Science of the Total Environment 458–460 (2013) 298–302

Contents lists available at SciVerse ScienceDirect

Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Assessment of pathogenic bacteria in treated graywater and irrigated soils

Maya Benami, Amit Gross ⁎, Moshe Herzberg, Ezra Orlofsky, Ahuva Vonshak, Osnat Gillor
Zuckerberg Institute for Water Research, Jacob Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben Gurion 84990, Israel

H I G H L I G H T S

• qPCR detected high amounts of pathogens in biologically treated GW.

• Comparable pathogen types were found in treated GW, and GW- and FW-irrigated soils.
• Treated GW irrigation has no effect on soil pathogen/indicator diversity or abundance.

a r t i c l e i n f o a b s t r a c t

Article history: Reuse of graywater (GW) for irrigation is recognized as a sustainable solution for water conservation. One
Received 19 February 2013 major impediment for reuse of GW is the possible presence of pathogenic microorganisms. The presence
Received in revised form 7 April 2013 and abundance of six pathogens and indicators were investigated in three GW recirculating vertical flow
Accepted 7 April 2013
constructed wetland treatment systems and their respective irrigated yard soils. The treated GW and soils
Available online 10 May 2013
were monitored once every two months for six months using real-time quantitative PCR. As a control,
Keywords:
samples from four soils irrigated with fresh water (FW) were similarly analyzed for pathogens and indicators.
Graywater Comparable types of pathogens and fecal indicator bacteria, including Escherichia coli, Klebsiella pneumoniae,
Soil Salmonella enterica, Pseudomonas aeruginosa, Enterococcus faecalis, and Shigella spp., were found in the
Pathogen treated GW, their corresponding irrigated soils and the FW-irrigated soils. Moreover, the abundance of
qPCR these bacteria in the GW- and FW-irrigated soils was of the same order of magnitude, suggesting that the
Irrigation source of the pathogens cannot be established. Our results suggest that GW irrigation has no effect on the
diversity and abundance of the tested pathogens and indicators in yard soils.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction Staphylococcus aureus, and Pseudomonas aeruginosa (Winward et al.,

Water scarcity calls for large-scale conservation and reuse. Combined
with an increasing population, the demand for water has prompted a
growing search for additional water sources. One such source is the
reuse of graywater (GW), which can account for as much as 50 to 80%
of the total water usage in a household (Christova-Boal et al., 1996).
GW is the non-toilet portion of domestic effluent, from bathing,
laundry, and kitchen use (Friedler et al., 2005). GW is often considered
a fairly safe water resource, and untreated GW is allowed for
constrained local reuse as irrigation water in several parts of Australia
and the USA. Other countries only allow the use of GW that complies
with certain treatment requirements, and a few ban its use entirely,
mainly due to public health concerns (Finley et al., 2009; Maimon et
al., 2010). GW might contain pathogens, such as fecal indicator bacteria
and opportunistic pathogens including fecal coliforms, Escherichia coli,
2008). Interestingly, there are still only limited data on the array of
pathogenic bacteria in treated GW (Birks and Hill, 2007; Winward et
al., 2008). Moreover, evaluations of the impact and associated risks, in
terms of viability and persistence of human pathogens in soil irrigated
with GW, have been neglected.
The principal aim of this study was to monitor and quantify an array
of bacterial pathogens and indicators in GW and the corresponding
irrigated soils after several years of treated-GW irrigation. We hypothe-
sized that there would be a correlation between pathogens found in GW
and the corresponding irrigated soil, and that FW-irrigated soils would
support a dissimilar array and concentrations of bacterial pathogens
and indicators.
2. Materials and methods
2.1. Field sites and sampling approach

⁎ Corresponding author at: Department of Environmental Hydrology & Microbiology,
Zuckerberg Institute for Water Research, Blaustein Institutes for Desert Research, Ben
Gurion University of the Negev, Sede Boqer Campus, Midreshet Ben Gurion 84990, Israel.
Tel.: +972 8 6596896; fax: +972 8 6596909.
E-mail address: amgross@exchange.bgu.ac.il (A. Gross).
Treated GW (excluding kitchen effluents) from recirculating vertical
flow constructed wetlands (RVFCW) (Gross et al., 2007) and corre-
sponding irrigated soil samples were taken once every two months
from December 2010 to May 2011 from three private domestic

