Article pubs.acs.org/est
Speciation and Frequency of Virulence Genes of Enterococcus spp. Isolated from Rainwater Tank Samples in Southeast Queensland, Australia W. Ahmed,*,†,‡ J. P. S. Sidhu,†,‡ and S. Toze†,§ †
CSIRO Land and Water, Ecosciences Precinct, 41 Boggo Road, Brisbane 4102, Australia Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore, DC, Qld 4558, Australia § School of Population Health, University of Queensland, Herston Road, Brisbane, 4006, Australia ‡
ABSTRACT: In this study, 212 Enterococcus isolates from 23 rainwater tank samples in Southeast Queensland (SEQ), Australia were identified to the species level. The isolates were also tested for the presence of 6 virulence genes associated with Enterococcus related infections. Among the 23 rainwater tank samples, 20 (90%), 10 (44%), 7 (30%), 5 (22%), 4 (17%), 2 (9%), and 1 (4%) samples yielded E. faecalis, E. mundtii, E. casselif lavus, E. faecium, E. hirae, E. avium, and E. durans, respectively. Among the 6 virulence genes tested, gelE and efaA were most prevalent, detected in 19 (83%) and 18 (78%) of 23 rainwater tank samples, respectively. Virulence gene ace was also detected in 14 (61%) rainwater tank samples followed by AS, esp (E. faecalis variant), and cylA genes which were detected in 3 (13%), 2 (9%), and 1 (4%) samples, respectively. In all, 120 (57%) Enterococcus isolates from 20 rainwater tank samples harbored virulence genes. Among these tank water samples, Enterococcus spp. from 5 (25%) samples harbored a single virulence gene and 15 (75%) samples were harboring two or more virulence genes. The significance of these strains in terms of health implications remains to be assessed. The potential sources of these strains need to be identified for the improved management of captured rainwater quality. Finally, it is recommended that Enterococcus spp. should be used as an additional fecal indicator bacterium in conjunction with E. coli for the microbiological assessment of rainwater tanks.
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INTRODUCTION Enterococcus spp. are widely accepted as fecal indicators to determine the microbiological quality of environmental waters.1 This group of bacteria is commonly found in the gastrointestinal tracts of warm-blooded animals including humans.2,3 The presence of Enterococcus in environmental waters indicates the possible occurrence of fecal pollution from animal or human associated wastewater. Although Enterococcus spp. have been used as fecal indicators, certain Enterococcus spp. can potentially be pathogenic and have become one of the primary causes of urinary tract infections, bacteremia, and hospitalacquired infection.4,5 Among the 19 species, Enterococcus faecalis and Enterococcus faecium are highly prevalent in human wastewater and among clinical isolates.6,7 Other Enterococcus spp. such as Enterococcus durans, Enterococcus avium, Enterococcus gallinarum, and Enterococcus casselif lavus are also reported to be associated with various human infections, however at a lesser extent than E. faecalis and E. faecium.8 Treatment of Enterococcus spp. related infections has been further complicated due to their high level of intrinsic and acquired antibiotic resistance.9,10 Several virulence genes have been identified in particular with E. faecalis associated infections,11−13 and their effects have been demonstrated in animal models and cultured cells.14−16 For © 2012 American Chemical Society
