Article pubs.acs.org/est
An Attempt to Identify the Likely Sources of Escherichia coli Harboring Toxin Genes in Rainwater Tanks 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 ‡
S Supporting Information *
ABSTRACT: In this study, 200 Escherichia coli isolates from 22 rainwater tank samples in Southeast Queensland, Australia were tested for the presence of 10 toxin genes (i.e., stx1, stx2, hlyA, ehxA, LT1, ST1, cdtB, east1, cnf1, and cvaC) associated with intestinal and extraintestinal pathotypes. Among the 22 rainwater tanks tested, 5 (28%), 7 (32%), 7 (32%), and 1 (5%) tanks contained E. coli harboring ST1, east1, cdtB, and cvaC genes, respectively. Of the 200 E. coli isolates from the 22 tanks, 43 (22%) strains from 13 (59%) tanks were harboring toxin gene. An attempt was made to establish a link between bird and possum fecal contamination and the presence of these potential clinically significant E. coli strains harboring toxin genes in rainwater tanks. Among the 214 E. coli isolates tested from birds, 30 (14%), 11 (5%) and 18 (8%) strains contained east1, cdtB, and cvaC toxin genes, respectively. Similarly, among the 214 possum E. coli isolates, 74 (35%) contained only the east1 toxin gene. All E. coli strains from rainwater tanks, bird and possum fecal samples harboring toxin genes were biochemically fingerprinted. Biochemical phenotypes (BPTs) of 14 (33%) E. coli strains from 7 rainwater tanks and 9 (21%) E. coli strains from 6 rainwater tanks were identical to a number of BPTs of E. coli strains isolated from bird and possum feces suggesting that these animals may be the sources of these E. coli in rainwater tanks. as a precautionary measure, it is recommended that rainwater should be treated prior to drinking. In addition, proper maintenance of roof and gutter hygiene and elimination of overhanging tree branches and other structures where possible to prevent the movement of possums are highly recommended.
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INTRODUCTION Roof-harvested rainwater is being used as an alternative water source for potable and nonpotable uses in many countries such as Australia, Canada, Denmark, Germany, India, Korea, New Zealand, Thailand, and the United States because it has the potential to replace significant volumes of water in and around domestic dwellings and industry if used for potable and nonpotable purposes. The most significant issue, however, in relation to roof-harvested rainwater use is the potential human health risks associated with pathogenic microorganisms found in the feces of birds, insects, mammals, and reptiles. Consequently, fecal matter from wild animals and other organic debris could be introduced to the tank via roof runoff. The microbiological quality of roof-harvested rainwater is generally assessed by monitoring fecal indicator bacteria which are commonly found in the gut of warm-blooded animals. Escherichia coli has traditionally been used as an indicator of fecal contamination in rainwater tanks.1−3 E. coli is often characterized as a harmless or commensal bacterium.4 Certain strains of E. coli, however, can be pathogenic and responsible for both intestinal and extra-intestinal infections.5,6 It has been reported that feces of some warm-blooded animals may contain high numbers of E. coli harboring virulence genes associated with intestinal and extra-intestinal infections.7 Published 2012 by the American Chemical Society
Pathogenic E. coli strains that are capable of causing diseases in humans and animals can be categorized as (I) intestinal pathogenic E. coli (InPEC) and (II) extra-intestinal pathogenic E. coli (ExPEC).8 E. coli pathotypes that are responsible for intestinal infections are known as enterotoxigenic (ETEC), enteropathogenic (EPEC), shiga-toxigenic (STEC), enteroinvasive (EIEC), enteroaggregative (EaggEC) and diffusely adherent (DAEC) E. coli.6 We have recently reported the presence of InPEC and ExPEC associated virulence genes in rainwater tanks in Southeast Queensland, Australia.9 Of the 20 virulence genes tested, eaeA, ST1, cdtB, cvaC, ibeA, kpsMT allele III, kpsMT allele K1, PAI, papAH and traT were detected in E. coli strains from 17 (77%) of the 22 rainwater tank samples. Among the 200 E. coli isolates tested, 8 (4%), 19 (9.5%), and 1 (0.5%) strains were positive for the ST1, cdtB, and cvaC toxin genes, respectively. Concerns have been expressed in terms of the presence and possible sources of these E. coli in rainwater tanks. Received: Revised: Accepted: Published: 5193
January 22, 2012 March 31, 2012 April 10, 2012 April 10, 2012 dx.doi.org/10.1021/es300292y | Environ. Sci. Technol. 2012, 46, 5193−5197
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Table 1. Escherichia coli Pathotypes and Associated Toxin Genes Tested in This Study
The primary aim of this study was to identify the likely sources of E. coli strains harboring toxin genes in rainwater tanks so that potential public health risks can be minimized. In our previous study, seven toxin genes (i.e., stx1, stx2, hlyA, LT1, ST1, cdtB, and cvaC) were tested, and in this study, the list was extended to 10 toxin genes by incorporating 3 additional toxin genes (i.e., ehxA, east1, and cnf1). E. coli isolates from bird and possum fecal samples were also tested for the toxin genes as these animals were identified as potential sources of fecal contamination. All E. coli strains isolated from bird and possum fecal samples harboring toxin genes were biochemically fingerprinted and compared with those strains found in rainwater tanks to establish a potential link with bird and possum fecal contamination.
