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Sep 10, 2012 - Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore, DC, Qld 4558, Australia. §. School of Popula...
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Escherichia coli and Enterococcus spp. in Rainwater Tank Samples: Comparison of Culture-Based Methods and 23S rRNA Gene Quantitative PCR Assays W. Ahmed,*,†,‡ K. Richardson,†,§ 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, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichia coli and Enterococcus spp. in water samples collected from rainwater tanks in Southeast Queensland, Australia. Among the 50 rainwater tank samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively. Similarly, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively. The mean E. coli (2.49 ± 0.85) log10 and Enterococcus spp. (2.72 ± 0.32) log10 numbers as measured by qPCR assays were significantly (P < 0001) different than E. coli (0.91 ± 0.80) log10 and Enterococcus spp. (1.86 ± 0.60) log10 numbers as measured by culture-based method. Weak but significant correlations were observed between both EPA Method 1603 and the E. coli qPCR assay (r = 0.47, P = 0.0009), and EPA Method 1600 and the Enterococcus spp. qPCR assay (r = 0.42, P = 0.002). Good qualitative agreement was found between the culture-based method and the Enterococcus spp. qPCR assay in terms of detecting fecal pollution in water samples from the studied rainwater tanks. More research studies, however, are needed to shed some light on the discrepancies associated with the culture-based methods and qPCR assays for measuring fecal indicator bacteria.

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INTRODUCTION Roof-captured rainwater has been used as alternative water sources in many countries.1−3 The most significant issue in relation to roof-captured rainwater for potable and nonpotable uses is the potential health risks associated with the exposure to bacterial and protozoa pathogens.4−8 The microbiological quality of rainwater tank samples is generally assessed by monitoring Escherichia coli, which are commonly found in the gut of warm-blooded animals.9−11 Stringent guidelines have been established to determine the acceptability of roof-captured rainwater for potable uses.12−14 Most guidelines stipulate that E. coli numbers should be 0 per 100 mL of water. Culture-based methods using membrane filtration (MF) and most-probable number (MPN) techniques are most commonly used for the measurement of E. coli and Enterococcus spp. in various types of water. These methods are widely accepted because of ease of use and being relatively inexpensive.15,16 The sample processing time, however, is lengthy, ranging from 18 to 96 h, which is not practical in a situation that demands quick assessment of water quality.17 Other limitations include underestimation of the bacterial numbers due to presence of injured or stressed cells, and the fact that certain microorganisms in environmental waters can be viable but not culturable (VBNC).18,19 The recent advances in quantitative PCR (qPCR) assays enable rapid, specific, and sensitive detection of various Published 2012 by the American Chemical Society

microorganisms in environmental water samples. The assays eliminate the incubation step by directly quantifying DNA from a target microorganism and can yield results within 2−4 h.20 Quantitative PCR, however, may overestimate target numbers because it quantifies DNA from both viable and nonviable cells.21,22 Several studies have found correlations between qPCR assays and corresponding culture-based methods used for measuring fecal indicator bacteria in environmental waters.20,23−25 In contrast, a recent study demonstrated that the method relationship may vary both spatially and temporally.26 Approximately 10% of Australian people currently use roofcaptured rainwater as a major source of their drinking water, and an additional 5% use rainwater as potable replacement for showering, toilet flushing, and clothes laundering.27 In a recent study, we have reported that household tap water fed from rainwater tanks in an Ecovillage in SEQ, Australia, used for drinking appears to be of poor microbiological quality. The presence of E. coli (as measured by EPA Method 1603) and Enterococcus spp. (as measured by EPA Method 1600) along with Campylobacter spp. and Giardia lamblia (as measured by Received: Revised: Accepted: Published: 11370

June 4, 2012 September 7, 2012 September 10, 2012 September 10, 2012 dx.doi.org/10.1021/es302222b | Environ. Sci. Technol. 2012, 46, 11370−11376

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Table 1. PCR Primers and Cycling Conditions Used in This Study target E. coli (23S rRNA gene)

Enterococcus spp. (23S rRNA gene)

a

size (bp)

qPCR amplification efficiency (%)

10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C

87

102

10 min at 95 °C, 40 cycles of 15 s at 95 °C, 2 min at 60 °C

86

97

primer sequencesa (5′−3′) F: GGT AGA GCA CTG TTT TGG CA R: TGT CTC CCG TGA TAA CTT TCTC P: FAM-TCATCCCGACTTACCAACCCGTAMRA F: AGA AAT TCC AAA CGA ACT TTG R: CAG TGC TCT ACC TCC ATC ATT P: FAM-TGG TTC TCT CCG AAA TAG CTT TAG GGC TA-TAMRA

qPCR cycling param.