0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.scitotenv.2013.04.023
 M. Benami et al. / Science of the Total Environment 458–460 (2013) 298–302 299

Table 1
Physical properties of the soils sampled in this study.
Region Soil Organic Bulk Porosity Particle size analysis
type carbon density % %
% g cm−3
Sand Silt
Sharon Hamra 0.54 1.24 49 87 7 6
loam
Negev Loess 0.27 1.20 55 64.3 15.6
desert
residences. Two of the residences were located in the Negev region
(desert) and the third was located in the Sharon region (Mediterranean
coastal plain), Israel. All residences housed adults and school-aged
children. From each household, 100 L of treated GW was collected in
four sterile 25-L plastic containers. Samples were delivered to the
laboratory and stored at 4 °C. GW was usually processed on the day of
collection and no more than 48 h after sampling.
The studied soils were Hamra loam in the Mediterranean Sharon
region and loess in the Negev region (Table 1). The FW-irrigated
soils (controls) were also loess, collected from households near the
GW irrigated households. One household was used as a principal
control (i.e. soil irrigated with FW) and monitored throughout the
experiment just like the treatment households (soils irrigated with
GW). To validate our results, we tested three additional control
households (soil irrigated with FW) at a single time point. The
physical properties of both GW and FW irrigated soils were similar.
It should be noted that all households sampled in this study did not
include infants, nor housed yard pets, and all were sampled at the
same time intervals.
The site in the Sharon region and one of the sites in the Negev
desert were drip-irrigated with GW for five years. The second desert
site was drip-irrigated with GW for over two years. The control sites
were drip-irrigated with FW for more than 10 years. Soil samples
were collected from each yard by randomly selecting five sampling
points next to the drippers. Each sample was collected by removing
approximately 100 g of soil at 0–5 cm depth with a sterile scoop,
after brushing away the soil crust and loose litter. The samples were
placed in individual sterile plastic bags (Whirl-Pak, Modesto, CA),
transported to the laboratory, stored at 4 °C and processed within
24 h. In the laboratory, samples were composited, homogenized
while removing plant debris and separated into subsamples for
chemical and microbial analyses. Samples for microbial analysis
were stored at − 80 °C and for chemical analysis they were dried at
65 °C.
and analyzed by the second derivative method for nitrate and Nessler
method for ammonium (APHA, 1998).
Surface
area 2.3. Microbial analyses of GW and soils
Clay m2 g−1
The use of an ultrafiltration (UF) membrane to concentrate multi-
38
ple microorganisms simultaneously from large water sample volumes
20 75 has been shown to be effective (Francy et al., 2009; Hill et al., 2007).
Hence the GW samples were concentrated directly from about 100 L
to approximately 100 mL using a disposable F200NR UF filter
(Fresenius Medical, Bad Homborg, Germany) based on protocols de-
scribed in Leskinen and Lim (2008) (see Supplementary information:
Figs. A.1 and A.2).
2.3.1. DNA and plasmid extractions
Genomic DNA was extracted from pure bacterial cultures using
the Accuprep Genomic DNA Extraction Kit (Bioneer, Seoul, South
Korea) according to the manufacturer's protocol. The primers used
for plasmid cloning are listed in Supplementary information Table
A.1. All plasmids were cloned according to the manufacturer's proto-
col using the InstaClone PCR cloning kit (Fermentas, Burlington, ON,
Canada) and extracted using the Accuprep plasmid mini extraction
kit (Bioneer).
DNA was extracted from the concentrated GW pellets and the soil
samples using PowerSoil DNA extraction kit (MoBio, Carlsbad, CA)
according to the manufacturer's instructions. DNA extraction from
environmental samples is notorious for sample variability and bias
of extraction toward specific microbial groups (Feinstein et al.,
2009). Yet, no optimal method has been found to date and so we
chose a kit that is known for its reproducibility, elimination of inhib-
itors (such as humic acids) and popularity among soil researchers
(Lauber et al., 2008; Ning et al., 2009; Lu et al., 2011). Extractions
were analyzed for purity and quantity in a Nanodrop 2000 spectro-
photometer (Thermo Scientific, Wilmington, DE). All DNA extracts
were stored at − 80 °C before further analysis by real-time quantita-
tive PCR (qPCR).
2.3.2. Reference bacteria
The reference bacteria listed in Supplementary information Table
A.1 were cultured in Luria–Bertani (LB) broth and agar (BD-Difco,
Sparks, MD) for E. coli, Klebsiella pneumoniae, Salmonella enterica, Shi-
gella spp., and P. aeruginosa. Brain heart infusion (BHI) (Sigma, St
Louis, MO) broth and agar plus 20% (w/v) dextrose was used for En-
terococcus faecalis. All bacteria were grown overnight (12–15 h) in a
shaking incubator (New Brunswick Scientific, Edison, NJ) at 37 °C
and 250 rpm.