pathotypes
toxin genes
description/function
EHEC
stx1 stx2 hlyAa exhAa LT1 ST1 cdtBa exhAa east1 cdtBa cnf1a cvaC hlyAa
shiga toxin i shiga toxin ii α-hemolysin enterohemolysin heat labile toxin 1 heat stable toxin 1 cytolethal distending toxin enterohemolysin EaggEC heat-stable enterotoxin cytolethal distending toxin cytotoxic necrotizing factor 1 colicin vV, conjugative plasmids α-hemolysin
ETEC EPEC EaggEC ExPEC
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MATERIALS AND METHODS Sources of E. coli. A total of 200 E. coli isolates were collected from 22 rainwater tanks from Brisbane and Gold Coast region in Southeast Queensland. A single water sample was collected from each rainwater tank within 3−7 days after a rain event (i.e., >80 mm). Water samples were collected in sterilized containers from the outlet tap located close to the base of the tank. Before the tank was sampled, the tap was wiped with 70% ethanol and allowed to run for 30 to 60 s to flush water from the tap.9 In addition, a total of 428 E. coli isolates were also collected from fresh fecal droppings of 38 birds (n = 214) and 40 brushtail possums (n = 214) from Brisbane and Gold Coast region. The bird species included plover (n = 3), wood duckling (n = 3), blue-faced honeyeater (n = 3), magpie (n = 3), crow (n = 4), ibis (n = 4), seagull (n = 3), topknot pigeon (n = 2), crested tern (n = 2), pacific baza (n = 3), fantail cuckoo (n = 3), rainbow lorikeet (n = 3), and tawny frogmouth (n = 2). All samples were transported to the laboratory, stored at 4 °C, and processed within 24 h. Isolation of E. coli, DNA Extraction and Confirmatory Test. The membrane filtration method was used to isolate E. coli from rainwater samples.9 Serial sample dilutions were made with phosphate buffer saline (PBS) and filtered through 0.45 μm pore sized (47 mm diameter) nitrocellulose membranes (Millipore, Tokyo, Japan) and placed on modified mTEC agar (Difco, Detroit, MI) for the isolation of E. coli. Possum and bird fecal samples were mixed with phosphate buffer saline (PBS) and streaked on modified mTEC agar plates for the isolation of E. coli. All modified mTEC agar plates were incubated at 35 °C for 2 h to recover stressed cells, followed by incubation at 44 °C for 22 h.10 All E. coli isolates were further streaked on mTEC agar plates to obtain pure colonies. Single, well-isolated colonies were picked from agar plates and inoculated into 1.5 mL screw-cap tubes containing 2 mL nutrient broth (Oxoid, Basingstoke, 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 E. coli by PCR amplification of the uidA gene as described elsewhere.11 PCR Detection of InPEC and ExPEC Toxin Genes. Confirmed E. coli isolates from rainwater tank, possum and bird fecal samples were tested for the presence of 10 E. coli toxin genes associated with intestinal and extra-intestinal diseases. The list of toxin genes and the corresponding pathotypes tested in this study is shown in Table 1. PCR detection of uidA gene,11 and toxin genes12−15 was undertaken using previously published primers and cycling parameters. PCR amplification of toxin genes was performed in 25 μL reaction mixtures using
a
Indicates genes shared by more than one E. coli pathotype.