F: Forward primer. R: Reverse primer. P: Probe.

Enumeration of Fecal Indicators. The membrane filtration (MF) method was used to process water samples for E. coli and Enterococcus spp. numbers following EPA Method 160332 and EPA Method 1600.33 Volumes of 100, 10, and 1 mL of each water sample were filtered through 0.45 μm pore sized (47 mm diameter) nitrocellulose membranes (Millipore, Tokyo, Japan). The membranes were placed on modified mTEC agar (Difco, Detroit, MI) and membraneEnterococcus indoxyl-β-D-glucoside (mEI) agar (Difco) for the isolation of E. coli and Enterococcus spp., respectively. 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, and mEI agar plates were incubated at 41 °C for 48 h according to the manufacturer’s instructions. Fecal indicators were enumerated from 100 mL of sample when the numbers of E. coli and Enterococcus spp. were 0.99 for both qPCR assays. The mean intra-assay and interassay CV values and standard deviations, respectively, were 0.89 ± 0.17% and 3.30 ± 1.09% (for E. coli assay), 0.99% ± 0.22% and 3.07% ± 0.98 (for Enterococcus spp. assay), indicating high reproducibility (Table 2). Quantitative PCR ALOD assays were performed using purified genomic DNA isolated from pure cultures of E. coli ATCC 35150 and E. faecalis ATCC 19433 strains. To determine the reproducibility of the assays, several replicates (n = 6) were tested with the qPCR. The qPCR ALOD were as low as 1 × 101 gene copies for both indicators. For distilled Table 2. Intra-assay and Interassay Coefficient of Variation (CV) for the qPCR Assays within the Range from 5 × 106 to 5 × 102 23S rRNA Genomic Copies of E. coli and Enterococcus spp. per Microliter of DNA Extract CV (%) E. coli qPCR no. genomic copies/μL DNA extract 5 5 5 5 5 5 11372

× × × × × ×

106 105 104 103 102 101

Enterococcus spp. qPCR

intraassay

interassay

intraassay

interassay

0.73 0.79 0.81 0.87 0.96 1.20

2.10 2.40 2.82 3.42 4.10 4.98

0.79 0.84 0.87 0.92 1.15 1.35

1.90 2.24 2.70 3.20 4.00 4.40

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PCR Inhibitors. All rainwater samples (n = 50) were checked for the potential presence of PCR inhibitory substances. For the HF183 spiked distilled water DNA samples, the mean CT value was 23.0 ± 0.4. For rainwater samples (n = 50), the mean CT value was 23.9 ± 0.98 (for the HF183 spiked undiluted DNA), suggesting the samples were potentially free of PCR inhibitors. Four (i.e., sample nos. 6, 17, 24, and 38) of 50 samples, however, gave CT values >3 units higher than those of the HF183 spiked distilled water DNA samples, suggesting PCR inhibition. These four samples were 10-fold serially diluted and spiked with 103 gene copies of the HF183 marker. The CT values were then compared to the HF183 spiked distilled water DNA samples. The mean CT values for the HF183 spiked 10-fold dilutions was 23.8 ± 0.32, suggesting the samples were free of PCR inhibitors after 10-fold dilution. E. coli and Enterococcus spp. Numbers in Rainwater Tank Samples. Among the 50 samples tested, 26 (52%) and 46 (92%) samples yielded E. coli numbers as measured by EPA Method 1603 and E. coli 23S rRNA gene qPCR assay, respectively (Figure 1a). Twenty-four (48%) samples did not yield any culturable E. coli. Of these, 22 samples yielded qPCR measurable signals. In contrast, three (6%) samples (i.e., sample nos. 24, 27, 39) did not yield any E. coli qPCR measurable numbers but yielded culturable E. coli. The numbers of E. coli in measurable samples ranged between 0.40 and 3.36 log10 (EPA Method 1603) and 1.34 and 4.04 log10 (qPCR assay) per 100 mL of water. The mean numbers of E. coli in measurable samples were (0.91 ± 0.80) log10 (EPA Method 1603) and

water samples, spiked with E. coli and Enterococcus spp., the qPCR SLOD assay resulted in the detection of E. coli and Enterococcus spp. up to dilution 10−4 (Table 3). At this dilution, no culturable E. coli and Enterococcus spp. were detected, as measured by EPA methods. Table 3. Sample Limit of Detection (SLOD) of E. coli and Enterococcus spp. Spiked into Tap Water Samples E. coli tap water sample sample 1

sample 2

sample 3

Enterococcus spp.

