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DOI 10.1007/s00253-009-2125-2
ENVIRONMENTAL BIOTECHNOLOGY
Received: 3 June 2009 / Revised: 29 June 2009 / Accepted: 30 June 2009 / Published online: 14 July 2009
# Springer-Verlag 2009
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
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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
tet(A)
210
20
Ng et al. 2001
171
50
278
70
280
10
202
4,000
tet(O)
tet(X)
intI1
16S rRNA
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
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
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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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