Antibiotic Resistant Gene Development in Wastewater Treatment Plants

Mariya Munir, Terence L. Marsh and Irene Xagoraraki

The increasing problem of the emergence of antibiotic resistant

genes (ARGs) and antibiotic resistant bacteria (ARB) is becoming a

major global health issue. Large amounts of antibiotics are released

into municipal wastewater because of either incomplete metabolism

in humans or disposal of unused antibiotics and finally find their way

into different natural environmental compartments. Human exposure

to these microbial contaminants (ARGs and ARB) can occur in

number of ways. We have detected high concentrations of ARGs and

ARB in biosolids (range of 5.61×106-4.32×109 copies/g and

3.17×104-1.85×109 CFU/g, respectively) in different wastewater

treatment plants (WWTPs) in Michigan. ARGs and ARB were also

detected in the final effluents (concentration range of Non-detect-

2.33x106 copies/100mL and 5.00×102-6.10×105 CFU/100mL,

respectively). WWTPs can be considered as important reservoirs for

the spread of antibiotic resistance to opportunistic pathogens and can

stimulate horizontal gene transfer among microbial species. The

objective of our current research is to understand the development of

ARGs within WWTPs with focus on bacteriophages as they can play

a major role in transferring ARGs via tranduction. An additonal goal

is to study WWTP operational conditions and engineering controls

that might influence the development of ARGs and ARB in the

environment.

ABSTRACT

Overall, the trends observed in concentration ranges at different sampling

points from all the wastewater treatment plants are:

raw influent > pre-disinfected effluent > post-disinfected effluent

Daily release loads of ARGs and ARB in the environment were found to

be higher through biosolids relative to effluents.

ARGs were detected in Phage DNA isolated from the sludge samples

CONCLUSIONS and FUTURE WORK

Acknowledgements We would like to thank the managers of all the wastewater treatment plants for providing the samples and information needed for this study.

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Pei, R., S.-C. Kim, K. H. Carlson, and A. Pruden. 2006. Effect of river landscape on the sediment

concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res. 40:2427–

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Genes in Mixed Microbial Populations via 59-Nuclease Assays. Appl. Environ. Microbiol. 66(11):4605-

4614.

Short Protocols in Molecular Biology : Ausubel, Brent, Kingston; 2nd Edition.

Colomer-Lluch, M., J. Jofre, and M. Muniesa. 2011. Antibiotic Resistance Genes in the

Bacteriophage DNA Fraction of Environmental Samples. PLoS ONE 6(3): e17549.

Positive control/ standard test

•Coliphage T4

•E.coli BREC607

(M9 supplement media)

ARGs detected

•Tet-W

•Tet-O

•Sul-I

Grow Pure Culture Plaque Assay

Environmental Sample

Phage Induction

Induced with Mitomycin C

(1µg/mL),

overnight shaking

Isolation of Phages

•Centrifugation

•Membrane Filtration (0.22µm)

PEG precipitation

Phage DNA Extraction

• Add DNase (100U/mL) and

Reaction Buffer , incubate @ 37°C

for 15mins

•Add Stop solution, incubate @ 70°C

for 10 mins

Real-time PCR

•PEG /NaCl mixed,

kept overnight @4°C

•Centrifugation

DNase Treatment

Activated Sludge

•Scrape soft agar with SM buffer

•Centrifugation

•Add chloroform in supernatant

•Store overnight @4°C

Titre phages Confirmation Plaque Assay

108 and 1010 pfu/mL

ARGs and ARB concentrations in the final effluent were found to be in the range of ND(non-

detectable)-2.33×106 copies/100mL and 5.00×102-6.10×105 CFU/100mL respectively.

Disinfection (Chlorination and UV) processes did not contribute in significant reduction of ARGs

and ARB (p>0.05).

Concentrations of ARGs and ARB in biosolids ranged from 5.61×106-4.32×109 copies/g and

3.17×104-1.85×109 CFU/g, respectively.