example, the cyl gene, which is involved in the production of cytolysin is reported to enhance the virulence factors of E. faecalis in animal models.17,18 The aggregation substance (AS) gene is a surface protein that is encoded by sex pheromone plasmids and promotes donor and recipient cell aggregation, leading to conjugative transfer of plasmids. 19 Other surface proteins such as ace (i.e., collagen-binding protein), efaA (i.e., a cell surface protein associated with endocarditis strains), and esp (i.e., an enterococcus surface protein involved in adhesion) have been described. The role of these genes, however, in the pathogenesis of Enterococcus spp. is not clearly understood. These genes are assumed to be involved in mechanisms by which the Enterococcus cells adhere to biotic and abiotic surfaces and in biofilm formation.13,20 Another possible virulence factor is gelE gene (a metalloproteinase that targets biomolecules) which is generally associated with endocarditis strains.21 Rainwater tanks have been used as a potential source for potable and nonpotable water supplies in many countries.22−24 The microbiological quality of rainwater tanks is generally Received: Revised: Accepted: Published: 6843
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Table 1. Characteristics and End Uses of Rainwater Tanks Tested in This Study
a
tank ID
location
size (L)
age (yr)
T1
peri-urban
20,000a
2
T2
peri-urban
20,000
5
T3
peri-urban
22,500a
1
T4
peri-urban
22,500
1
T5
peri-urban
22,500a
2
T6
peri-urban
22,500a
1
T8
peri-urban
30,000a
1
T9
peri-urban
20,000a
1
T10
peri-urban
22,500a
3
T11
peri-urban
20,000a
T12
peri-urban
22,500
a
1
T13
peri-urban
10,000a
2
T14
peri-urban
20,000a
T15
peri-urban
15,000 a
presence of overhanging treesb
evidence of wildlife droppings on the roofb
N
Y
never
N
N
4 yr ago
Y
Y
never
Y
N
never
N
N
never
N
N
never
N
N
never
galvanized steel polyethylene
N
Y
never
Y
N
never
N
N
never
N
N
never
Y
N
never
2
galvanized steel galvanized steel galvanized steel polyethylene
N
N
never
3
colorbond
N
Y
1 year
3
galvanized steel galvanized steel polyethylene galvanized steel polyethylene polyethylene polyethylene polyethylene polyethylene polyethylene polyethylene galvanized steel polyethylene
N
N
never
N
Y
never
N N
Y Y
2 yr ago never
N N Y Y Y Y Y Y
Y N Y Y Y Y Y N
never 1 mo ago never 1 yr ago 1 yr ago never never 2 yr ago
N
Y
3 yr ago
T17
peri-urban
10,000
T25
peri-urban
15,000
3
T28 T29
urban peri-urban
10,000 20,000a
3 1
T30 T31 T32 T33 T34 T36 T37 T38
urban urban urban urban urban urban urban urban
5,000 5,000 20,000 10,000 5,000 5,000 5,000 22,000a
3 3 4 3 3 3 3 5
T39
urban
10,000
5
material galvanized steel galvanized steel galvanized steel galvanized steel galvanized steel galvanized steel colorbond
desludging
end uses potable, nonpotable potable potable, nonpotablec potable, nonpotable potable, nonpotable potable, nonpotable potable, nonpotablec potablec potable, nonpotable potable, nonpotablec potable, nonpotablec potable, nonpotablec potable, nonpotable potable, nonpotablec potable, nonpotable potable, nonpotable nonpotable potable, nonpotable nonpotable nonpotable potable d nonpotable nonpotable nonpotable nonpotable potable, nonpotablec potable, nonpotablec
First flush diverter installed. bY, yes; N, no. cUnder sink filtration and UV installed.
contamination in rainwater tanks, none of the studies has characterized the Enterococcus spp. detected in rainwater tank samples. In this present study, a collection of Enterococcus isolates were obtained from rainwater tank samples in Southeast Queensland, Australia. The distribution of these Enterococcus isolates to the species level and the occurrence of six virulence genes within the collection were determined. This was done to obtain information on the source and ecology of Enterococcus spp. isolated from rainwater tank samples.