2.2. Physicochemical characterization of GW and soils 2.3.3. Real-time qPCR

qPCR was performed for a suite of pathogens (see Supplementary
Treated GW samples were analyzed for turbidity, electrical con- information: Table A.1). Plasmid DNA and genomic DNA of pure cul-
ductivity (EC), optical density (OD254) and pH by respective meters, tures were used as a reference for calibrating the bacterial quantities
total suspended solids (TSS) by the gravimetric method, biological (Loddenkötter et al., 2004; Shanks et al., 2009). The extracted DNA
oxygen demand (BOD) by standard BOD bottles, total organic
carbon (TOC) by TOC analyzer (Tekmar, Mason, OH), total nitrogen
Table 2
(N) and total phosphorus (P) by persulfate digestion followed by
Treated GW quality data. Results are average ± SD in mg L−1 unless otherwise stated.
vanadomolybdate analysis for phosphorus and UV analysis for
nitrogen. Analyses followed standard procedures (APHA, 1998). Soil Parameter Negev 1 Negev 2 Sharon
analyses followed the procedures outlined by the Soil and Plant Turbidity (NTU) 3.5 ± 0.81 3.4 ± 1.7 2.4 ± 1.12
Analysis Council (1999), including water content, saturation, EC and TSS 7.03 ± 8.23 4.03 ± 2.2 2.7 ± 0.36
pH by respective meters in a 1:1 soil-to-water ratio. Cations (Na, K, BOD5 1.4 ± 0.7 1 ± 0.7 1.6 ± 0.3
COD 24.3 ± 13.07 23.5 ± 17.1 17.3 ± 9.2
P) were extracted by the double acid solution followed by inductively Total P 1 ± 0.4 2 ± 2.3 1 ± 1.5
coupled plasma (ICP) analysis (model 720-ES, Varian Inc., Melbourne, Total N 6.6 ± 3.3 19.1 ± 13.3 11.5 ± 10.8
Australia) and the sodium adsorption ratio (SAR) was calculated. Total B 0.5 ± 0.02 0.5 ± 0.04 0.2 ± 0.06
Percent organic matter was analyzed by the dichromate oxidation UV (OD254) 0.1 ± 0.03 0.2 ± 0.04 0.1 ± 0.02
EC 1.0 ± 0.1 1.0 ± 0.2 1.9 ± 0.3
method. For nitrate and ammonium analyses, soils were extracted
pH 8.2 ± 0.5 8.1 ± 0.3 8.2 ± 0.4
in 2 M potassium chloride (KCl) and the supernatant was filtered
300 M. Benami et al. / Science of the Total Environment 458–460 (2013) 298–302

