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Antibiotic Resistance Gene Testing of Recycled Water Samples in Flagstaff, AZ

Summary Report to Dr. Robin Silver

Prepared by:

Dr. Amy PrudenAssociate ProfessorCivil and Environmental EngineeringVirginia TechBlacksburg, VAMaureen O’BrienBS CandidateEnvironmental Science and EngineeringColorado School of MinesMark MazzochetteMS CandidateCivil and Environmental EngineeringVirginia TechDr. Nicole FahrenfeldPost-doctoral ResearcherCivil and Environmental EngineeringVirginia Tech

Updated: August 20, 2012

Executive SummaryRationale:

Antibiotic resistance is a growing problem and is a major challenge to humanmedicine 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 theirDNA by antibiotic resistance genes (ARGs). ARGs have been found in wastewater treatmentsystems, which receive antibiotics and resistant gastrointestinal flora excreted by humans. ARGshave been observed to persist in water and sediment where treated water is discharged. This hasraised the question about the persistence of ARGs in recycled water, which is treated to a higherstandard 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 intreated recycled water prior to distribution and at the point of use at several irrigation points inFlagstaff, AZ. ARGs corresponding to five classes of antibiotics were examined, including:tetracyclines (

tet

O), sulfonamides (

sul

2), macrolides (

erm

F), glycopeptides(

van

A, resistance to a last-resort antibiotic), and methicillin (

mec

A gene that is present in MRSAand some other bacteria). The study was intended as a screen: presence of ARGs indicates valueof further research. Quantitation of ARGs by Q-PCR allowed comparison of the levels of ARGsin recycled water relative to other environments.

Results:

The main finding of this study is that although ARGs were relatively diminished intreated recycled water (only

sul

2 and

erm

F were detected), the ARGs dramatically increased atthe point of use. This is likely due to the growth of biofilms in the purple pipes. The fact thatthe ARGs increased in the purple pipe demonstrates viable cells carrying the ARGs capable of growth in the distribution system.

Interpretation:

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 fallshort in assessing risks of many pathogens, especially in recycled water. The results of this studysupport efforts to develop more comprehensive and accurate assessment of microbiologicalsafety of reclaimed water that addresses next-generation challenges in the control of antibioticresistance and emerging pathogens. It is true that some levels of ARGs exist in the backgroundin nature, but it is also clear that human activities, especially wastewater collection andtreatment, 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

3water to determine if they are present and potentially stimulating antibiotic resistance in thepurple pipes. Together, this information may help inform effective treatment and distributionmanagement strategies.

This research team would be amenable to working with themunicipality in pro-actively evaluating and testing a treatment and management plan thatcould minimize potential risks of antibiotic resistance in recycled water.

Risk assessmentmodels 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 plantsprovide provides recycled water for twelve elementary schools, seventeen publicparks/landscaping areas, and various golf courses and cemeteries. Recycled wastewater samplesexamined in this study were collected by Dr. Robin Silver and shipped overnight on ice toVirginia Tech for analysis. The eleven water samples were collected from a range of areas thatthe reclamation plants serve, including baseball fields, parks, and soccer fields (see Table 1 forcomplete list of sample names and locations).

Table 1: Recycled Water Samples

Sample ID Sample TypeSample LocationRdF1

Water Thorpe Park, Baseball Field North

RdF2

Water Bushmaster Park

RdF3

Water Thorpe Park

RdF4

Water Rio de Flag Construction Outlet

RdF5

Water Wildcat WWTP Outlet

RdF6

Water Rio de Flag WWTP Outside Faucet

RdF7

Water Thorpe Park, Baseball Field South

RdF8

Water Coconino HS, Baseball Field

RdF9

Water Foxglen Soccer Field

RdF10

Water Thorpe Soccer Field North

RdF11

Water Wheeler Park

Sample processing

50 mL sub-samples were concentrated to a powder by freeze-drying. DNA was extractedfrom the powder using standard procedures in preparation for quantitative polymerase chainreaction (Q-PCR). Q-PCR enabled direct quantification of ARGs, without cell culturing. This isan 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 thewater samples that could be compared with other studies. ARGs targeted in this study are listedin Table 2.