assessed by monitoring fecal indicator bacteria such as Escherichia coli, and lesser extent Enterococcus spp. E. coli has traditionally been used as indicator of fecal contamination in rainwater tanks.25−27 A recent study, however, suggested that E. coli may be of limited use to assess the microbial quality of rainwater tank samples due to the fact that a number of samples yielded culturable Enterococcus spp., but no E. coli. 28 The authors suggested that rainwater tank samples should be tested for multiple indicators where possible, to obtain multiple lines of evidence on the potential occurrence of fecal contamination.28,29 Several studies also reported that Enterococcus spp. are more prevalent in rainwater tank samples than E. coli.25,27,30 and thus may be a better indicator for assessing fecal contamination. Despite increasing evidence that Enterococcus spp. are more prevalent and may be a better indicator for assessing fecal
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MATERIALS AND METHODS
Survey of Rainwater Tanks and Sampling. Twentyseven rainwater tanks were selected for this study, representing seven suburbs in Brisbane and the Gold Coast region in Southeast Queensland, Australia (Table 1). These tanks were located in peri-urban and urban areas and were selected on the
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basis of the end uses. Among the 27 tanks, 20 were used for both potable and nonpotable uses while the remaining seven were used solely for nonpotable purposes. A sanitary survey was undertaken to identify physical characteristics of the rainwater tank systems such as size of the tanks, age of the tanks, tank material, and factors that may contribute to the fecal contamination of the tanks such as the presence of overhanging trees on the roof. The roofs were also surveyed for the presence of possible wildlife fecal contamination. A single 1-L water sample was collected from each rainwater tank, within 3−7 days after a rain event (i.e., >60−80 mm over 2 days). Water samples were collected in sterilized containers from the outlet taps located close to the base of the tanks. Before the tank was sampled, the tap was wiped with 70% ethanol and allowed to run for 30−60 s to flush water from the tap and pipe. Samples were transported on ice to the laboratory, and processed within 2−4 h. Enumeration and Isolation of Enterococcus spp. The membrane filtration method was used to process the water samples for Enterococcus enumeration.31 Volumes of 100, 10, and 1 mL from each water sample were filtered through 0.45μm pore sized (47-mm diameter) nitrocellulose membranes (Millipore, Tokyo, Japan), and placed on membrane-enterococcus indoxyl-β-D-glucoside (mEI) agar (Difco, Detroit, MI) for the isolation of Enterococcus. Agar plates were incubated at 41 °C for 48 h. 31 For Enterococcus enumeration, all volumes (i.e., 100, 10, and 1 mL) from each water sample were tested in triplicate yielding a total of 9 agar plates per water sample. DNA Extraction and Confirmatory Test. Up to 10 Enterococcus isolates were selected from replicate agar plates, and further streaked on ChromoCult Enterococci Agar (Merck) to obtain pure cultures. This gave a total number of 212 Enterococcus isolates from 23 rainwater tank samples. Enterococcus could not be detected in water samples from the remaining 4 of 27 tanks. Single well-isolated colonies were picked from the agar plates and inoculated into 2-mL screw-cap tubes containing 1.5 mL of brain heart infusion (BHI) broth (Oxoid, UK). The tubes were kept in an