dilutions

CFU per 100 mL

PCR positive results

CFU per 100 mL

PCR positive results

10−1 10−2 10−3 10−4 10−5 10−1 10−2 10−3 10−4 10−5 10−1 10−2 10−3 10−4 10−5

180 20 2 ND ND 187 23 3 ND ND 210 21 2 ND ND

+ + + + ND + + + + ND + + + + ND

230 25 3 ND ND 245 21 2 ND ND 250 25 1 ND ND

+ + + + ND + + + + ND + + + + ND

Figure 1. (a) Log10 numbers of E. coli per 100 mL of water as measured by EPA Method 1603 vs qPCR for rainwater tank samples. (b) Log10 numbers of Enterococcus spp. per 100 mL of water as measured by EPA Method 1600 vs qPCR for rainwater tank samples. 11373

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(2.49 ± 0.85) log10 (qPCR assay) per 100 mL of water (Figure 2). The mean numbers of E. coli as measured by qPCR assay

24) did not yield any Enterococcus spp. qPCR measurable numbers but yielded Enterococcus spp. as measured by culturebased method. The numbers of Enterococcus spp. in measurable samples ranged between 0.40 and 3.40 log10 (EPA Method 1600) and 1.60 and 4.07 log10 (qPCR assay) per 100 mL of water. The mean numbers of Enterococcus spp. in measurable samples were (1.86 ± 0.60) log10 (EPA Method 1600) and (2.72 ± 0.32) log10 (qPCR assay) per 100 mL of water (Figure 2). The mean Enterococcus spp. numbers as measured by qPCR also significantly (P < 0001) differed than culturable Enterococcus spp. numbers. Forty-six (92%) samples were in agreement with respect to the measurement of Enterococcus spp. as measured by both EPA Method 1600 and qPCR assay. A weak but significant correlation (r = 0.42, P = 0.002) was also observed between EPA Method 1600 and qPCR assay (Figure 3b). Significant correlations were also obtained for E. coli EPA Method 1603 vs Enterococcus spp. EPA Method 1600 (r = 0.49, P = 0.0002), and E. coli qPCR vs Enterococcus spp. qPCR assays (r = 0.58, P = 0.0001).

Figure 2. Mean log10 numbers of E. coli and Enterococcus spp. as measured by EPA methods (1603 for E. coli and 1600 for Enterococcus spp.) vs qPCR methods for rainwater tank samples.

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significantly (P < 0001) differed from the mean numbers of E. coli as measured by the EPA Method 1603. Twenty-three (46%) samples were in agreement with respect to the violation of a single sample standard (i.e., >0 cells per 100 mL of water) as measured by both EPA Method 1603 and qPCR assay. A weak but significant correlation (r = 0.47, P = 0.0009) was observed between EPA Method 1603 and qPCR assay despite the variability observed in their mean numbers (Figure 3a).

DISCUSSION Drinking water guidelines have been used to determine the microbiological quality of roof-captured rainwater for potable use. For most guidelines, this entails the numbers of culturable E. coli should be zero per 100 mL.12−14 It has been reported that Enterococcus spp. are more prevalent in rainwater tank samples than E. coli and thus may be a better indicator for assessing fecal contamination.4,11,31,38 The data in the present study is in agreement with our previous findings that Enterococcus spp. are more prevalent in rainwater tank samples than E. coli.4,31 The numbers of E. coli and Enterococcus spp. measured by qPCR assays were 1 to 2 orders of magnitude higher than culture-based methods for most of the water samples. Previous method comparison studies also reported high numbers of qPCR Enterococcus spp. cell equivalents compared to culture-based numbers in recreational waters.25,26,34,39 Despite the variability, weak but significant correlations were observed between culture-based methods and qPCR assays for both E. coli (r = 0.47, P = 0.0009) and Enterococcus spp. (r = 0.42, P = 0.002). The correlation coefficient value did not improve when the nondetect culturebased E. coli (r = 0.48, P = 0.012) and Enterococcus spp. (r = 0.44, P = 0.016) results were removed from the data analysis. The high numbers of E. coli and Enterococcus spp. in rainwater tank samples as determined by qPCR assays could be due to the quantification of DNA from both viable and nonviable cells. Currently, we are undertaking a study on the inactivation of E. coli and Enterococcus spp. in ambient rainwater tank condition using culture-based method and qPCR assays. One-log reduction of E. coli was achieved in 5 and 20 days as measured by culture-based method and qPCR assay, respectively (data not shown). Another sewage microcosm study reported that the numbers of culturable Enterococcus spp. fell below the detection limit within 5 days, but the qPCR signals persisted for 28 days.40 Another possible explanation for such discrepancy between culture-based methods and qPCR assays is that E. coli and Enterococcus spp. can enter VBNC state when subjected to adverse environmental conditions.19,41 The qPCR SLOD assay resulted in the detection of both indicators up to a dilution of 10−4. At this dilution, no culturable E. coli or Enterococcus spp. were detected. Such data suggest the higher sensitivity of the qPCR SLOD at least 1 order of magnitude over culture-based

