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Appl Microbiol Biotechnol (2009) 84:791796

DOI 10.1007/s00253-009-2125-2

ENVIRONMENTAL BIOTECHNOLOGY

The role of anaerobic digestion in controlling the release


of tetracycline resistance genes and class 1 integrons
from municipal wastewater treatment plants
Sudeshna Ghosh & Sara J. Ramsden &
Timothy M. LaPara

Received: 3 June 2009 / Revised: 29 June 2009 / Accepted: 30 June 2009 / Published online: 14 July 2009
# Springer-Verlag 2009

Abstract In this study, the abilities of two anaerobic


digestion processes used for sewage sludge stabilization
were compared for their ability to reduce the quantities of
three genes that encode resistance to tetracycline (tet(A), tet
(O), and tet(X)) and one gene involved with integrons
(intI1). A two-stage, thermophilic/mesophilic digestion
process always resulted in significant decreases in the
quantities of tet(X) and intI1, less frequently in decreases of
tet(O), and no net decrease in tet(A). The thermophilic
stage was primarily responsible for reducing the quantities
of these genes, while the subsequent mesophilic stage
sometimes caused a rebound in their quantities. In contrast,
a conventional anaerobic digestion process rarely caused a
significant decrease in the quantities of any of these genes,
with significant increases occurring more frequently. Our
results demonstrate that anaerobic thermophilic treatment was
more efficient in reducing quantities of genes associated with
the spread of antibiotic resistance compared to mesophilic
digestion.

S. Ghosh : S. J. Ramsden : T. M. LaPara (*)


Department of Civil Engineering, University of Minnesota,
500 Pillsbury Drive SE,
Minneapolis, MN 55455-0116, USA
e-mail: lapar001@umn.edu
Present Address:
S. Ghosh
Department of Civil and Environmental Engineering,
University of Michigan,
Ann Arbor, MI, USA
Present Address:
S. J. Ramsden
Barr Engineering,
Minneapolis, MN, USA

Keywords Anaerobic digestion . Antibiotic resistance .


Municipal wastewater treatment . Tetracycline resistance

Introduction
While the proliferation of antibiotic resistance has been
recognized as an eminent problem for the last few decades
and efforts have been made to curtail the overuse of
antibiotics, resistance to antibiotics continues to increase
(Livermore 2003; Alanis 2005; Smith et al. 2005; Falagas
and Bliziotis 2007). One of the causes of the proliferation
of antibiotic resistance is the lateral transfer of genes that
confer resistance among bacteria, such that environmental
bacteria can potentially serve as vectors for genes conferring resistance and transfer them to pathogenic bacteria
(Summers 2002; Wright 2007).
The long-term goal of our research is to identify
environmental reservoirs of antibiotic resistance and to
develop novel and effective strategies to ameliorate these
reservoirs. There are numerous scientific publications to
suggest that municipal wastewater is a pertinent reservoir of
antibiotic-resistant bacteria (for examples, see Auerbach
et al. 2007; Schluter et al. 2007; Zhang et al. 2009a, b).
Because municipal wastewater is typically passed through a
treatment facility designed to prevent adverse environmental impacts (Tchobanoglous et al. 2003), we believe that
municipal wastewater treatment facilities could be easily
adapted to be used as tools to help slow the proliferation of
antibiotic resistance.
The primary avenue by which resistant bacteria could
escape from municipal wastewater treatment facilities is
with the residual solids. These wastewater solids are
preferably disposed by stabilizing the solids (i.e., reducing
their organic content) and then applying them to land for

792

use as a fertilizer and a soil conditioner (Tchobanoglous


et al. 2003). In the USA, these residual solids from
municipal wastewater treatment facilities are classified
according to the extent of organic stabilization and
pathogen inactivation. Current regulations recognize stabilization technologies as Class A when these processes
achieve a high level of stabilization and drastically reduce
pathogen levels. Alternatively, Class B processes achieve
a similar extent of stabilization but have a less stringent
requirement for pathogen inactivation (US EPA 1994).
In this study, we determined the effectiveness of two
full-scale anaerobic digestion systems (originally designed
to stabilize wastewater solids) to reduce the quantities of
three different genes encoding for resistance to tetracycline (tet(A), tet(O), and tet(X)) and one gene involved
with class 1 integrons (intI1). This research was needed
because, although there have been numerous studies
detailing the extent of pathogen removal achieved by
different stabilization technologies (for examples, see
Aitken et al. 2005; Grewal et al. 2006; Iranpour and Cox
2006; Lang and Smith 2008), there is very little data about
the reduction of genes conferring antibiotic resistance
during stabilization.

