Table S1. Locations, dates, water temperatures and water sources of sediment samples
Locations
AL1
AL2
AR
AZ1
AZ2
AZ3
CA1
CA2
CA3
IL
NC
NJ1
NJ2
OH1
OH2
PA1
PA2
TN
Date of sampling
6/23/2012
6/5/2013
10/15/2012
11/6/2012
1/14~1/18/2014
1/15/2014
3/15/2013
3/15/2013
1/21/2014
9/23/2013
11/9/2012
4/25/2014
4/14/2014
9/25/2012
9/26/2012
6/26/2012
1/15/2014
12/12/2012
Temperature (°C)
28.9
26.7
17.2
23.9
12.2
11.7
17.8
17.8
11.1
15.6
8.3
15.5
20.2
10.6
10.6
16.1
5.6
2.2
Source of water
surface
ground
surface
ground
ground
ground
surface
surface
surface
surface
ground
ground
ground
ground
surface
ground
ground
ground
Table S2. Sediment parameters across seven sampling sites
Particle Size
Total
Tank
Exchange
pH
%
Location % Clay
Capacity
% Silt
Sand
(ME/100g)
TOC
(%)
Organic
Matter
(%)
Source
water
TN
0.4
1.06
98.54
8.2
3
0.42
0.43
ground
NC
7.63
23.39
68.98
7.6
110.8
3.11
5.45
ground
OH1
1.36
6.7
91.94
7.1
26.7
0.25
0.89
ground
AL1
2.73
2.33
94.94
7.8
12.2
0.42
0.88
surface
AR
3.91
14.44
81.65
6.7
12.17
2.78
11.45
surface
OH2
1.68
21.67
76.65
6.6
57.34
2.09
5.9
surface
AZ1
7.35
34.34
58.31
6.7
154.14
9.42
4.08
ground
Table S3. Screening analyses and methods performed on sediment samples
Test parameter
Method
Total organic carbon
Automated instrumental analysis of carbon and
nitrogen in plant and soil samples (1) (Comparable
to EPA Method 9060A) (2).
Estimation of soil organic matter by weight,
loss on ignition (3) (Comparable to EPA
Method 160.4 (4).
ASTM D422 (5) (sieve/hydrometer)
EPA Method 9045C rev 3 (6)
Total organic matter
Particle size analysis (sand, silt, and clay)
pH
Total exchange capacity
U.S. EPA, EPA Method 9080, Cation
Exchange Capacity in Soils, Rev. 0, 1986 (7).
References
1. McGeehan, S.L., and D.V. Naylor. 1988. Automated instrumental analysis of carbon and
nitrogen in plant and soil samples. Commun. Soil Sci. Plant Anal. 19:493-505.
2. U.S. EPA, EPA Method 9060A, Total Organic Carbon, Rev. 1, November 2004.
3. Schulte, E.E., and B.G. Hopkins. 1996. Estimation of soil organic matter by weight LossOn-Ignition. P. 21-32; in: Soil Organic Matter: Analysis and interpretation. (ed.) F.R.
Magdoff, M.A. Tabatabai, and E.A. Hanlon, Jr. Special publication No. 46. Soil Sci. Soc.
Am. Madison, WI.
4. U.S. EPA, EPA Method 160.4, Volatile Residue, Rev. 1, 1971 in Methods for Chemical
Analysis of Water and Wastes, EPA/600/4-79/020, March 1983.
5. ASTM Standard D422, 1998, “Standard Test Method for Particle-Size Analysis of Soils,”
ASTM International, West Conshohocken, PA, www.astm.org.
6. U.S. EPA, EPA Method 9045C, pH in Liquid and Soils, Rev. 1, 2000.
7. U.S. EPA, EPA Method 9080, Cation Exchange Capacity in Soils, Rev. 0, 1986. 8.
Table S4. Oligonucleotide taqMan qPCR primer sequences used to screen pathogens and humanfecal indicator Bacteroidetes and PCR primer sequences for clone sequencing
Target
Human-specific
HF183
Bacteroidales
(qPCR)
Campylobacter
spp. (qPCR)
Oligo
name
Sequences (5'-3')
Tm
(°C)
HF183F
ATCATGAGTTCACATGTCCG
60
BthetP1
FAMCTGAGAGGAAGGTCCCCCACATTG
GA-TAMRA
CGTAGGAGTTTGGACCGTGT
BthetR1
CampF2
CampR2
CampP2
Campylobacter
spp. (PCR)
Legionella spp.