Figure 3. (a) Log10 E. coli numbers as measured by EPA Method 1603 vs qPCR (r = 0.47, P = 0.0009). (b) Log Enterococcus spp. numbers as measured by EPA Method 1600 vs qPCR (r = 0.42, P = 0.002).

Among the 50 samples tested, 49 (98%) and 47 (94%) samples yielded Enterococcus spp. numbers as measured by EPA Method 1600 and Enterococcus spp. 23S rRNA gene qPCR assay, respectively (Figure 1b). One sample did not yield any culturable Enterococcus spp. but yielded qPCR measurable number. In contrast, three samples (i.e., sample no. 13, 20 and 11374

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ACKNOWLEDGMENTS This research was undertaken and funded as part of the Queensland Urban Water Security Research Alliance, a scientific collaboration between the Queensland government, CSIRO, The University of Queensland, and Griffith University. We thank residents of Southeast Queensland who provided access to their houses for collecting samples.

methods. The 23S rRNA gene qPCR assays used in this study target multiple gene copies compared to a single gene copy assay. Such assays can be advantageous for water samples where target numbers are low.42,43 In this study, samples were collected preceding dry weather when the contamination level is generally low in rainwater tank samples. Improved agreement (for E. coli) and better correlation (for both E. coli and Enterococcus spp.) can be found in samples collected immediate after rainfall events when fecal indicator bacteria are transported to the tank via roof runoff. A small number of samples did not yield any qPCR signals but yielded culturable E. coli and Enterococcus spp. Similar results have been reported in a previous method comparison study for environmental waters in California, U.S.A.;44 qPCR false negative results can be an issue because the results do not provide information on whether the water is safe for its designated use. The presence of PCR inhibitors in rainwater tank samples can be ruled out based on the spiking experiment. The DNA amount and quantity were measured using a spectrophotometer, and qPCR negative samples were amplified using a universal bacteria primer set suggesting the presence of DNA in these samples (data not shown). The numbers of culturable E. coli (1.28 to 1.33) log10 and Enterococcus spp. (0.4 to 1.0) log10 in these samples were low. It is possible that the qPCR did not pick up any signals, since 5 μL DNA was used from 200 μL extracts for the qPCR analysis. The reason for underestimation may also be attributed to growth of nontarget species on the agar plates.44 For example, EPA Method 1600 has previously been reported to have 17% to 40% false positive rates in environmental samples.45 In conclusion, good qualitative agreement was found between culture-based method and qPCR assay for Enterococcus spp. in terms of detecting fecal pollution in rainwater tank samples. Based on our results, it appears that Enterococcus spp. may be a better fecal indicator for assessing microbial contamination in rainwater tanks, although some Enterococcus spp. can be associated with plants, soil, and nonhuman animal hosts.46 In a recent study, we reported the presence of E. mundtii and E. casseiliflavus in a number of rainwater tank samples.30 These environmental associated species comprised 26% of all isolates tested and were detected along with E. faecalis and E. faecium in most of the tank water samples. The numbers of E. coli and Enterococcus spp. measured by qPCR assays were 1−2 orders of magnitude higher than numbers obtained by culture-based methods. Despite the variability observed, weak but significant correlations were found between culture-based methods and qPCR assays for both fecal indicators. More research studies are needed in order to understand the uncertainties associated with the culture-based methods and qPCR assays. For the accurate measurement of fecal indicator bacteria in rainwater tank samples, an approach such as propidium monoazide (PMA)-qPCR may be useful and will be worth investigating in comparison with culture-based and qPCR assays.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +617 3833 5582. Fax: +617 3833 5503. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 11375

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