Materials and methods


Site descriptions
The Western Lake Superior Sanitary District (WLSSD)
wastewater treatment facility (Duluth, MN, USA) is designed
to treat 48 million gallons per day (182,000 m3 day1). This
facility treats its residual biosolids via a two-stage,
temperature-phased anaerobic digestion process that includes
treatment by thermophilic anaerobic digestion (typical
operating temperatures=5060C) followed by conventional
anaerobic digestion (typical operating temperature=35
37C). The Empire wastewater treatment plant (Farmington,
MN, USA) is designed to treat up to 12 million gallons of
wastewater per day (45,000 m3 day1). The Empire facility
utilizes single-stage, conventional mesophilic anaerobic
digestor (typical operating temperatures=3537C) to treat
its residual biosolids.
Sample collection
Samples (approximately 50 mL) were aseptically collected
in triplicate from the untreated wastewater solids as well as
directly from the digestors at WLSSD and Empire (samples
were collected from both thermophilic and mesophilic
digestors at the WLSSD facility) on three separate days.
Samples were transported to the laboratory on ice and
processed within 12 h of collection.

Appl Microbiol Biotechnol (2009) 84:791796

Genomic DNA extraction


Bacterial suspensions in lysis buffer underwent three
consecutive freeze-thaw cycles and an incubation of 90 min
at 70C. Genomic DNA extraction and purification using the
FastDNA Spin Kit (Qbiogene; Vista, CA, USA) was
performed according to manufacturers instructions. DNA
extractions were stored at 20C until needed.
Real-time PCR
Real-time polymerase chain reaction (PCR) was used to
quantify the presence of tet(A), tet(O), tet(X), and intI1
genes as well as 16S rRNA genes (as a measure of bacterial
biomass). We targeted these three genes for tetracycline
resistance because they encode for proteins that represent
each of the three known mechanisms of resistance (tet(A):
efflux pump; tet(O): ribosomal protection protein; tet(X):
enzymatic modification; Chopra and Roberts 2001). We
also targeted the intI1 gene because of its general role in the
molecular ecology of antibiotic resistance (Mazel 2006),
and our observation that a large fraction of tetracyclineresistant bacteria isolated from municipal wastewater
treatment facilities harbored a class 1 integron (Firl 2006).
Real-time PCR was conducted using an ABI 7900HT
thermocycler (Applied Biosystems; Foster City, CA, USA).
Each gene was quantified in duplicate from each of the
triplicate genomic DNA extractions. The quantity of target
DNA in unknown samples was calculated based on a standard
curve generated using known quantities of template DNA.
PCR conditions and primer concentrations were optimized to eliminate the formation of primer-dimers and nonspecific products using a dissociation curve (data not
shown). A typical PCR run consisted of a 10 min initial
denaturation at 95C, followed by forty cycles of denaturation
at 95C for 15 s and anneal/extension for 1 min at a
temperature specific for the target gene (Table 1). A 25-L
reaction mixture contained 12.5 L of 2 Power SYBR
Green Master Mix (Applied Biosystems), 25 g bovine
serum albumin, optimized quantities of forward and reverse
primers, and approximately 1 ng of template DNA.
Standards for quantitative PCR were prepared by PCR
amplification of genes from positive control strains,
followed by ligation into pGEM-T Easy vectors following
manufacturers instructions (Promega; Madison, WI, USA),
and transformation into Escherichia coli DH5. Plasmids
were purified using the alkaline lysis procedure (Sambrook
et al. 1989). Plasmid DNA was quantified by staining with
Hoechst 33258 dye and measured on a TD-700 fluorometer
(Turner Designs, Sunnyvale, CA, USA) using calf thymus
as a DNA standard. Tenfold serial dilutions of plasmid
DNA were prepared and run on the thermal cycler to
generate standard curves.

