Antibiotics in the Environment.
Erik J. Rosenfeldt, PhD, PE
Assistant Professor
University of Massachusetts, Amherst
Department of Civil and Environmental
Engineering
Me
• BS in Chemical Engineering
– Washington University in St. Louis, MO
• MS in Civil and Environmental Engineering
– Duke University, Durham, NC
– Thesis: Destruction of Endocrine Disrupting Compounds in
Water with Direct UV and UV/H2O2 Advanced Oxidation.
• PhD in Civil and Environmental Engineering
– Duke University, Durham, NC
– Dissertation: UV and UV/H2O2 Advanced Oxidation – A
theoretical, practical, and comparative examination of UV
processes used to treat emerging contaminants of concern
in drinking water.
Research Interests
• Mitigating threats from Endocrine Disrupting Chemicals (EDCs),
Pharmaceuticals and Personal Care Products (PPCPs) in drinking
water, wastewater, and reuse water, by combining novel adsorption
methods and advanced oxidation processes.
• Quantifying advanced oxidation potential in drinking water,
wastewater, and reuse water.
• UV AOP and alternative oxidation technologies for direct potable
reuse applications.
• Evaluate strong oxidizing processes to replace traditionally energy
intensive regeneration methods for spent zeolite adsorption media.
• Assess the ability of several natural zeolite products for removal of
water contaminants
• Assessing distribution system microbial quality utilizing flow
cytometry methods
More about Me
• Hobbies include running, hiking, basketball,
playing with my wife daughter, son and dog
Today’s Agenda
• 8:45 – 9:00 Introduction
• 9:00 – 10:30 Antibiotics in the environment
– 9:00 – 9:45: Analyzing for antibiotics in water (presentation)
– 9:45 – 10:30: Setting up the lab portion of the course
– 10:30: Samples need to be incubating (need 2 hours)
• 10:30 – 10:45 Break
• 10:45 – 11:45 Issues with Emerging Contaminants in Water
(presentation)
• 11:45 – 12:00 Break
• 12:00 – 1:00 Data analysis and discussion
– Take pictures of samples, process pictures
– Data Analysis
– Discussion
Analyzing for Antibiotics in Water
Erik Rosenfeldt, PhD, PE
Assistant Professor
University of Massachusetts, Amherst
The story begins…
• Penicillin – 1928
– Alexander Flemming found antimicrobial
properties of substance exuded from Penicillium
notatum (mold)
– “might have therapeutic
value if it could be produced
in quantity.”
The story now…
• 100,000 – 200,000 tons of antibiotics used
worldwide
• US Production = 22.7 million kilograms
– In US, 70% of dispensed antibiotics are given to
healthy livestock to prevent infections and/or
promote growth
– This practice is outlawed in EU countries
Categories of Antibiotics
Mode of Action
Examples
Inhibit Bacterial Cell Wall
Biosynthesis
b-lacatams, cephalosporins,
Human therapeutic and animal
glycopeptides, everninomycins therapeutic/ subtherapeutic
Block 30S or 50S
Ribosomes (inhibit protein
synthesis)
Macrolides, streptogramins,
chloramphenicol, fusidic acid,
tetracyclines, lincosamides,
aminoglycosides,
oxazolidinones,
Quinolines, rifamycines
Human therapeutic and animal
therapeutic/ subtherapeutic
Disrupt Integrity of Cell
Membranes
Antifungal azoles, polyenes
Treat fungal infections, topical
application for Eczema
Antagonize Metabolic
Processes
Sulfonamides,
trimethoprim (TMP)
Humans, livestock,
aquaculture; TMP and
sulfamethoxazole often
prescribed together
Block Replication of DNA
Typical Uses
Treat urinary tract, systemic,
and respiratory infections
Antibiotics in the Environment
Antibiotic
Manufacturers
Livestock Feed
Manufacturers
Veterinarians
Phamacies
Livestock Use
Human Use
Injestion/
Excretion