incubator shaker at 100 rpm overnight. DNA was extracted from 1 mL of pure culture using a DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Strains were confirmed as Enterococcus by PCR amplification as described elsewhere.32 PCR Speciation and Detection of Virulence Genes. PCR confirmed Enterococcus isolates were identified to the species level using previously published primers (Table 2). PCR cycling conditions have been described elsewhere.33 Enterococcus spp. identified in this study included E. faecalis, E. faecium, E. casseilif lavus, E. durans, E. hirae, E. avium, and E. mundtii. All isolates were further tested for the presence of 6 Enterococcus associated virulence genes namely AS, ace, gelE, efaA, esp (i.e., E. faecalis variant), and cylA. Detection of virulence genes was undertaken using previously published primers (Table 2). PCR cycling conditions have been described elsewhere.34−36 PCR amplification was performed in 20 μL of reaction mixture using Sso Fast EvaGreen Supermix (Bio-Rad Laboratories, CA, USA). The PCR mixture contained 10 μL of Supermix, 300 nM each primer, DNase- and RNase-free deionized water, and 3 μL of template DNA. For each PCR experiment, corresponding positive (i.e., target DNA) and negative controls (sterile water) were included. The PCR was performed using Bio-Rad iQ5 (Bio-Rad Laboratories). To separate the specific product from nonspecific products, DNA melting curve analysis was performed for each PCR assay. To
Table 2. Primers Used in This Study for the Identification of Enterococcus to the Species Level and Virulence Genes Associated with Enterococcus spp. Isolated from Rainwater Tanks target E. faecalis
E. faecium
E. casseliflavus
E. hirae
E. durans
E. avium
E. gallinarum
E. mundtii
AS
ace
gelE
efaA
esp
cylA
a
primera F: ACT TAT GTG ACT AAC TTA ACC R: TAA TGG TGA ATC TTG GTT TGG F: GAA AAA ACA ATA GAA GAA TTAT R: TGC TTT TTT GAA TTC TTC TTTA F: TCC TGA ATT AGG TGA AAA AAC R: GCT AGT TTA CCG TCT TTA ACG F: CTT TCT GAT ATG GAT GCT GTC R: TAA ATT CTT CCT TAA ATG TTG F: CCT ACT GAT ATT AAG ACA GCG R: TAA TCC TAA GAT AGG TGT TTG F: GCT GCG ATT GAA AAA TAT CCG R: AAG CCA ATG ATC GGT GTT TTT F: TTA CTT GCT GAT TTT GAT TCG R: TGA ATT CTT CTT TGA AAT CAG F: CAG ACA TGG ATG CTA TTC CAT CT R: GCC ATG ATT TTC CAG AAG AAT F: CCA GTA ATC AGT CCA GAA ACA ACC R: TAG CTT TTT TCA TTC TTG TGT TTG TT F: AAA GTA GAA TTA GAT CCA CAC R: TCT ATC ACA TTC GGT TGCG F: AGT TCA TGT CTA TTT TCT TCAC R: CTT CAT TAT TTA CAC GTT TG F: CGT GAG AAA GAA ATG GAG GA R: CTA CTA ACA CGT CAC GAA TG F: TTA CCA AGA TGG TTC TGT AGG CAC R: CCA AGT ATA CTT AGC ATC TTT TGG F: ACT CGG GGA TTG ATA GGC R: GCT GCT AAA GCT GCG CTT
amplicon size (bp)
melting temp °C
360
83.0
215
81.5
288
84.0
187
81.9
295
83.0
368
83.9
173
83.0
98
78.5
406
81.5
320
78.5
402
80.5
499
82.0
913
82.0
688
80.5
F: Forward primer; R: Reverse primer.
minimize PCR contamination, DNA extraction, PCR setup, and gel electrophoresis were performed in separate laboratories.
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RESULTS Survey results. The size of the tanks (n = 27) surveyed ranged from 5000 to 30,000 L and they were installed 1−5 6845
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a
6846
n = 23.
T1 T2 T3 T4 T5 T8 T9 T10 T11 T13 T14 T17 T25 T28 T29 T30 T31 T33 T34 T36 T37 T38 T39 totala
tank
21 12 91 3 3 40 4 17 28 3 54 2 5 4 2 13 16 16 450 29 123 44 60
no. of Enterococcus (CFU/100 mL) 10 10 10 7 10 10 10 10 10 8 10 8 10 10 3 8 10 10 10 10 10 8 10 212
no. of Enterococcus isolated from each tank E. faecalis
27/212 (13)
12/212 (6)
