(2009). the role of anaerobic digestion in controlling the release

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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.

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 confer-

ring 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 environmen-

tal 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

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

Department of Civil Engineering, University of Minnesota,

500 Pillsbury Drive SE,

Minneapolis, MN 55455-0116, USAe-mail: [email protected]

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

Appl Microbiol Biotechnol (2009) 84:791 – 796

DOI 10.1007/s00253-009-2125-2



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 stabi-

lization 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 tetracy-

cline (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 day−1). 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=50 – 60°C) followed by conventional

anaerobic digestion (typical operating temperature=35 –

37°C). The Empire wastewater treatment plant (Farmington,

MN, USA) is designed to treat up to 12 million gallons of

wastewater per day (45,000 m3

day−1

). The Empire facility

utilizes single-stage, conventional mesophilic anaerobic

digestor (typical operating temperatures=35 – 37°C) 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.

Genomic DNA extraction

Bacterial suspensions in lysis buffer underwent three

consecutive freeze-thaw cycles and an incubation of 90 min

at 70°C. Genomic DNA extraction and purification using the

FastDNA Spin Kit (Qbiogene; Vista, CA, USA) was

performed according to manufacturer ’s instructions. DNA

extractions were stored at −

20°C 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 tetracycline-

resistant 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 opti-

mized to eliminate the formation of primer-dimers and non-

specific products using a dissociation curve (data not

shown). A typical PCR run consisted of a 10 min initial

denaturation at 95°C, followed by forty cycles of denaturation

at 95°C for 15 s and anneal/extension for 1 min at a

temperature specific for the target gene (Table 1). A 25-μ L

reaction mixture contained 12.5 μ L of 2× Power SYBR

Green Master Mix (Applied Biosystems), 25 µg 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

manufacturer ’s instructions (Promega; Madison, WI, USA),

and transformation into Escherichia coli DH5α . 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.

792 Appl Microbiol Biotechnol (2009) 84:791 – 796



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 Tukey’s honest significant difference

(HSD) with P <0.05. HSD used a stringent Type I error rate

and the studentized range distribution to construct simulta-

neous 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 85 – 99% and 80 – 95%, respectively,

which was statistically significant during all three sample

events. Similarly, the quantities of tet (A) and tet (O)

decreased by 50 – 80%, 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. Statisti-

cally 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 quanti-

ties 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 bac-

teria 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

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 (5′→3′) Amplicon size (bp) Detection limit (gene copies) Reference

tet (A) GCT ACA TCC TGC TTG CCT TC 210 20 Ng et al. 2001

CAT AGA TCG CCG TGA AGA GG

tet (O) ACG GAR AGT TTA TTG TAT ACC 171 50 Aminov et al. 2002

TGG CGT ATC TAT AAT GTT GAC

tet (X) AGC CTT ACC AAT GGG TGT AAA 278 70 Ng et al. 2001; Ghosh 2007TTC TTA CCT TGG ACA TCC CG

intI1 CCT CCC GCA CGA TGA TC 280 10 Davelos et al. 2004

TCC ACG CAT CGT CAG GC

16S rRNA CCT ACG GGA GGC AGC AG 202 4,000 Muyzer et al. 1993

ATT ACC GCG GCT GCT GG

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

Appl Microbiol Biotechnol (2009) 84:791 – 796 793

http://www.stat.umn.edu/macanova

http://www.stat.umn.edu/macanova



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 antibiotic-

resistant 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 resis-

tance by reducing the “genetic cost ” of harboring a

resistance gene (Mazel 2006). We envisioned, therefore,

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 signifi-

cant 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)




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 inten-

tionally designed to slow the proliferation of antibiotic-

resistant 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.

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 decreasecompared the untreated

wastewater solids

Appl Microbiol Biotechnol (2009) 84:791 – 796 795



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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