Table 3 3. Results
Soil characteristics and quality. Results are average ± SD in mg kg−1 unless otherwise
stated.
3.1. GW and soil characteristics
Parameter GW-irrigated soil FW-irrigated
soil The treated GW was fairly uniform throughout the study with low
Loess Loess Hamra Loess average levels of total suspended solids and turbidity, biological oxy-
(Negev 1) (Negev 2) (Sharon) (Negev) gen demand of less than 10 mg L −1, chemical oxygen demand
Saturation point (%) 39 ± 4.2 39 ± 8.5 34.5 ± 2.12 N/A around 20 mg L −1, total phosphorus between 1 and 2 mg L −1, total
pH 7.5 ± 0.2 7.3 ± 0.1 7.2 ± 0.2 7.5 ± 0.12 nitrogen averaging 6.6 mg L −1, total boron b 0.5 mg L −1, UV absorp-
EC (dS m−1) 3.2 ± 1.7 4.1 ± 6.2 2.8 ± 1.6 2.1 ± 0.7 tion ratio ranging between 0.1 and 0.2, electrical conductivity under
Na (mg kg−1) 263 ± 67 430 ± 524 151 ± 2.6 N/A
SAR ratio 6.4 ± 1.3 7.3 ± 7.2 3.8 ± 5.5 N/A
2 dS m −1, and pH of 8.2 (Table 2).
Nitrate (mg kg−1) 8.0 ± 2.2 44.1 ± 48 52.6 ± 54.2 51.2 ± 65.7 Soil parameters were typical (Travis et al., 2010) except for some-
Ammonium (mg kg−1) 14.2 ± 6 30.8 ± 12.7 14.7 ± 10.4 61.3 ± 40.7 what elevated average phosphorus levels in the Negev 1 site
P (mg kg−1) 148 ± 53 495 ± 177 98.2 ± 35.1 80.8 ± 50.4 (Table 3). A saturation point of less than 40% was recorded for all
Soluble K 30.1 ± 12.2 30 ± 12.3 44.9 ± 28.4 41.2 ± 26.6
soils. The average concentration of sodium was 281 mg kg −1 but
varied within a large range and corresponded to an average sodium
absorption ratio of 6. All sampled locations had a fairly neutral pH
and electrical conductivity ranged between 2.1 and 4.1 dS m −1.
was analyzed with the primers and probes listed in Supplementary Inorganic nitrogen (i.e. nitrate and ammonium) averaged from 8 to
information Table A.1. To determine the detection sensitivity of the 61 mg kg −1, potassium was fairly similar in all soils averaging
qPCR assay, preliminary amplification and cycle threshold (CT) tests 36.5 mg kg −1, and phosphorus varied between samples (Table 3).
were performed with a series of four 10-fold dilutions of each of the
reference bacterial genomic DNA (starting at 1 pg per reaction to 1 fg 3.2. Pathogens
per reaction). The highest detection sensitivity of our qPCR assay was de-
termined to be in the region of 1 fg for each bacterium tested using pure P. aeruginosa and E. faecalis were detected in all GW samples,
plasmid genomic DNA, corresponding to approximately 300 copies of a while E. coli, K. pneumoniae, Shigella spp. and S. enterica were not
given gene. A standard curve was generated for each reaction and used detected in at least one of the samples. Bacterial quantities averaged
to estimate the number of species that could be detected in each sample's from 1 to 3 log10 gene copies per 100 mL by the qPCR method, where-
extracted DNA. Bacteria were quantified in pathogenic gene copies, with as there was a log difference lower between the E. coli quantities
one gene copy representing one bacterium. detected by qPCR and by traditional plating methods (Fig. 1).
Multiplex qPCR was performed with fluorescent probes It is important to note that recoveries of the seeded bacteria from
(Metabion, Martinsried, Germany) and was conducted in at least the UF membranes as detected by qPCR were less than 100% and
three independent replicate assays, each performed in triplicate. The slightly variable among the seeded bacteria. The UF membranes
overall PCR product size was smaller than 120 bases. As different concentrated S. enterica, E. coli, and E. faecalis to the same log10
probe dyes could be detected at distinct wavelengths, we confirmed magnitude, whereas K. pneumoniae, Shigella spp., and P. aeruginosa
that the copy numbers of each strain (see Supplementary informa- were recovered within 1 log10 magnitude of the original spiking
tion: Table A.1) matched and were similar in single and multiplex re- amount after 100 × concentration. The recovery efficiencies reported
actions (data not shown). here coincide with numerous previous studies (e.g. Francy et al.,
The following reaction mix was used for each reaction: 5 μL tem- 2009; Hill et al., 2007; Knappett et al., 2011). We consider these
plate genomic DNA (from 4 ng μL −1), 0.8 μL bovine serum albumin numbers to indicate that most of the bacteria were recovered within
(BSA), 0.62 μL of 5 μM of each set of primers and probe, 12.5 μL Abso- 1 log10 magnitude and little was lost to the UF concentration process.
lute Blue qPCR mix (Thermo Scientific) and 4.84 μL DDW, to a final vol- Therefore, since the recoveries of bacterial DNA copies in the GW
ume of 25 μL. All qPCRs were executed in a real-time PCR machine were always less than 100%, we refer to the qPCR results as underes-
(Corbett Research Rotor Gene 6000, Thermo Scientific) following en- timation of the overall bacterial quantities (Table 4).
zyme activation at 95 °C for 15 min, 40 cycles of denaturation at 95 °C Interestingly, the pathogen and indicator diversity in the GW was
for 10 s, annealing at 55 °C for 15 s, and extension at 60 °C for 45 s. similar to the one found in both irrigated GW and FW irrigated soils.
To account for recovery efficiency in the GW samples, we seeded a Moreover, similar quantities were observed, averaging 3–6 log10 gene
known amount of each of the bacteria, 1 mL of 0.6 OD600 (approximate- copies per 1 g soil. P. aeruginosa and E. faecalis were detected in all
ly 108–109 cells), in 10 L of simulated GW (Travis et al., 2010) in a man- soil samples, while Shigella spp., E. coli, K. pneumoniae and S. enterica
ner identical to the prospective GW samples prior to the concentration were found in some soil samples. Like in the GW samples, qPCR
step. qPCR was applied following seeding and GW concentration to analysis of the soils consistently revealed over a log difference higher
determine recovery of each bacterium (Table 4). The number of quantity of E. coli than the plating method (Fig. 2).
organisms found by qPCR in seeded retentates was determined from
standard curves and then normalized to gene copies per 100 mL. 4. Discussion