(qPCR)
CampF
CampR
Leg F1c
Leg R1c
Legprobe
Legionella spp.
(PCR)
LegF
LegR1
LegR3
mipF1a
L. pneumophila
(qPCR)
mipR1
mipProbe
L. pneumophila
(CYBR-Green
qPCR)
L. pneumophila
(CYBR-Green
qPCR)
L. pneumophila
(CYBR-Green
qPCR)
L. pneumophila
(CYBR-Green
qPCR)
L. pneumophila
(qPCR)
rpsLf
rpsLr
rtxAf
rtxAr
sidFf
sidFr
cegC1-F
cegC1-R
P65f
P66r
CACGTGCTACAATGGCATAT
GGCTTCATGCTCTCGAGTT
FAMCAGAGAACAATCCGAACTGGGACA
-TAMRA
GGA TGA CAC TTT TCG GAG C
CAT TGT AGC ACG TGT GTC
TAGTGGAATTTCCGGTGTA
CCAACAGCTAGTTGACATC
6FamCGGCTACCTGGCCTAATACTGATamra
GATTAGCCTGCGTCCGATTAG
GAAATTCCACTACCCTCTCCCA
AGTGTCAGTATTAGGCCAGGTAGC
AGGATAAGTTGTCTTATAGCA
TTAAGAACGTCTTTCA
TTTG
6FAMTAATCCGGAAGCAATGGCTAATamra
GAAAGCCTCGTGTGGACGTA
CAACCTTACGCATAGC
TGAGTTA
ATTGCGCCTGGCAAACTTTAGGTG
GGCGCAAATCGTTTCCACCTTGTA
ATTGTTCGCGAGGGTATGAAAGC
G
TCTTTCCAAGACAGACTCTCGCGT
TGCCTAAACGGTATGACCGCATCA
GGCATATGCACCAAAC
CACCGAAT
CAAAGGGCGTTACAGT
CAAACC
CAAACACCCCAACCGT
AATCA
Detection
limit (CE
per
reaction)
2
Reference
Bernhard and
Field (2000)
Shanks et al.
(2008)
Converse et
al. (2009)
58
1
Lund et al.
2004
60
2
Linton, 1996
50
2
This study
64
2
This study
60
2
This study
64
2
Lu, et al.
2013
64
2
Lu, et al.
2013
64
2
Lu, et al.
2013
64
2
Faucher et al.
2011
60
2
Lp-lg1,75bp,
60C, Mérault
2011
Salmonella spp.
(qPCR)
Mycobacterium
spp. (ID)
Mycobacterium
spp. (qPCR)
sg1-pb
invA_176
F
invATx_208
invA_291
R
T-39
T-13
23Smyco
F
23Smyco
R
23Smyco
Probe
FAMTCTTGGGATTGGGTTG
GGTTATTTTAACTCCTBHQ
CAACGTTTCCTGCGGT
ACTGT
FAMCTCTTTCGTCTGGCATTATCGATCA
GTACCA-TAMRA
CCCGAACGTGGCGATAATT
TGCACACAGGCCACAAGGGA
GGG GTGTGGTGTTTGAG
62
1
Bruijnesteijn
van
Coppenraet,
2004
60
2
Riviere et al.
2006
56
2
Kuiper et al.
2006
60
2
Schroeder et
al. 2001
60
2
Hadfield et al
2011
60
2
Hill et al.
2007
60
2
Guy, 2003
63
1
Qvarnstrom
et al. 2006
Vermamoeba
vermiformis
(qPCR)
Acanthamoeba
spp. (PCR)
Hv1227F
Hv1728R
JDP1
GGCCCAGATCGTTTACCGTGA A
JDP2
CRU18Sf
TCTCACAAGCTGCTAGGGAGTCA
GAG GTA GTG ACA AGA AAT AAC
AAT ACA GG
CTG CTT TAA GCA CTC TAA TTT
TCT CAA AG
6FAM-TAC GAG CTT TTT AAC TGC
AAC AA-BHQ
ATG ACG GGT AAC GGG GAA T
CCA ATT ACA AAA CCA AAA AGT
CC
CGC GCC TGC TGC CTT CCT TAG
ATG
CATCCGCGAGGAGGTCAA
JVA18S f
r
p
Giardia spp.
qPCR
Naegleria
fowleri
β-Giardin
P241 F
R
P
NaegIF19
2
NaegIR3
44
NfowlP
Talaat et al.
(1997).