Injestion/
Excretion
Treatment of
Manure
Treated
Discharge
Land Application
of Solids
Topical
application
Flushing unused
Municipal WW
Individual WW
(ie septic)
Runoff
Treated
Discharge
Leaching to
Groundwater
Surface Water
Contamination
Potential
Drinking Water
Sources
Human Inputs
• Excretion of therapeutic antibiotics and metabolites
– Many discreted directly in amounts > 40% of ingested dose
• Disposal of unused antibiotics
– Flushing down the toilet
• Washing topically applied antibiotics
• All go to septic systems or municipal WWTPs
– Inefficient removal
– Breeding grounds for antibiotic resistant organisms (ARO)
Veterinary Inputs
• Antibiotics used in livestock operations since the early
1950s
• Used to treat infections, or to improve growth and feed
efficiency
• Four “types” of AB use in livestock operations
– Therapy: Antibiotics for treatment of frank clinical disease
– Control: Antibiotics administered to a herd or flock in
which morbidity and/or mortality has exceeded baseline
norms
– Prevention: Antibiotics used in animals considered “at
risk”, but where individuals do not show signs of disease
– Growth Promotion: Antibiotics administered over a period
of time, usually as a feed additive, to growing animals
Veterinary Impacts
• Antibiotics impact aquatic environment
through manure
– Large fraction of the medicines present
unchanged in animal waste
– Direct runoff or collection and use as fertilizer
Impacts – Human health concern?
• Found at 1 ng/L – 1 ug/L levels in environment
• Typical therapeutic doses = 100 – 250 mg per
dose (3x per day?)
• Need to drink
• Not likely an issue
Impacts – Microbial Resistance?
• Environmental levels have been found to
enhance the ability of microorganisms to
develop antibiotic resistance
– Onan and Lapara (2003), FEMS Microbiology Letters 220: 15 – 20
• Evidence of antibiotic resistant
microorganisms has been found in surface
wastewater, groundwater, drinking water.
– Schwartz et al. (2003), FEMS Microbiology Ecology 43, 325 - 335
Revisit human health concerns?
• MRSA (Methicillin Resistant Staphylococcus
aureus)
– Very difficult to treat
– First appeared as hospital derived infection
– 1990s – Community associate outbreaks among high
risk populations
– Today, colonization rates in general population ~ 1.5%
– Infections account for majority of skin and soft tissue
infections treated in US Emergency Rooms.
– In 2005, MRSA caused more deaths in US than AIDS
Antibiotic Resistance as a human
health concern
• Enteric pathogens such as Salmonella and
EHEC (Enterohaemorrhagic Escherichia coli)
causing illness and treatment failure
• Other multi-drug resistant organisms:
– Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa.
• Relatively few pathogens in nature, but…
– Antibiotic resistance is transferable!!!
How to detect antibiotics in water?
• Very sensitive, low level detection available
through GC/MS/MS and LC/MS/MS
– Example Minimum Reporting Limits (MRLs):
•
•
•
•
Cyprofloxacin: 20 ng/L
Erythromycin: 50 ng/L
Sulfamethoxazole: 0.25 ng/L
Trimethoprim: 140 ng/L
– Approximately $500 - $700 quoted per sample!!!!!
What if we could detect overall
“antibiotic activity”
• Advantages:
–
–
–
–
–
Much cheaper (<$1 per sample)
Faster (may get results in a few hours, instead of weeks)
Don’t have to ship samples
Anyone can do it!
Activity might be the only thing that matters
• Questions?
– Do we really care which antibiotics are in solution?
• Different antibiotics act on different parts of the microbe cell
• Different antibiotics possess different “strength”
– Are there other “antimicrobial agents” in solution?
