 11/16/2005 ------+++ 61 0 cells series 2 11/16/2005 ------+++ 62 0.1 cell/ml series 1 11/22/2005 ------+++ 63 0.1 cell/ml series 2 11/22/2005 ------+++ 64 1 cells/ml series 1 11/22/2005 ------+++ 65 1 cells/ml series 2 11/22/2005 ------+++ 66 10 cells/ml series 1 11/22/2005 + + + - - - + + +

67 67 10 cells/ml series 2 11/22/2005 + + + - - - + + + Table 7. PCR Results for Limit of Detection Experiment (continued).

E. coli 16s Sample Number Sample Name Results PCR Negative Control PCR Positive Control Date abcabcabc 54 0 cells 11/14/2005 + + + - - - + + + 55 0.1 cells 11/14/2005 + + + - - - + + + 56 1 cells 11/14/2005 + + + - - - + + + 57 10 cells 11/14/2005 + + + - - - + + + 58 filter material rep 1 8/29/2005 ------+++ 59 filter material rep 2 8/29/2005 ------+++ 60 0 cells series 1 9/2/2005 + + + - - - + + + 61 0 cells series 2 9/2/2005 + + + - - - + + + 62 0.1 cell/ml series 1 9/2/2005 + + + - - - + + + 63 0.1 cell/ml series 2 9/2/2005 + + + - - - + + + 64 1 cells/ml series 1 9/2/2005 + + + - - - + + + 65 1 cells/ml series 2 9/2/2005 + + + - - - + + + 66 10 cells/ml series 1 9/2/2005 + + + - - - + + + 68 67 10 cells/ml series 2 9/2/2005 + + + - - - + + + Table 7. PCR Results for Limit of Detection Experiment (continued).

E. coli O157:-- Sample Number Sample Name Results PCR Negative Control PCR Positive Control Date abcabcabc 54 0 cells 8/22/2005 ------+++ 55 0.1 cells 8/22/2005 ------+++ 56 1 cells 8/22/2005 ------+++ 57 10 cells 8/22/2005 ------+++ 58 filter material rep 1 8/29/2005 ------+++ 59 filter material rep 2 8/29/2005 ------+++ 60 0 cells series 1 8/22/2005 ------+++ 61 0 cells series 2 8/22/2005 ------+++ 62 0.1 cell/ml series 1 8/22/2005 ------+++ 63 0.1 cell/ml series 2 8/22/2005 ------+++ 64 1 cells/ml series 1 8/22/2005 ------+++ 65 1 cells/ml series 2 8/22/2005 ------+++ 66 10 cells/ml series 1 8/22/2005 ------+++ 69 67 10 cells/ml series 2 8/22/2005 ------+++ Table 7. PCR Results for Limit of Detection Experiment (continued).

E. coli --:H7 Sample Number Sample Name Results PCR Negative Control PCR Positive Control Date abcabcabc 54 0 cells 8/23/2005 ------+++ 55 0.1 cells 8/23/2005 ------+++ 56 1 cells 8/23/2005 ------+++ 57 10 cells 8/23/2005 ------+++ 58 filter material rep 1 8/29/2005 ------+++ 59 filter material rep 2 8/29/2005 ------+++ 60 0 cells series 1 8/23/2005 ------+++ 61 0 cells series 2 8/23/2005 ------+++ 62 0.1 cell/ml series 1 8/23/2005 ------+++ 63 0.1 cell/ml series 2 8/23/2005 ------+++ 64 1 cells/ml series 1 8/23/2005 ------+++ 65 1 cells/ml series 2 8/23/2005 ------+++ 66 10 cells/ml series 1 8/23/2005 ------+++ 70 67 10 cells/ml series 2 8/23/2005 ------+++ Table 8. Summary of PCR PositiveN. fowleri Environmental Samples.

Initial Groundwater Dissolved Final Meter Volume Sample Sample Identification Sample HPC Cond. Sample Meter Filter Size Analyzed Temperature pH Oxygen Reading Filtered Pump Type Number Name Date/Time (CFU/ml) (µS) Date/Time Reading (µm) by PCR (oC) (mg/L) (gal) (gal) (gal)

5 TON1C 9/1/2005 <1.0 47 8.4 - 968 9/1/2005 21941.8 22260.1 318.3 1 submersible Yes 7:27 7:45-13:37

6 TON1D 9/1/2005 2 48.1 8.3 - 921 9/1/2005 21941.8 22260.1 318.3 0.5 submersible Yes 7:28 7:45-13:37

28 SAF3.1C 9/1/2005 8 35.9 8.6 - 352 9/1/2005 13436 13588 152 1 submersible Yes 10:29 11:10-15:15

29 SAF3.1D 9/1/2005 1 35.6 8.6 - 343 9/1/2005 13436 13588 152 0.5 submersible Yes 10:30 11:10-15:15 71

43 SC85A 8/31/2005 7 31.2 8.4 - 365 8/31/2005 21665.4 21932.2 266.8 1 oil lube Yes 6:49 7:07-11:40 turbine

44 SC85B 8/31/2005 15 31.4 8.2 - 470 8/31/2005 21665.4 21932.2 266.8 0.5 oil lube Yes 6:50 7:07-11:40 turbine Table 9. Summary of Environmental Samples Analyzed by Full Cycle 16s rRNA Approach.

Groundwater Sample Sample Identification HPC PCR Positive Filter Size Analyzed Temperature Pump Type Number Name (CFU/ml) for N. fowleri (µm) by PCR (oC)

3 TON1A 2 43.9 No 1 submersible Yes

4 TON1B 3 44.2 No 0.5 submersible Yes

5 TON1C <1.0 47 Yes 1 submersible Yes 72

28 SAF3.1C 8 35.9 Yes 1 submersible Yes

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