CTCCCACGTCCTTCATC
TaqAcF1
TaqAcR1
TaqAcP1
P
GonzalezEscalona, N.,
et al. 2009
50
Acanthamoeba
spp. (qPCR)
r
4
GCGAACGGGTGAGTAACACG
6-carboxyfluoresceinTGGATAGTGGTTGCGAGCATCBHQ1
CGACCAGCGATTAGGAGACG
CCGACGCCAAGGACGAC
FAMTGAATACAAAACACCACCATCGGC
GC-TAMRA
TTA CGA GGT CAG GAC ACT GT
GAC CAT CCG GAG TTC TCG
Cryptosporidium
spp. (qPCR)
60
GCAGCCATGGTGTCGATCT
FAM/AAGTCCGCCGACAACATGTA
CCTAACGA/BHQ-1
GTG CTG AAA CCT AGC TAT TGT
AAC TCA GT
CAC TAG AAA AAG CAA ACC TGA
AAG G
HEX-AT AGC AAT ATA TTC AGG
GGA GCT GGG C-BHQ1
References
Bernhard A.E. and Field, K.G. (2000) A PCR assay to discriminate human and ruminant feces on
the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl Environ
Microbiol 66:4571–4574. http: //dx.doi.org/10.1128/AEM.66.10.4571-4574.2000.
Converse, R.R., Blackwood, A.D., Kirs, M., Griffith, J.F. and Noble, R.T. (2009) Rapid QPCRbased assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational
waters. Water Res 43, 4828–4837.
Lu, J., Struewing, I., Buse, H.Y., Kou, J., Shuman, H.A., Faucher, S.P. and Ashbolt, N.J. (2013)
Legionella pneumophila transcriptional response following exposure to CuO nanoparticles. Appl
Environ Microbiol 79: 2713-2720.
Gonzalez-Escalona N., et al. (2009) Detection of live Salmonella sp. cells in produce by a
TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA. Appl
Environ Microbiol 75:3714–3720.
Talaat, A.M., Reimschuessel, R. and Trucksis, M. (1997) Identification of mycobacteria
infecting fish to the species level using polymerase chain reaction and restriction enzyme
analysis. Vet Microbiol 58:229-37.
Bruijnesteijn van Coppenraet, E.S., Lindeboom, J.A., Prins, J.M., Peeters, M.F.,E. Claas, C.J.
and Kuijper, E.J. (2004) Real-time PCR assay using fine-needle aspirates and tissue biopsy
specimens for rapid diagnosis of mycobacterial lymphadenitis in children. J Clin Microbiol
42:2644
Riviere, D., Szczebara, F.M., Berjeaud, J.M., Frere, J. and Hechard, Y. (2006) Development of a
real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts. J Microbiol
Methods 64, 78–83.
Schroeder, J.M., Booton, G.C., Hay, J., Niszl, I.A., Seal, D.V., Markus, M.B.,Fuerst, P.A. and
Byers, T.J. (2001) Use of subgenic 18S ribosomal DNA PCR and sequencing for genes and
genotype identification of acanthamoebae from human with keratitis and sewage sludge. J Clin
Microbiol 39:1903–1911.
Hadfield, S.J., Robinson, G, Elwin, K., Chalmers, R.M. (2011) Detection and differentiation of
Cryptosporidium spp. in human clinical samples by use of realtime PCR. J Clin Microbiol 49:
918–924.
Hill, V.R., Kahler, A.M., Jothikumar, N., Johnson, T.B., Hahn, D., et al. (2007) Multistate
evaluation of an ultrafiltration-based procedure for simultaneous recovery of enteric microbes in
100-liter tap water samples. Appl Environ Microbiol 73: 4218–4225.
Qvarnstrom, Y., Visvesvara, G.S., Sriram, R. and da Silva, A. J. (2006). Multiplex real-time
PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and
Naegleria fowleri. J Clin Microbiol 44, 3589-3595.
