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Microsoft word - progress report to robin silver final aug 20 2012.docx

Summary Report to Dr. Robin Silver
Prepared by:
Updated: August 20, 2012
Executive Summary
Rationale: Antibiotic resistance is a growing problem and is a major challenge to human
medicine because it results in drugs losing their effectiveness for treating bacterial infections.
Bacteria are able to fight antibiotics through many mechanisms, all of which are encoded in their
DNA by antibiotic resistance genes (ARGs). ARGs have been found in wastewater treatment
systems, which receive antibiotics and resistant gastrointestinal flora excreted by humans. ARGs
have been observed to persist in water and sediment where treated water is discharged. This has
raised the question about the persistence of ARGs in recycled water, which is treated to a higher
standard than conventional wastewater treatment, and then distributed via purple pipe for non-
potable use, such as irrigation of recreational parks.
Purpose and Approach: The purpose of this study was to examine the occurrence of ARGs in
treated recycled water prior to distribution and at the point of use at several irrigation points in
Flagstaff, AZ. ARGs corresponding to five classes of antibiotics were examined, including:
tetracyclines (tetA, tetW, tetO), sulfonamides (sul1, sul2), macrolides (ermF), glycopeptides
(vanA, resistance to a last-resort antibiotic), and methicillin (mecA gene that is present in MRSA
and some other bacteria). The study was intended as a screen: presence of ARGs indicates value
of further research. Quantitation of ARGs by Q-PCR allowed comparison of the levels of ARGs
in recycled water relative to other environments.

The main finding of this study is that although ARGs were relatively diminished in
treated recycled water (only sul2 and ermF were detected), the ARGs dramatically increased at
the point of use. This is likely due to the growth of biofilms in the purple pipes. The fact that
the ARGs increased in the purple pipe demonstrates viable cells carrying the ARGs capable of
growth in the distribution system.

Currently, there are no water quality standards defining a “safe” level of ARGs.
Water quality standards are primarily based on coliform testing (less than 23 coliform organisms
/ 100 ml for a single sample in Arizona for Class A+ Reclaimed Water), which are known to fall
short in assessing risks of many pathogens, especially in recycled water. The results of this study
support efforts to develop more comprehensive and accurate assessment of microbiological
safety of reclaimed water that addresses next-generation challenges in the control of antibiotic
resistance and emerging pathogens. It is true that some levels of ARGs exist in the background
in nature, but it is also clear that human activities, especially wastewater collection and
treatment, can drive sharing of ARGs among bacteria and measureable increases in ARG levels.

Recommended Next Steps:
Future efforts are merited for more rigorous monitoring of ARGs,
as well as re-sampling for living resistant organisms. Assays verifying the activity levels of
ARGs would also be beneficial. It may also be useful to measure the levels of antibiotics in the
water to determine if they are present and potentially stimulating antibiotic resistance in the
purple pipes. Together, this information may help inform effective treatment and distribution
management strategies. This research team would be amenable to working with the
municipality in pro-actively evaluating and testing a treatment and management plan that
could minimize potential risks of antibiotic resistance in recycled water.
Risk assessment
models tailored to ARGs and antibiotic resistance would also be beneficial.

Research Methods

Site Description
Samples were collected from a representative range of areas irrigated by the City of Flagstaff’s two wastewater reclamation facilities, Rio de Flag and Wildcat. The two plants provide provides recycled water for twelve elementary schools, seventeen public parks/landscaping areas, and various golf courses and cemeteries. Recycled wastewater samples examined in this study were collected by Dr. Robin Silver and shipped overnight on ice to Virginia Tech for analysis. The eleven water samples were collected from a range of areas that the reclamation plants serve, including baseball fields, parks, and soccer fields (see Table 1 for complete list of sample names and locations). Table 1: Recycled Water Samples
Sample ID Sample Type
Sample Location

Sample processing
50 mL sub-samples were concentrated to a powder by freeze-drying. DNA was extracted from the powder using standard procedures in preparation for quantitative polymerase chain reaction (Q-PCR). Q-PCR enabled direct quantification of ARGs, without cell culturing. This is an advantage because the vast majoring of bacteria (99%) do not grow on a Petri dish. Therefore, Q-PCR quantification provided comprehensive detection of ARGs present in the water samples that could be compared with other studies. ARGs targeted in this study are listed in Table 2. Table 2: Antibiotic Resistance Genes (ARGs) Targeted in this Study
Gene Name Antibiotic Resistance
Example Carrier
Pathogen Effects
common cause of hospital infections that can spread to the heart, bones, ermF
lungs, and bloodstream with fatal results Can cause infections in blood, urinary tract and heart, and can be life- vanA
threatening. Vancomycin is a last-resort antibiotic. tetA
Data Collection & Analysis

All Q-PCR tests were done in triplicate, with negative controls in each run to confirm absence of contamination. The qPCR measurement provides a measure of the number of target genes in the test tube. In order to calculate the number of copies in the original samples, the triplicates measurements were averaged and then corrected to account for dilution of the DNA extract (1:50 for all assays, except tetO and ermF were undiluted) and the concentration of the original sample (50 mL). The final units are reported as “gene copies per milliliter of water”. It should be noted that mecA and tetW were assayed as well. The first test, using the 1:50 dilution of the DNA sample, showed no detection of these ARGs. For mecA, the qPCR test was
optimized to determine a better annealing temperature, but the gene still was not detected. Both
mecA and tetW are therefore not presented below, but further optimization of test conditions for
these two genes are still being attempted.