Many studies have shown that biological treatment of GW is es-

2.4. Statistical analysis
Results of microbial quantities in the GW and soils were plotted in
box plots demonstrating means, medians, and standard deviations
using SigmaPlot2000 software (Systat Software Inc., Erkrath,
Germany). A comparison of culture-dependent and independent
methods for E. coli along with comparisons between each microbe's
quantities in GW- and FW-irrigated soils were performed by t-test
(pb0.05) using STATISTICA software (Statsoft, Tulsa, OK) and
Sigmaplot 2000.
sential to lessening the contaminant load of raw GW for reuse appli-
cations other than potable water (Birks and Hill, 2007; Nolde, 2000).
In this study, we confirmed that GW quality after treatment in
RVFCW (Table 2) meets the standards of “very high quality water”
(Halperin and Aloni, 2003) and can be used for unlimited irrigation
except for microbial gene copies, where gene copies correlate with
microbial presence (when validating microbial gene copies using
qPCR). These results were similar across households and seasons,
and they also concurred with previous reports on the performance
of the RVFCW (Gross et al., 2007; Travis et al., 2010), demonstrating
 M. Benami et al. / Science of the Total Environment 458–460 (2013) 298–302 301

8
8 A
7
Log10 Gene Copies per 100 mL

Log10 Gene Copies per 1 g

7 Median
Mean 6
6
5
5
4
4
3
3
2
2
1
1
0
0

p. 8
sa ica ali
s
sp iae oli ure B
gino nter a ec l la m on E . c cult
f e i
eru S.
e
E. ig eu ol 7
a Sh pn E.c

Log10 Gene Copies per 1 g

P. K.
6
Bacteria
5
Fig. 1. Detection of pathogenic bacterial gene copies in treated GW. The E. coli culture 4
(last column on the right) was analyzed by a culture-dependent method and reported
in log10 CFU 100 mL–1. 3