Table S5. Legionella strains used to test PCR coverage
Name
ATCC #
Designation
Source
Assay
(F2/R1)
Legionella
cincinnatiensis
43753
72-OH-0
human lung tissue,
Cincinnati, OH
+
Legionella dumoffii
33279
NY23
Cooling tower, New
York
+
Legionella feeleii
35849
91-WI-H,
human lung tissue,
Wisconsin
+
Legionella gormanii
33297
LS-13
[ALLO3],
soil from creek bank,
Atlanta, GA
+
Legionella hackeliae
35250
Lansing 2
human bronchial
biopsy, Ann Arbor, MI
+
Legionella
jamestowniensis
35298
JA-26-G1-E2
wet soil, Jamestown,
NY
+
Legionella longbeacheae
33462
Long Beach 4
human lung
+
Legionella maceachernii
35300
PX-1-G2-E2
water in home
evaporator cooler,
Phoenix, AZ
+
L. micdadei [Tatlockia
micdadei]
33218
TATLOCK
blood-human (human
blood via yolk sac)
+
Legionella oakridgensis
33761
Oak Ridge 10
industrial cooling tower +
water, PA
Legionella quinlivanii
43830
1442-AUS-E
water in bus air
conditioner, Austrailia
+
Legionella rubrilucens
35304
WA-270A-C2
tap water, Los Angeles,
CA
+
Legionella sainthelensis
35248
MSH-4
spring water, Mt. St.
Helens, WA
+
Legionella wadsworthii
33877
L. pneumophila strain
Lp02
Wadsworth 81- sputum, South Carolina
716A
Dept of Health and
Environmental Control
+
n/a
+
L. pneumophila subsp.
pneumophila strain
Philadelphia-1
33152
human lung
+
L. pneumophila subsp.
pneumophila strain
Bloomington-2
33155
creek water
+
L. pneumophila subsp.
fraseri strain Dallas 1E
33216
cooling tower
+
L. pneumophila; subsp.
pneumophila strain
Chicago 2
33215
human lung biopsy
+
Table S6 Bacterial strains used to check the specificity of the PCR and qPCR assays for Legionella
Strains
Source
Strains
Source
Acinetobacter baumannii
ATCC 19606
Micrococcus luteus
ATCC 10240
Aeromonas hydrophila
ATCC 7966T
Proteus mirabilis
ATCC 12453
Aeromonas hydrophila
ATCC 7966
Proteus vulgaris
ATCC 29905
Aeromonas cavae
ATCC 15468
Pseudomonas aeruginosa
ATCC 10145
Bacillus cereus
ATCC 10876
Salmonella enteriditis
ATCC 13076
Burkholderia cepacia
ATCC 25416
Serratia marcescens
ATCC 14756
Burkholderia cepacia
ATCC 25416
Serratia marcescens
ATCC 13880
Campylobacter jejuni
ATCC 29428
Shewanella putrefaciens
ATCC 49138
Citrobacter freundii
ATCC 4391
Shigella sonnei
ATCC 25931
Citrobacter freundii
ATCC 8090
Shigella sonnei
ATCC 9290
Enterobacter aerogenes
ATCC 13048
Staphylococcus aureus
ATCC 25923
Enterobacter aerogenes
ATCC 13048
Staphylococcus aureus
ATCC 25923
Enterobacter cloacae
ATCC 13047
Streptococcus pyogenes
ATCC 19615
Enterococcus faecalis
ATCC 29302
Yersinia enterocolitica
ATCC 23715
Enterococcus faecalis
ATCC 19433
Escherichia coli
EPA isolate 8
Enterococcus faecium
ATCC 19434
E. coli
EPA isolate AD#1
Lactobacillus acidophilus
ATCC 314
E. coli
EPA isolate 58
Klebsiella oxytoca
ATCC 13182
E. coli
EPA isolate 59
Klebsiella pneumoniae
ATCC 13882
E. coli
EPA isolate 62
Klebsiella pneumoniae
ATCC 31488
Legionella pneumophila
strain Lp02
33152
Listeria monocytogenes
Scott O2
L. pneumophila subsp.
pneumophila strain
Philadelphia-1
Table S7. Total clone numbers, unique OTUs and dominant sequences in those sediments
showing positive for Legionella spp.
Site
AL1
Total
clone
67
OTU#
6
AZ1
23
7
AZ2
24
4
IL
NC
NJ1
58
25
62
16
12
36
NJ2
36
9
OH2
17
13
PA2
TN
9
15
6
6
Dominant
L impletisoli str OA1 1 (AB233209)
unknown
L impletisoli str OA1 1 (AB233209)
unknown
L impletisoli str OA1 1 (AB233209)
L adelaidensis (Z49716)
unknown
unknown
unknown
L pneumophila subsp pneumophila str Philadelphi
L anisa strain
L rubrilucens
L rubrilucens
L brunensis
%
22
72
30
26
29
21
17
93
64
2
31
42
64
11
L impletisoli
L cincinnatiensis
L brunensis
S. lyticum
L pneumophila subsp pneumophila str Philadelphi
18
12
18
89
100