Fig.1 Log concentration (copies/100 mL) of tetracycline resistant genes (tetW, tetO), sulfonamide resistant

gene (SulI) and 16s rRNA gene abundance at different sampling points of all the five wastewater utilities.

Occurrence in WWTPs

Phage isolation protocol method was tested and optimized to get high

concentration recovery

Phage was successfully recovered and isolated from the high titer T4

coliphage suspension, followed by CsCl gradient for further purification

Fig.2 Concentration (copies/100 mL) of tetracycline resistant genes (tetW), sulfonamide resistant gene (SulI) abundance in phage DNA isolated from

activated sludge , East Lansing Wastewater Treatment Plant. Note: y-axis is in logarithmic scale.

To monitor gene transfer rate in activated sludge with different solid

retention times and temperatures.

Detection of ARGs in bacteriophage and bacteria from activated sludge

Concentration of ARGs in Phage DNA from returned activated sludge (RAS)

and primary sludge (PS) were found to be 3.84x102 and 8.14x103 copies/100mL

for Tet-W gene and 5.89x104and 7.9x104 copies/100mL for Sul-I gene,

respectively.

There was ~4-5 log difference between concentration of ARGs in phage DNA

and in bacterial DNA.

Tet-O gene was not detected in these samples.

Bacterial DNA extraction Real-time PCR

EAST

LANSING IMLAY ROMEO

TRAVERSE

CITY LANSING

Sludge

treatment Dewatering

Gravity

Thickening

Anaerobic

Digestion

Anaerobic

Digestion

Lime

Stabilization

Disposal of

sludge Landfill

Agricultural

land

Agricultural

land

Agricultural

land

Agricultural

land

Disposal rate

(dry tons per

year)

3596 118 125 850 4380

% solid 18.05% 1.49% 7.98% 4.85% 9.20%

Table 2: Biosolids Treatment Characteristics.

EAST

LANSING IMLAY ROMEO

TRAVERSE

CITY LANSING

Wastewater

treatment process

(Biological

treatment)

Activated

Sludge

(AS)

Oxidation

Ditch

(OD)

Rotating

Biological

Contactors

(RBCs)

Membrane

Biological Reactor

(MBR)

Activated

Sludge

(AS)

Capacity 18.8 MGD 0.9 MGD 2.1 MGD 17.0 MGD 37.0 MGD

Average flow 13.4 MGD 0.4 MGD 0.8 MGD 8.5 MGD 20.0 MGD

Discharge Rate 14.1 MGD 0.02 MGD 0.8 MGD 4.0 MGD 19.0 MGD

Disinfection Chlorine

(Cl)

Ultra-Violet

(UV)

Chlorine

(Cl)

Ultra-Violet

(UV)

Ultra-Violet

(UV)

Table 1: Wastewater Treatment Characteristics.

Target Primers Sequences

(5’-3’)

Annealing

temperatu

re (°C)

Amplico

n Size

(bp)

References

Tet-W

Tet-O

Sul-I

Bacteria

16srRNA

tet(W)-FV

tet(W)-RV

tet(O)-FW

tet(O)-RV

sul(I)-FW

sul(I)-RW

BACT1369F

PROK1492R

TM1389F

(Probe)

GAGAGCCTGCTATATGCCAGC

GGGCGTATCCACAATGTTAAC

ACGGARAGTTTATTGTATACC

TGGCGTATCTATAATGTTGAC

CGCACCGGAAACATCGCTGCAC

TGAAGTTCCGCCGCAAGGCTCG

CGGTGAATACGTTCYCGG

GGWTACCTTGTTACGACTT

CTTGTACACACCGCCCGTC

64

60

55.9

56

168

171

163

143

Aminov et al

2001

Aminov et al

2001

Pei et al

2006

Suzuki et al

2000

Table 3: Primers and Probes used in this study.