Results & Discussion

Flagstaff, AZ

Five out of the eight ARGs tested were detected among the eleven water samples from the Flagstaff area. tetW and mecA were not detectable. The 16S rRNA gene was included as an indicator of the total bacterial density in each sample. Figure 1 shows the quantification of the genes for the Flagstaff samples, including the 16S rRNA gene. Figure 2 shows the same ARG measurements normalized to the 16S rRNA genes for each sample. Dividing by the 16S rRNA gene provides an indicator of the portion of the total bacteria carrying ARGs (but note this is not a 1:1 relationship, because some bacteria may carry several copies of the ARGs, and also the 16S rRNA gene assay is not guaranteed to detect 100% of bacteria). Figure 1: Gene Quantification of Flagstaff Recycled Water Samples
Figure 2: Gene Quantification of Flagstaff Recycled Water Samples
Normalized to 16S rRNA Genes
Examination of the ARG data reveals a striking trend. Samples collected from the
point of use contain dramatically more kinds of ARGs relative to samples collected directly
from WWTP outlets
(RdF4, RdF5, RdF6). This difference suggests that bacteria carrying and
producing ARGs are multiplying in the purple pipes.

Relative Comparison

Levels of tet and sul ARGs in the distributed recycled water ranged from 102 to 104 genes per mL, while vanA and ermF ARGs ranged from 102 to 103 genes per mL. Levels of 16S rRNA genes (total bacterial marker) were about 105 genes per mL. To our knowledge, this is the only
quantitative survey of antibiotic resistance genes in recycled water,
which makes comparison
difficult. Table 3 therefore provides comparison to other water types from other studies, such as
drinking water, wastewater, livestock lagoons, manure, river water, and pristine river water. For
example, the levels of 16S rRNA genes (~105 gene copes per mL) seems reasonable given that
we recently measured 104 16S rRNA genes per mL in first draw drinking water samples
collected from household taps (10).


We acknowledge the funding of the National Science Foundation through NSF/REU Site Grant EEC-1062860. Any opinions, findings, and conclusions or recommendations expressed in
this paper are those of the author(s) and do not necessarily reflect the views of the National
Science Foundation.
1. Czekalski N, Berthold T, Caucci S, Egli A, and Bürgmann H. (2012). Increased levels of
multiresistant bacteria and resistance genes after wastewater treatment and their dissemination into Lake Geneva, Switzerland. Frontiers in Microbiology. Vol. 3 article 106 doi: 10.3389/fmicb.2012.0016. 2. Kim, S., H. Park and K. Chandran (2010) Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: A mathematical modeling approach. Chemosphere. 78(2010), 1071-1077. 3. McKinney, C.W., Loftin, K.A., Meyer, M.T., Davis, J.G., and A. Pruden. (2010). tet and sul Antibiotic Resistance Genes in Livestock Lagoons of Various Operation Type,
Configuration, and Antibiotic Occurrence. Environmental Science & Technology. 44 (16):
4. Negreanu Y, Pasternak Z, Jurkevitch E, Cytryn E (2012) Impact of Treated Wastewater Irrigation on Antibiotic Resistance in Agricultural Soils. Environmental Science & Technology 46 (9):4800-4808. doi:10.1021/es204665b 5. Pruden, A., Pei, R., Storteboom, H.N., and K.H. Carlson. (2006). Antibiotic Resistance Genes (ARG) as Emerging Contaminants: Studies in Northeastern Colorado. Environmental Science and Technology. 40(23): 7445-7450. 6. Pruden, A., Arabi, M., and Storteboom, H.N. Correlation of upstream human activities with riverine antibiotic resistance genes. Submitted to Environmental Science & Technology June 2012. 7. Smith MS, Yang RK, Knapp CW, Niu Y, Peak N, Hanfelt MM, Galland JC, Graham DW. (2004) Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR. Applied Environmental Microbiology. 70(12):7372-7. 8. Thames, C., Knowlton, K., Jones, R., and A. Pruden. (2012). Excretion of Antibiotic Resistance Genes by Dairy Calves Fed Milk Replacers With Varying Doses of Antibiotics. Frontiers in Microbiology. 3:139. doi: 10.3389/fmicb.2012.00139. 9. Xi, CW, Zhang, Y.L, Marrs, C.F., Ye, W., Simon, C., Foxman, B., and Nriagu, J. (2009) Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Applied Environmental Microbiology. 75(17), 5714-5718. 10. Wang, H., Edwards, M.A., Falkinham III, J.O., and A. Pruden. (2012). Molecular Survey of Occurrence of Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa and Amoeba Hosts in Two Chloraminated Drinking Water Distribution Systems. Applied and Environmental Microbiology. 78 (17): doi:10.1128/AEM.01492-12. (in press)
Table 3: Relative Comparisons of ARGs Measured in this Study and Other Studies (Log
number of ARGs per mL (10X per mL)

ermF Reference

WW disnf.


Water Plant

Water Tap


Key: WW= wastewater; WWin = influent wastewater; WW AS = wastewater activated
sludge; WWout = treated wastewater; WW disnf. = treated and disinfected wastewater;
WW discharge = surface water to which wastewater is discharged; *ND = Tested, but not

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CURRICULUM VITAE Texas A&M Health Science Center (TAMU 1266) E-ma EDUCATION: University of Houston, Houston, Texas Professor, Department of Health Policy and Management, College of Rural Public Health, Texas A&M Health Science Center, College Station, Texas. Scientific Advisor, U.S. Health Economics, Oxford Outcomes, Ltd., Morristown, New Jersey. Professor, Department of Health Man

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