6/10 (60)
115/212 (54)
1/3 (33) 3/8 (37) 3/10 (30) 3/10 (30)
5/10 (50)
5/7 (71) 4/10 (40)
E. casselif lavus
3/10 (30) 1/10 (10) 3/8 (38)
2/10 (20)
E. faecium
10/10 (100) 1/10 (10) 10/10 (100) 10/10 (100) 8/8 (100) 2/10 (20) 6/8 (75) 6/10 (60) 3/10 (30) 2/3 (66) 4/8 (50) 2/10 (20) 6/10 (60) 8/10 (80) 5/10 (50) 2/10 (20) 3/8 (38)
8/10 (80) 9/10 (90) 10/10 (100)
1/212 (0.5)
1/7 (14)
E. durans
3/10 (30) 12/212 (6)
2/10 (20) 1/10 (10)
6/10 (60)
E. hirae
1/8 (13) 1/10 (10) 2/212 (1)
E. avium
no. of Enterococcus classified into species level/no. of Enterococcus tested (%)
Table 3. Numbers of Enterococcus Isolates Tested from Rainwater Tanks and Their Distribution into Species Level
E. mundtii
28/212 (13)
7/10 (70)
2/10 (20)
3/10 (30)
2/10 (10) 1/10 (10)
8/10 (80)
2/10 (20)
1/7 (14)
1/10 (10) 1/10 (10)
others
1/8 (13) 6/10 (60) 15/212 (7)
2/10 (20)
1/8 (13)
2/8 (25)
2/10 (20)
1/10(10)
Environmental Science & Technology Article
dx.doi.org/10.1021/es300595g | Environ. Sci. Technol. 2012, 46, 6843−6850
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Table 4. Occurrence of Virulence Genes in Enterococcus Isolates from Rainwater Tanks distribution of virulence genes in Enterococcus isolates from tank water/no. of Enterococcus tested (%)
a
tank
no. of Enterococcus harboring virulence genes/no. Enterococcus tested (%)
T1 T2 T3 T4 T5 T8 T9 T10 T11 T13 T14 T17 T25 T28 T29 T30 T31 T33 T34 T36 T37 T38 T39 totala
8/10 (80) 9/10 (90) 10/10 (100) 0/7 (0) 6/10 (60) 10/10 (100) 0/10 (0) 10/10 (100) 10/10 (100) 8/8 (100) 1/10 (10) 6/8 (75) 6/10 (60) 3/10 (30) 2/3 (66) 4/8 (50) 2/10 (20) 6/10 (60) 9/10 (90) 5/10 (50) 2/10 (20) 3/8 (37.5) 0/10 (0) 120/212 (57)
AS
ace
gelE
efaA
3/10 (30)
5/10 (50) 1/10 (10) 10/10 (100)
7/10 (70) 9/10 (90) 10/10 (100)
7/10 (70) 9/10 (90) 10/10 (100)
9/10 (90)
10/10 (100)
10/10 (100)
10/10 (100) 10/10 (100) 8/8 (100) 1/10 (10) 6/8 (75) 6/10 (60) 3/10 (30) 2/3 (66) 4/8 (50) 2/10 (20) 6/10 (60) 9/10 (90) 4/10 (40) 2/10 (20) 3/8 (38)
10/10 (100) 10/10 (100) 8/8 (100)
112/212 (53)
111/212 (52)
2/10 (20) 7/10 (70)
10/10 (100) 8/8 (100) 1/8 (13) 5/10 (50) 1/10 (10)
1/10 2/10 3/10 1/10 1/10
12/212 (6)
(10) (20) (30) (10) (10)
58/212 (27)
6/8 (75) 6/10 (60) 3/10 (300) 2/3 (66) 4/8 (50) 2/10 (20) 6/10 (60) 9/10 (90) 5/10 (50) 1/10 (10) 3/8 (38)
esp
cylA
2/10 (20)
1/3 (33) 4/8 (50)
5/212 (2)
2/212 (1)
n = 23.
Table 5. Frequency of Virulence Genes Per Enterococcus Isolates in Rainwater Tanks frequency of virulence genes per Enterococcus isolate/no. of Enterococcus tested (%)
a
tank
no. of Enterococcus harboring virulence genes/no. of Enterococcus tested (%)
T1 T2 T3 T4 T5 T8 T9 T10 T11 T13 T14 T17 T25 T28 T29 T30 T31 T33 T34 T36 T37 T38 T39 totala
8/10 (80) 9/10 (90) 10/10 (100) 0/7 (0) 6/10 (60) 10/10 (100) 0/10 (0) 10/10 (100) 10/10 (100) 8/8 (100) 1/10 (10) 6/8 (75) 6/10 (60) 3/10 (30) 2/3 (66) 4/8 (50) 2/10 (20) 6/10 (60) 9/10 (90) 5/10 (50) 2/10 (20) 3/8 (38) 0/10 (0) 120/212 (57)
0
1
2
3
4
2/10 (20) 1/10 (10)
1/10 (10)
3/10 (30) 6/10 (60)
4/10 (40) 2/10 (20) 10/10 (100)
1/10 (10)
1/10 (10)
9/10 (90)
7/10 (70)
2/10 (20) 3/10 (30) 8/8 (100)
5/8 (63) 1/10 (10) 2/10 (20) 1/3 (33)
1/8 (13) 5/10 (50) 1/10 (10) 1/3 (33) 4/8 (50) 1/10 (10) 2/10 (20) 3/10 (30) 1/10 (10) 1/10 (10)
7/7 (100) 4/10 (40)
6/10 (60)
10/10 (100)
9/10 (90) 2/8 (25) 4/10 (40) 7/10 (70) 1/3 (33) 4/8 (50) 8/10 (80) 4/10 (40) 1/10 (10) 5/10 (50) 8/10 (80) 5/8 (63) 10/10 (100) 92/212 (43)
1/10 (10) 7/10 (10)
1/10 (10)
1/10 (10) 1/10 (10)
1/10 4/10 6/10 3/10
(10) (40) (60) (30)
3/8 (38) 10/212 (5)
43/212 (20)
58/212 (27)
9/212 (4)
n = 20.