SYBR Green iQ Supermix (Bio-Rad Laboratories, Calif). The PCR mixture contained 12.5 μL SuperMix, 300 nM of each primer, and 2 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 the Bio-Rad iQ5 (Bio-Rad Laboratories). To separate the specific product from nonspecific products, DNA melting curve analysis was performed for toxin genes. Biochemical Fingerprinting of E. coli Harboring Toxin Genes. The principle of the biochemical fingerprinting method using the PhPlate system (PhPlate AB, Stockholm, Sweden) has been previously described. 16,17 This system uses quantitative measurements of the kinetics of several biochemical reactions of bacteria in microtiter plates with dehydrated substrates.17 The typing reagents used in this method are specifically chosen for different groups of bacteria to give an optimal discriminatory power and reproducibility.17,18 For each bacterial isolate, it yielded a biochemical fingerprint made of several quantitative data which are used with the PhPlate software to calculate the level of similarity between the tested isolates. Prepared microtiter plates contained 11 different substrates in each row and allowed the testing of eight isolates per plate.In this study, PhP-RE plates were used for typing of E. coli strains. Reagents used in the PhPRE plates have been described previously.16,19 Sterile toothpicks were used to pick the pure E. coli strains from the mTEC agar plates and suspended into the first well of each row containing 350 μL of growth medium. Aliquots of 20 μL of bacterial suspension were transferred from this first well into each of the other 11 wells containing 150 μL growth medium. Plates were then incubated at 37 °C and absorbance (A620) was measured at 7, 24, and 48 h using a flatbed scanner. Data Analysis. After the final reading, the mean value for all three readings was calculated for each strain to obtain a biochemical phenotype (BPT).18 The BPTs were compared pairwise and the resulting similarity matrix was clustered according to the unweighted pair group method (UPGMA) method.20 An identity (ID) level of 0.95 was established for the system based on testing 10 strains in duplicate. BPTs showing similarity to each other above the ID-level were regarded as identical. All data handling, including optical readings, calculations of similarities among fingerprints of strains as well as clustering and printing dendrograms, was performed 5194
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Table 2. Occurrence of Escherichia coli Harbouring Toxin Genes in Rainwater Tanks, Bird, and Possum Fecal Samplesa distribution of E. coli harboring toxin genes into intestinal and extra-intestinal pathotypes (%) ETEC
a
EaggEC
EPEC/ExPEC
ExPEC
samples
no. of E. coli tested
no. of E. coli isolates harboring toxin genes (%)
ST1
east1
cdtB
cvaC
rainwater tanks birds possums
200 214 214
43 (22) 55 (23) 74 (35)
8 (4) ND ND
25 (13) 30 (14) 74 (35)
19 (10) 11 (5) ND
1 (0.5) 18 (8) ND
ND: Not detected.
using the PhPlate software version 4001 (Bactus AB, Stockholm, Sweden).
Four BPTs from the 3 rainwater tanks (i.e., T42, T48 and T49) were identical to both bird and possum BPTs. In contrast, 22 (51%) BPTs from rainwater tanks were not identical to BPTs from either birds or possums.
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RESULTS Occurrence of InPEC and ExPEC Toxin Genes in Rainwater Tanks. Among the 10 toxin genes tested, 4 genes (i.e., ST1, east1, cdtB, and cvaC) were detected in 43 of 200 E. coli strains from 13 (59%) of the 22 rainwater tank samples (Table 2). The remaining toxin genes stx1, stx2, hlyA, exhA, LT1, and cnf1 could not be detected in any of isolates tested from any of the 22 rainwater tanks. ST1 belonging to the ETEC pathotype was detected in E. coli strains from 5 (23%) of the 22 tanks. The toxin gene east1 belonging to the EaggEC was detected in strains from 7 (32%) of 22 tanks. Toxin genes cdtB and cvaC belonging to EPEC/ExPEC were detected in strains from 7 (32%) and 1 (5%) of the 22 tanks, respectively. Among the 200 isolates tested, 8 (4%), 25 (13%), 19 (10%), and 1 (0.5%) strains were positive for the ST1, east1, cdtB, and cvaC, respectively. Occurrence of InPEC and ExPEC Toxin Genes in Possum and Bird Fecal Samples. Among the 10 toxin genes tested, 3 genes (i.e., east1, cdtB, cvaC) were detected in 55 E. coli strains from 16 (42%) of the 38 bird fecal samples (Table 2). The remaining toxin genes could not be detected. Toxin gene east1 (belonging to EaggEC) cdtB (belonging to EPEC/ ExPEC) and cvaC (belonging to ExPEC) were detected in strains from 5 (13%), 3 (8%) and 11 (29%) of 38 bird fecal samples, respectively. Among the 214 isolates tested from birds, 30 (14%), 11 (5%) and 18 (8%) strains were harboring east1, cdtB, and cvaC toxin genes, respectively. E. coli toxin genes were, however, less prevalent in possum fecal samples. Only east1 toxin gene was detected in E. coli strains from 17 (43%) of the 40 possum fecal samples. The remaining toxin genes could not be detected in any of the isolates tested from possum fecal samples. Among the 214 isolates tested, 74 (35%) strains were harboring the east1 toxin gene. Biochemical Fingerprinting Analysis of E. coli Harboring Toxin Genes. A number of strains from rainwater tanks (n = 43), bird (n = 55) and possum (n = 74) fecal samples harboring toxin genes were typed with the biochemical fingerprinting method. E. coli strains harboring toxin genes in rainwater tanks were more diverse (Di = 0.92) than those found in bird (Di = 0.90) and possum (Di = 0.81) fecal samples. Cluster analysis was used to compare the BPTs of the 43 strains from rainwater tanks with those isolated from bird and possum fecal samples. Of the 43 BPTs, 14 (33%) strains from 7 rainwater tanks were identical to single or multiple BPTs from birds with 6 BPTs harboring the same virulence genes (Table 3). Similarly, of the 43 BPTs, 9 (21%) strains from 6 rainwater tanks were identical to single or multiple BPTs of possums with 4 BPTs harboring same virulence genes.