Antibiotic Activity
Mode of Action
Examples
Typical Uses
Inhibit Bacterial Cell Wall
Biosynthesis
b-lacatams, cephalosporins,
glycopeptides, everninomycins
Human therapeutic and animal
therapeutic/ subtherapeutic
Block 30S or 50S Ribosomes
(inhibit protein synthesis)
Macrolides, streptogramins,
chloramphenicol, fusidic acid,
tetracyclines, lincosamides,
aminoglycosides, oxazolidinones,
Human therapeutic and animal
therapeutic/ subtherapeutic
Block Replication of DNA
Quinolines, rifamycines
Treat urinary tract, systemic, and
respiratory infections
Disrupt Integrity of Cell
Membranes
Antifungal azoles, polyenes
Treat fungal infections, topical
application for Eczema
Antagonize Metabolic
Processes
Sulfonamides,
trimethoprim (TMP)
Humans, livestock, aquaculture;
TMP and sulfamethoxazole
often prescribed together
We look at other compounds through
“activity”
• Yeast Estrogen Screen (YES)
YES
• Advantages:
–
–
–
–
Cheap (<$1 per sample)
Fast (Get results in a few days, instead of weeks)
Easy for a grad student to do.
Activity might be the only thing that matters
(Rosenfeldt et al, 2007)
• Questions:
– Do we really care about which EDCs are in solution?
– Synergistic / Competitive effects?
YES Outputs
YES Calibration Curves
Compound
EC50 (nM)
E2eq
E2
~0.3
1.0
E1
~0.21
1.4
EE2
~0.21
1.4
NP
~1050
0.0003
My Research Question
• Is it possible to make a YES-style assay for the
detection of Antibiotics in water?
– “Inexpensive”
– Rapid and convenient
– Give us good information
for occurrence and
treatment studies??
The Antibiotic Challenge (ABC) Assay
• Smith et al, 2007 “The development of a rapid
screening technique to measure antibiotic
activity in effluents and surface water
samples”
• Utilized the fact that the meat and dairy
industry regularly (in EU) and semi-regularly
(in US) tests for antibiotic residues.
– Commercially available kits have been created to
ease this process for meat and dairy producers (ie
farmers, not cows)
Smith et al, 2007
• Utilized the Premitest, from DSM food
industries (Netherlands)
– Designed to test meat for the presence of
antibiotics
• Made slight modifications to test water
samples
– Add a little “synthetic meat”
– Extract and concentrate samples
How does the Premitest work?
• Uses a rapidly growing, thermophilic bacteria as the
indicator
– Bacillus stearothermophilus
• Responsive to all of the most commonly used antibiotics (ie easy to
kill)
• Provides a measurable color change in the agar substrate when not
exposed to antibiotics
• As Bacillus grow, they release acid products
– Acid makes color change (just like pH indicators)
• If Bacillus growth inhibited, less grow, less acid, less color
change
Smith et al, 2007 (cont)
• Identified a qualitative “calibration curve”
Color
Antibiotic effect (%)
Yellow Yellow with 50/50
some purple
Purple with
some yellow
Purple
0
25
50
75
100
Comparative
erythromycin
potency (mg/L)
<25
40
50.7
63
>100
Comparative
sulfamethoxazole
potency (mg/L)
<40
40
100
155
>250
Smith et al, 2007 (cont.)
• Calibration curves
• Surface Water Samples
Other similar antibiotic activity assays
Antibiotic
Manufacturer Time per test
Detection
Assay
Delvotest SP DSM
2.5 hours
Kit
Premitest
DSM
3.5 hours
Copan Italia 2.5 – 3 hours
SpA
Charm Farm Charm
2.5 – 3 hours
Test
Sciences
Charm AIM – Charm
4 hours
96
Sciences
Copan test
Antibiotic
Tested
Reaction
Type
Broad
Spectrum
Broad
Spectrum
Broad
Spectrum
Broad
Spectrum
Broad
Spectrum
Color Change
Color Change
Color Change
Color Change/
pH Change
Color Change
Our Procedure
• “Delvotest P-Mini”
– Sensitive to many antibiotics
– Initial kit comes with several boxes worth of tests
along with a block heater for ~ $100.