Appl Microbiol Biotechnol (2009) 84:791796

793

Table 1 Polymerase chain reaction (PCR) primer sequences and other pertinent information related to the use of real-time PCR in this study
Gene

PCR primer sequence (53)

Amplicon size (bp)

tet(A)

GCT ACA TCC TGC TTG CCT TC


CAT AGA TCG CCG TGA AGA GG
ACG GAR AGT TTA TTG TAT ACC
TGG CGT ATC TAT AAT GTT GAC
AGC CTT ACC AAT GGG TGT AAA
TTC TTA CCT TGG ACA TCC CG
CCT CCC GCA CGA TGA TC
TCC ACG CAT CGT CAG GC
CCT ACG GGA GGC AGC AG
ATT ACC GCG GCT GCT GG

210

20

Ng et al. 2001

171

50

Aminov et al. 2002

278

70

Ng et al. 2001; Ghosh 2007

280

10

Davelos et al. 2004

202

4,000

Muyzer et al. 1993

tet(O)
tet(X)
intI1
16S rRNA

Detection limit (gene copies)

Reference

The annealing temperature for all of these PCR reactions was 60C except for tet(O) (57C)

Data analysis
Differences in the quantities of tet(A), tet(O), tet(X), and
intI1 genes normalized by the quantity of 16S rRNA genes
in different samples were analyzed by analysis of variance.
Data were also analyzed by pairwise comparison of
resistance levels using Tukeys honest significant difference
(HSD) with P<0.05. HSD used a stringent Type I error rate
and the studentized range distribution to construct simultaneous confidence intervals for differences of all pairs of
means. These statistical analyses were performed using
MacAnova software (version of 5 February 2003 Win32s
[BCPP5.0], Department of Applied Statistics, University of
Minnesota [http://www.stat.umn.edu/macanova]).

Results
The thermophilic stage at the WLSSD generally led to
statistically significant reductions in the quantities of tet(A),
tet(O), tet(X), and intI1 (Fig. 1). The quantities of tet(X)
and intI1 decreased 8599% and 8095%, respectively,
which was statistically significant during all three sample
events. Similarly, the quantities of tet(A) and tet(O)
decreased by 5080%, which was statistically significant
during two of the three sample events.
The second, mesophilic stage at WLSSD, however,
was generally ineffective in reducing the quantities of the
tet(A), tet(O), tet(X), and intI1 (Fig. 1). In fact, the levels
of all four genes were often higher in the mesophilic
digestor compared to the thermophilic digestor. Statistically significant decreases in the quantities of tet(O) and
tet(X) occurred during only one of the sample events.
Increases in the quantities of tet(A) and intI1 were noted,
of which, the increases in intI1 quantities were statistically
significant during two of the three sample events.
Similarly, statistically significant increases in the quantities of tet(O) and tet(X) in the mesophilic digestor relative

to the thermophilic digestor were noted during one of the


sample events.
In contrast, the mesophilic anaerobic digestion process at
the Empire wastewater treatment plant was generally
unable to reduce the quantities of tet(A), tet(O), tet(X),
and intI1 (Fig. 2). Statistically significant reductions were
observed on two occasions for tet(A) and on one occasion
for intI1. Curiously, statistically significant increases were
observed in the quantities of tet(O) and tet(X) during two of
the three sample events.

Discussion
Municipal wastewater is known to be a substantial reservoir
of antibiotic-resistant bacteria as well as genes that encode
for antibiotic resistance. The technical literature, however,
contains relatively little information on the effectiveness of
existing treatment operations to inactivate resistant bacteria
and/or reducing the quantities of genes that encode for
resistance. The largest reservoir of antibiotic-resistant
bacteria within a municipal wastewater treatment plant is
undoubtedly the residual wastewater solids, which include
the particulate material collected in the primary clarifier as
well as the excess biomass that is grown in the aeration tank
and collected in the secondary clarifier. The results
presented herein, therefore, represent an important step
towards considering municipal wastewater treatment as an
opportunity to slow and control the proliferation of
antibiotic-resistant bacteria.
We anticipated that the temperature-phased anaerobic
digestion process at the WLSSD facility would achieve
substantially better reductions in tetracycline-resistant bacteria because it is well-established that high temperature
treatment processes are effective at inactivating pathogenic
bacteria (Aitken et al. 2005; Berg and Berman 1980; Han et
al. 2009; Wagner et al. 2008). We did not anticipate,
however, a recovery in the quantities of two of these