2
the robustness of the GW treatment. Measured parameters of the soil 1
irrigated with the treated GW did not differ between sites (p>0.05; Median
0 Mean
Table 3). Similarly, previous studies that analyzed soils irrigated
with treated GW (Al-Hamaiedeh and Bino, 2010; Travis et al., 2010)
suggested that a GW irrigation regime may have little, if any, effect sa a ali
s p. iae co
li re
ino ric ec sp on ltu
g nte fa e lla m E. cu
on the soil. aeru S.
e
E. hig eu .co
li
P. S pn E
The tested GW contained an array of pathogens at high concentra- K.
tions (Fig. 1). Similar pathogenic and indicator bacteria were detected
Bacteria
in the GW- and FW-irrigated soils and a t-test revealed no statistical
difference between these same detected bacteria in the two soils Fig. 2. Detection of pathogenic bacterial gene copies in soils irrigated with (A) treated
(p>0.05). The bacterial quantities of E. coli detected by both plating GW and (B) FW. The E. coli culture (last column on the right) was analyzed by a
and qPCR methods (Fig. 2) in both soils were also similar, although culture-dependent method and reported in log10 CFU g–1.

there was a statistically significant difference in quantification

between the detection methods (pb0.05). broad range of environments (it has been found in soil, water, and
The GW, and GW- and FW-irrigated soils contained large amounts plants) and under various pH and temperature conditions (Gilmore,
of P. aeruginosa and E. faecalis (Figs. 1 and 2). P. aeruginosa is an 2002). Specifically, E. faecalis strains are frequently isolated from
opportunistic pathogen that attaches to the skin and mucous mem- environmental waters (He and Jiang, 2005). Yet the presence of this
branes, and not surprisingly, is found in significant quantities in GW fecal indicator has never been tested in treated GW or GW-irrigated
from washing the human body. Other studies also confirmed the soils. Here we demonstrate that both P. aeruginosa and E. faecalis are
presence of P. aeruginosa in most of their GW samplings (Friedler ubiquitous in GW, and in GW- and FW-irrigated soils.
and Gilboa, 2010; Winward et al., 2008), although no studies checked The pathogens S. enterica and Shigella spp. were found in some of
its occurrence in GW- or FW-irrigated soils. E. faecalis is a resilient, the GW, and in the soils irrigated with GW and FW. Both pathogens
facultative anaerobe, a gram-positive coccus that can survive in a have been previously reported in GW samples (Kotut et al., 2011;
Rose, 1991). Similarly, they have been detected in treated wastewater
Table 4 and found to vary depending on the environmental conditions, time
Recovery measurements from seeding experiments using the UF units and ×100 con- of sampling, and the type of contaminants entering the system
centrated. Results are in log10 ± SD.
(Shannon et al., 2007). Some amounts of S. enterica and Shigella spp.
Bacteria Initial seed Expected recovery Actual recovery Difference might contaminate the GW system as fecal remnants, but their pres-
quantified amount after ×100 after ×100 (CFU 100 ence in GW- and especially FW-irrigated soils is puzzling. They may
by qPCR detected by concentration concentration mL−1)
have been introduced into the soil via the fecal pellets of animals
qPCR (CFU 100 mL−1) (CFU 100 mL−1)
(CFU 100
hosted in the household yards (Barak and Liang, 2008). K.
mL−1) pneumoniae, a thermotolerant coliform bacterium, was detected in
fewer samples of GW, and GW- and FW-irrigated soils. As the fecal in-
Salmonella 104 106 106 ± 0.9 ~Full
enterica recovery dicators E. coli and E. faecalis, E. faecalis were found in large amounts
Escherichia coli 10 5
10 7 7
10 ± 0.8 ~Full in the tested matrices, encountering K. pneumoniae—a common coli-
recovery form indicator organism—was not unexpected in the GW, and GW-
Enterococcus 103 105 105 ± 0.7 ~Full
and FW-irrigated soils, and it could also have been introduced into
faecalis recovery