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may be a better indicator for assessing fecal contamination. In this study, Enterococcus isolates were identified to the species level to obtain information on their potential sources and ecology in rainwater tank samples in Southeast Queensland. Among the 212 isolates from the 23 rainwater tanks, the predominant Enterococcus spp. identified was E. faecalis, followed by E. mundtii, E. casseilaf lavus, and E. faecium. The presence of high numbers of E. faecalis (20 out of 23 tanks) and a lesser extent E. faecium (5 out of 23 tanks) in rainwater tanks suggests the presence of fecal strains due to their high prevalence in warm blooded animals.7,37 It has been reported that E. faecalis and E. faecium are predominant in human feces and because of that several authors have proposed these two species as potential candidates for microbial source tracking (MST) studies in environmental waters.2,38,39 The presence of E. faecalis in rainwater tank samples suggests fecal contamination from wildlife or other nonpoint sources as the chance of human wastewater contaminating rainwater tanks is minimal. The high prevalence of E. faecalis in rainwater tank samples, however, suggests that these two species may be ubiquitous in nature or perhaps E. faecalis may not have limited host specificity as previously reported. 38 E. faecalis are reported to be present in the feces of animals including wildlife.40−42 During the sanitary survey, we identified wild animals such as birds, possums, and reptiles as the most likely sources of fecal contamination in rainwater tanks in Southeast Queensland as these animals have access to the roof surface. The presence of E. faecalis and E. faecium in bird fecal samples has been reported. 43 Among the 40 bird fecal samples tested, 15% and 25% samples carried E. faecalis and E. faecium, respectively. Other research studies have also reported the presence of E. faecalis in owl, seagulls, and pelicans.39,44 The species distribution of Enterococcus in possum fecal samples is not known. In this study, we did not speciate the Enterococcus spp. isolated from possum and bird fecal samples. However, we investigated the presence of E. faecalis and E. faecium in total fecal DNA isolated from a small number of possum (n = 20) and bird (n = 20) fecal samples. Among the 20 possum fecal samples tested, 3 (15%) and 2 (10%) were positive for the E. faecalis and E. faecium, respectively. Similarly, among the 20 bird fecal samples, 7 (35%) and 6 (30%) were positive for the E. faecalis and E. faecium, respectively. The prevalence of E. faecalis and E. faecium in possum and bird fecal samples indicates that these animals may have contributed Enterococcus in rainwater tanks. It is also possible that a portion of these E. faecalis and E. faecium may have originated from other sources such as rats, lizards, frogs, or fruit bats which were not tested in this study. A recent study also reported the presence of E. coli and pathogenic microorganisms in airborne particulate matter and in water samples from rainwater tanks in the tropical atmosphere in Singapore, which may account for another potential source of Enterococcus in rainwater tanks in subtropical Southeast Queensland.45 The presence of E. mundtii and E. casselif lavus in a number of rainwater tanks (n = 10, E. mundtii; n = 7, E. casselif lavus) samples is not unexpected due to their documented association with plants, soil, and nonhuman animal hosts.46 In this study, these environmental associated species comprised 26% of all isolates tested and were detected along with E. faecalis and E. faecium in most of the tanks. Such results indicate the importance of identification of Enterococcus into species level as fecal indicators. Virulence genes gelE and efaA were most prevalent in rainwater tank samples followed by ace. Among the 23 tanks, 19
years ago (Table 1). Among the 27 tanks surveyed, 10 (37%) had overhanging trees and 14 (52%) tanks had visible sign of fecal droppings on the roof. Among the 27 tanks, 14 (52%) had first flush diverter installed, 9 (33%) treated the water before consumption, and 20 (74%) were never desludged since installation (i.e., 1−5 years). Among the 27 tanks, 20 (74%) were used for both potable and nonpotable uses and the remaining 7 (26%) were used for only nonpotable purposes. Number of Enterococcus in Rainwater Tank Samples. Among the 27 rainwater tank samples tested, 23 (85%) had culturable Enterococcus spp. The numbers of Enterococcus in these samples ranged between 2 ± 1 to 450 ± 43 CFU per 100 mL of water. Eight (35%) tanks had