– Cost of 25 ampules = ~ $30 - $35
– Color Change takes ~ 2 – 2.5 hours
• Advantages over Premitest
– Initial tests indicate no additional materials needed
• Comes with “nutrient pellets”
Procedure
• Step 1: Preparation
Cut off the required number of ampoules with a pair
of scissors. Be careful not to damage the foil of the
remaining ampoules.
• Step 2: Open the seal
Open ampoule(s) by punching a hole in the aluminum
foil with the syringe. Mark the ampoules for sample
identification.
• Step 3: Take a milk water sample
Attach a new disposable pipette to the syringe. Depress the
plunger completely, dip the tip in the milk sample and allow
the plunger to return slowly under pressure of the spring.
Procedure
• Step 4: Inoculation
Empty the syringe into the correspondingly
marked ampoule by slowly depressing the
plunger of the syringe. Use a fresh disposable
pipette for each sample.
• Step 5: Incubation
Check the temperature of the incubator (64ºC
±0.5ºC). Put the ampoule(s) into the incubator. Record
the time and set timer for 3 hours or use control time.
• Step 6: Results
After 3 hours, remove the samples from the
incubator. Read the colour of the lower 2/3 part of
the solid agar in the ampoule(s) after the required
incubation time.
Data Analysis
• Collect digital image of the vials
– Digital Photo
– Image Scanner
• Create .pdf file
• Analyse image for color using “colors.exe”
– http://www.isao.com/pica.html
– Otaka I, Kumagai K, Inagaki Y, Shimoyama M, Saegusa K, Hara T (2002) Simple and inexpensive
software designed for the evaluation of color American Journal of Ophthalmology 133 (1): 140
– 142.
• Vector Analysis to quantify response
Digital Images
Positive
Control
Negative
Control
“Real Images”
• Full Test (Amoxicillin in DI water)
• Images of groups of vials
0.5, 1, 1 and 4 µg/L
6, 8, 10 and 20 µg/L
Positive control
Data Analysis
Data Analysis
• Trim the image and analyze the R, G, B
contribution of the average image
• ie, R = 0.74, G = 0.59, B = 0.63
Data Analysis
• Create a “3-D” plot of the data point, with
Red, Green, Blue as the axes
• Vector Analysis to analyze distance from
– Negative Control to the Data Point (Segment 1)
– Data Point to Positive Control (Segment 2)
Red
Seg 2
Seg 1
Negative
Control
Positive
Control
Data
Point
Blue
Green
Data Analysis
• Comparison of Response curve of Pennicillin
dissolved in lab water and Quabbin water
1
0.9
0.8
y = 3.9578x
R² = 0.947
Fractional Response
0.7
y = 3.648x
R² = 0.9935
0.6
Very minimal
interference in
“pristine, natural
water”
0.5
0.4
Penicillin in DI
0.3
Penicillin in Natural Water
0.2
0.1
0
0
0.05
0.1
0.15
Disolved Penicillin (mg/L)
0.2
0.25
Today’s Experiment
• 3 groups
• 1 group will be creating a calibration curve
– Positive (50 mg/L Penicillin), Negative (0 Penicillin)
– 100, 250, 400, 500, 700, 1000 ng/L
• 1 group will investigate potentially negative activity
impact of synthetic estrogen
– Positive (50 mg/L Penicillin), Negative (0 Penicillin)
– 1000 ng/L Penicillin + 50, 100, 250, 500, 1000, 2000 ng/L
EE2)
• 1 group will investigate potential false positives (H2O2)
– Positive (50 mg/L Penicillin), Negative (0 Penicillin)
– 0 ng/L Penicillin + 2, 5, 10, 50, 100, 250 mg/L H2O2
Hypotheses?
• Addition of a compound with unrelated
activity can negatively impact activity of an
antibiotic
• Addition of an inorganic antimicrobial agent
(H2O2) will not interfere with assay at “low”
concentrations
– Optimistic??