Shigella spp. 104 106 107 ± 0.1 1 log the yard environment through fecal remnants (Shannon et al., 2007).
Pseudomonas 103 105 104 ± 0.2 1 log The presence of pathogens and indicators in the FW-irrigated soils
aeruginosa was unexpected and suggests that the contribution of treated GW to
Klebsiella 103 105 104 ± 0.1 1 log soil pathogenic diversity is not significant as compared to other
pneumoniae
possible routes of introduction, such as the application of composts
302 M. Benami et al. / Science of the Total Environment 458–460 (2013) 298–302
in gardens and the presence of animals (e.g. dogs, cats, and passing
birds). As all households sampled during this study often hosted
passing mammals in their yards, and enteric pathogens may have
been introduced into the soil with their fecal pellets. It has been
suggested that once introduced, pathogens and indicators can persist
in the soil if key resources are available (Jiang et al., 2001; van Elsas et
al., 2011). Interestingly, the presence/absence of the tested bacteria in
the GW was not affected by site or season, suggesting that all
pathogens were found in all seasons and households (data not
shown). However, as only three sites were used, these findings
require more research to validate our observations.
5. Conclusions
We showed that treated GW, the soils irrigated with this GW, and
FW-irrigated soil all hold a wide array of comparable pathogen types.
P. aeruginosa and E. faecalis were found in all matrices in all samples.
E. coli, K. pneumoniae, Shigella spp. and S. enterica were detected in
some samples. FW-irrigated soils harbored the same array of patho-
gens and indicators as the GW-irrigated soils. These observations
indicate that the variability and introduction of pathogenic bacteria
in the soil are affected by several factors, and that GW may not be a
major contributor to bacteria detected in yard soils. Since qPCR—the
method used here to identify bacteria in soils—cannot distinguish
between viable and nonviable pathogens, further research into the
viability of these bacteria in these environments is called for to
more accurately identify risk of infection.
Acknowledgments
The authors acknowledge Ashraf al-Ashab and the families who
contributed GW and soil to this research. This study was supported
by the Rosenzweig-Coopersmith Foundation and the Zuck Macabi
Fund.
AppendixA. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.scitotenv.2013.04.023.
References
Al-Hamaiedeh H, Bino M. Effect of treated grey water reuse in irrigation on soil and
plants. Desalination 2010;256:115–9.
APHA. Standard methods for the examination of water and wastewater. 20th ed.
Washington, DC: American Public Health Association; 1998.
Barak JD, Liang AS. Role of soil, crop debris, and a plant pathogen in Salmonella enterica
contamination of tomato plants. PLoS One 2008;3:e1657.
Birks R, Hill S. Characterisation of indicator organisms and pathogens in domestic
greywater for recycling. Environ Monit Assess 2007;129:61–9.
Christova-Boal D, Eden RE, McFarlane S. An investigation into greywater reuse for
urban residential properties. Desalination 1996;106:391–7.
Feinstein LM, Sul WJ, Blackwood CB. Assessment of bias associated with
incomplete extraction of microbial DNA from soil. Appl Environ Microbiol
2009;75:5428–33.
Finley S, Barrington S, Lyew D. Reuse of domestic greywater for the irrigation of food
crops. Water Air Soil Pollut 2009;199:235–45.
Francy DS, Bushon RN, Brady AMG, Bertke EE, Kephart CM, Likirdopulos CA, et al.
Comparison of traditional and molecular analytical methods for detecting
biological agents in raw and drinking water following ultrafiltration. J Appl
Microbiol 2009;107:1479–91.
Friedler E, Gilboa Y. Performance of UV disinfection and the microbial quality of
greywater effluent along a reuse system for toilet flushing. Sci Total Environ
2010;408:2109–17.
Friedler E, Galil NI, Kovalio R. On-site greywater treatment and reuse in multi-storey