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DISCUSSION A number of E. coli strains from rainwater tanks tested in this study were positive for InPEC and ExPEC pathotypes associated toxin genes. The toxin gene east1 (25 strains from 7 tanks) and cdtB (25 strains from 7 tanks) were more prevalent among E. coli strains than ST1 (i.e., 8 strains from 5 tanks) toxin genes. The presence of these toxin genes in E. coli strains from rainwater tanks, nonetheless, can be of concern as toxins are the most obvious virulence factors in pathogenic E. coli. For example, E. coli strains harboring east1 toxin gene alone have been reported to be shown a clear association with diarrhea in Japan and Spain.21,22 Similarly, E. coli strains harboring the cdtB or ST1 toxin genes are known to cause extra-intestinal as well as intestinal infections.23,24 We acknowledge that in the absence of any in vivo study, it was not possible to determine whether strains harboring toxin genes in rainwater tanks were in fact capable of expressing pathogenicity and because of that these strains can be considered as potential pathogenic strains. Birds and possums were identified as potential sources of roof-harvested rainwater contamination. Among the 214 isolates tested from the 38 bird fecal samples, 14%, 5%, and 8% strains were indeed harboring east1, cdtB, and cvaC toxin genes, respectively. Toxin gene east1 was detected only in E. coli strains from 17 of the 40 possum fecal samples. Among the 214 isolates tested, 35% strains were harboring the east1 toxin gene. Although, toxin genes stx1, stx2, hlyA, exhA, LT1, and cnf1 could not be detected in any of the tested isolates from rainwater tanks and animal fecal samples (i.e., birds and possums), these toxin genes have been detected in E. coli isolated from avian fecal samples.25−27 The presence of ST1 gene in E. coli isolates from pigeon/geese feces has been reported.27,28 The presence of stx1, stx2, and ST1 in total DNA isolated from bird and possum fecal samples has also been reported.9 To identify the likely sources of these potential clinically significant E. coli in rainwater tanks, a source-tracking approach was undertaken. A biochemical fingerprinting method was used for typing of E. coli strains. The method has a high discriminatory ability and reproducibility and is shown to be comparable with genotypic methods in comparative studies.19,29 This method has been successfully used to identify the sources of E. coli and enterococci in environmental waters in Southeast Queensland, Australia.16 In this study, it was postulated that BPTs of E. coli strains from bird and possum fecal samples and rainwater samples harboring clinically significant toxin genes can be compared with each other to 5195
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identify the likely sources of these strains in rainwater tank samples. One important feature of such an approach is that the analysis is focused on strains carrying toxin genes rather than commensal E. coli of little significance. In addition, identical BPTs along with the presence of single or multiple toxin genes in two or more compared strains can increase the confidence level that the sources in rainwater tanks may have been correctly identified. Of the 43 strains from rainwater tank samples, 14 (from 7 tanks) and 9 (from 6 tanks) had identical BPTs to those found in bird and possum fecal samples, respectively. Five strains from 4 rainwater tanks were identical to those isolated from both bird and possum fecal samples. The remaining 22 strains could not be identified. This may be due to the fact that the numbers of bird and possum fecal samples tested in this study were not sufficient enough to capture the diversity of E. coli in rainwater tanks. This is partially supported by the fact that the E. coli BPTs were more diverse in rainwater tanks compared to those found in bird and possum fecal samples (see Supporting Information Figures S1, S2, and S3). It is also possible that a portion of these unidentified BPTs might 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 tank in the tropical atmosphere in Singapore, which may account for another potential source of unidentified BPTs in rainwater tanks in subtropical Southeast Queensland.30 While, 9 strains from 4 rainwater tanks (i.e., T15, T42, T45, and T47) had identical BPTs and similar toxin