794

Appl Microbiol Biotechnol (2009) 84:791796

Fig. 1 Quantities of three genes


that encode for resistance to
tetracycline and one gene
involved in integrons through a
temperature-phased anaerobic
digestion process. Error bars
represent one standard deviation
about the mean. Arrows
designate a statistically significant increase or decrease compared the preceding step (i.e.,
the thermophilic digestor
compared to the untreated
wastewater solids or mesophilic
digestor compared to the
thermophilic digestor)

genes in the second, mesophilic stage of the digestion


system. Similarly, the quantities of genes encoding for
tetracycline resistance generally remained unchanged or
apparently increased in the mesophilic anaerobic digestor
at Empire.
An increase in gene quantities within an anaerobic
digestor would suggest either that the organisms harboring
genes encoding for resistance are multiplying within the
anaerobic digestor or that the quantities of genes are
increasing via lateral gene transfer. Lateral gene transfer
within anaerobic digestors seems plausible given that these
reactors contain very high densities of biomass, which is a
known prerequisite for lateral gene transfer to occur
(Snyder and Champness 2007). The possibility of extensive
lateral gene transfer within the municipal wastewater

treatment process is of concern because it suggests that


anaerobic digestors could be a source of new antibioticresistant bacteria. This would be a substantial paradigm
shift from our original viewpoint, which was that municipal
wastewater was an important reservoir of resistant bacteria
and that municipal wastewater treatment processes could be
used to decrease the size of this reservoir of resistance.
In this study, we also quantified the effect of anaerobic
digestion on the quantities of class 1 integrons. Integrons
are genetic elements that incorporate open reading frames
and regulate the expression of these exogenous genes;
integrons are therefore believed to be pertinent in the
molecular ecology of the proliferation of antibiotic resistance by reducing the genetic cost of harboring a
resistance gene (Mazel 2006). We envisioned, therefore,

Appl Microbiol Biotechnol (2009) 84:791796

795

Fig. 2 Quantities of three genes


that encode for resistance to
tetracycline and one gene
involved in integrons through a
conventional anaerobic
digestion process. Error bars
represent one standard deviation
about the mean. Arrows
designate a statistically
significant increase or decrease
compared the untreated
wastewater solids

that municipal wastewater treatment could be used to


counteract the proliferation of integrons by reducing the
quantities of organisms that harbor them.
Our study used a cultivation-independent approach,
which avoids the known biases of bacterial cultivation
(Amann et al. 1995). Our cultivation-independent approach,
however, has its own limitations and caveats that need
consideration. First, it is impractical to simultaneously
quantify all of the different genes that are known to encode
for antibiotic resistance (for example, there are more than
40 different genes known to encode for resistance to
tetracycline; Chopra and Roberts 2001). While we looked
at a specific set of genes, these three genes do not
necessarily reflect the trajectory of all genes conferring
resistance to tetracycline during the anaerobic digestion of

residual wastewater solids. Similarly, we are unable to


comment if the genes quantified were associated with
viable bacteria or with bacteria capable of expressing them.
Given that wastewater treatment plants are not intentionally designed to slow the proliferation of antibioticresistant bacteria, it is pertinent to consider alternative
process designs that could further prevent the release of
antibiotic-resistant bacteria. We recommend the widespread
implementation of Class A treatment technologies, which
are specifically designed for their ability to inactivate
pathogenic bacteria (Tchobanoglous et al. 2003). We
simultaneously recommend a reevaluation of the process
used at WLSSD, as the two-stage thermophilic/mesophilic
process studied herein exhibited a rebound in quantities of
resistance genes during the mesophilic stage.

796
Acknowledgements This research was supported by a grant from
the Center for Urban and Regional Affairs to TML and a fellowship
from Geomatrix Consultants to SJR.

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