buildings. Water Sci Technol 2005;51:187–94.
Gilmore M. The enterococci: pathogenesis, molecular biology, and antibiotic resistance.
Washington, DC: American Society for Microbiology Press; 2002.
Gross A, Shmueli O, Ronen Z, Raveh E. Recycled vertical flow constructed wetland
(RVFCW)—a novel method of recycling greywater for irrigation in small
communities and households. Chemosphere 2007;66(5):916–23.
Halperin R, Aloni U. Standards for treated wastewater reuse in the city, for recreation
and in industry (in Hebrew). Jerusalem: Israel Ministry of Health; 200315.
He JW, Jiang S. Quantification of enterococci and human adenoviruses in environmental
samples by real-time PCR. Appl Environ Microbiol 2005;71(5):2250–5.
Hill VR, Kahler AM, Jothikumar N, Johnson TB, Hahn D, Cromeans TL. Multistate evalu-
ation of an ultrafiltration-based procedure for simultaneous recovery of enteric
microbes in 100-L tap water samples. Appl Environ Microbiol 2007;73:4218–25.
Jiang X, Morgan J, Doyle MP. Fate of Escherichia coli O157:H7 in manure-amended soil.
Appl Environ Microbiol 2001;68:2605–9.
Knappett SKP, Layton A, McKay LD, Williams D, Mailloux BJ, Huq MR, et al. Efficacy of
hollow-fiber ultrafiltration for microbial sampling in groundwater. Ground Water
2011;49(1):53–65.
Kotut K, Nganga VG, Kariuki FW. Physico-chemical and microbial quality of
greywater from various households in Homa Bay Town. Open Environ Eng J
2011;4:162–9.
Lauber CL, Strickland MS, Bradford MA, Fierer N. The influence of soil properties on the
structure of bacterial communities across land-use types. Soil Biol Biochem
2008;40(9):2407–15.
Leskinen SD, Lim DV. Rapid ultrafiltration concentration and biosensor detection of
enterococci from large volumes of Florida recreational water. Appl Environ
Microbiol 2008;78:4792–8.
Loddenkötter B, Becker K, Hohoff C, Brinkmann B, Bajanowski T. Real-time quantitative
PCR assay for the detection of Helicobacter pylori: no association with sudden
infant death syndrome. Int J Legal Med 2004;119:202–6.
Lu J, Ryu H, Santo Domingo JW, Griffith JF, Ashbolt N. Molecular detection of Campylobacter
spp. in California gull (Larus californicus) excreta. Appl Environ Microbiol 2011;77:
5034–9.
Maimon A, Tal A, Friedler E, Gross A. Safe on-site reuse of greywater for irrigation — a
critical review of current guidelines. Environ Sci Technol 2010;44(9):3213–20.
Ning J, Liebich J, Kastner M, Zhou J, Schaffer A, Burauel P. Different influences of
DNA purity indices and quantity on PCR-based DGGE and functional gene
microarray in soil microbial community study. Appl Microbiol Biotechnol
2009;82(5):983–93.
Nolde E. Greywater reuse systems for toilet flushing in multi-storey buildings—over
ten years experience in Berlin. Urban Water 2000;1:275–84.
Rose JB. Microbial quality and persistence of enteric pathogens in graywater from
various household sources. Water Res 1991;25:37–42.
Shanks OC, Kelty CA, Sivaganesan M, Varma M, Haugland RA. Quantitative PCR for
genetic markers of human fecal pollution. Appl Environ Microbiol 2009;75:
5507–13.
Shannon KE, Lee DY, Trevors JT, Beaudette LA. Application of real-time quantitative PCR
for the detection of selected bacterial pathogens during municipal wastewater
treatment. Sci Total Environ 2007;382:121–9.
Soil and Plant Analysis Council. Soil analysis handbook of reference methods. Boca
Raton: CRC Press LLC; 1999.
Travis MJ, Wiel-Shafran A, Weisbrod N, Adar E, Gross A. Greywater reuse for irrigation:
effect on soil properties. Sci Total Environ 2010;408:2501–8.
van Elsas JD, Semenov AV, Costa R, Trevors JT. Survival of Escherichia coli in the
environment: fundamental and public health aspects. Int Soc Microb Ecol J
2011;5:173–83.
Winward GP, Avery LM, Frazer-Williams R, Pidou M, Jeffrey P, Stephenson T, et al. A
study of the microbial quality of greywater and an evaluation of treatment
technologies for reuse. Ecol Eng 2008;32:187–97.