genes to those isolated from bird and possum fecal samples suggesting these animals may be the likely sources of these strains in rainwater tanks. Other strains isolated from rainwater tanks had identical BPTs to possum or birds isolates but were carrying different toxin genes. For example, strains from T10 were harboring east1 toxin gene which had identical BPTs to strains from birds which were harboring cdtB toxin gene. This could be due to the fact that some toxin genes are carried on plasmid and therefore, these genes can be lost or gained. It is also possible that strains carrying similar virulence genes have different biochemical fingerprints. The rainwater tanks that contained E. coli harboring toxin genes were surveyed. Of the 13 tanks, 12 tanks had either visible fecal droppings on the roof or overhanging trees or the both (see Supporting Information Table S1). It has been suggested that rainwater tanks should be appropriately maintained, including ensuring the cleanliness of the roofs and gutters periodically, while the receiving tanks should be cleaned at least two times per year to improve the quality of water.31 These results showing the presence of E. coli harboring toxin genes in bird and possum fecal samples demonstrates the need for good maintenance of roof and gutter and elimination of overhanging tree branches to protect potential public health risks. In conclusion, in the present study, our data suggest that the presence of potential clinically significant E. coli in rainwater tanks may have been originated from bird and possum feces. The presence of E. coli strains harboring toxin genes may pose a health risk mainly to users directly consume the rainwater. In view of this, it is recommended that rainwater should be treated with effective treatment procedures such as filtration, ultraviolet disinfection or simply boiling the water prior to drinking. Maintenance of good roof and gutter hygiene and elimination
Table 3. Comparison of Biochemical Phenotypes (BPTs) of Escherichia coli Isolates Harbouring Toxin Genes in Rainwater Tank Samples, Possum and Bird Fecal Samplesa rainwater BPTs identical to bird and possum BPTs rainwater tanks ID T1
T10
T12
T15
T33 T34
T42
T44 T45 T46 T47
BPTs ID 54d 56d 58d 7c 8c 9c 11c 13d 15d 18d 140c 148c 103c,d 105c,d 110c,d 112c,d 113c,d 114c,d 115c,d 116c,d 117c,d 119c,d 150c 154b 155c
bird BPTs ID
B-78d B-78d 9c B-89
d
13d 15d 18d B-34d B-46c, B-106c, B116d, B-123d, B-164e 148c 103c,d 105c,d 110c,d 112c,d 113c,d 114c,d 115c,d 116c,d 117c,d 119c,d 150c B-27c, B-37c B-27c, B-37c B-181e B-27c, B-37c
200b 122d
B-181e B-34d, B-43d, B-46c, B106c, B-116d, B-123d, B-164e, B-169e B-34d, B-43d, B-46c, B106c, B-116d, B-123d, B-164e, B-169e B-24c, B-32c, B-40c, B69c, B-72d
T48
137b
T49
140d 162b
n = 13
163b 165b 167b n = 43
unknown BPTs 54d 56d 58d
156c 157c 158c 159c 171d,e 185c 187c 198b 199c
123d
possum BPTs ID
P-86c, P-95c, P105c, P-115c, P139c, P-140c
159c B-78
d
B-31c, B-101c, B-102c, B-182e, B-189e
P-71c P-71c P-71c P-86c, P-95c, P105c, P-115c, P139c, P-140c P-71c
P-71c
P-181c, P-191c 163b 165b
14/43 (33%)
P-18c, P-19c 9/43 (21%)
22/43 (51%)
a
Identical BPTs and toxin genes found in isolates from rainwater tanks, possum and bird fecal samples are bolded. bST1 toxin gene. c east1 toxin gene. dcdtB toxin gene. ecvaC toxin gene. 5196
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of overhanging tree branches and other structures where possible to prevent the flocking of possums and birds should be considered for improved quality of the water.
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
* Supporting Information S
One additional table and three figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author
*CPhone: (617) 3833 5582; fax: (617) 3833 5503; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was undertaken and funded as part of the Urban Water Security Research Alliance, a scientific collaboration in Southeast Queensland, Australia, between the Queensland government, CSIRO, The University of Queensland and Griffith University. We thank residents of Southeast Queensland who provided rainwater samples. We also thank “Peter the Possum Man” and Currumbin Wildlife Sanctuary Hospital for providing possum and bird fecal samples.
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dx.doi.org/10.1021/es300292y | Environ. Sci. Technol. 2012, 46, 5193−5197
