Antibiotics in the Environment by cuiliqing

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									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                         Typical Uses
Inhibit Bacterial Cell Wall    b-lacatams, cephalosporins,   Human therapeutic and animal
  Biosynthesis                 glycopeptides, everninomycins therapeutic/ subtherapeutic
Block 30S or 50S               Macrolides, streptogramins,      Human therapeutic and animal
  Ribosomes (inhibit protein   chloramphenicol, fusidic acid,   therapeutic/ subtherapeutic
  synthesis)                   tetracyclines, lincosamides,
                               aminoglycosides,
                               oxazolidinones,
Block Replication of DNA       Quinolines, rifamycines          Treat urinary tract, systemic,
                                                                and respiratory infections
Disrupt Integrity of Cell      Antifungal azoles, polyenes      Treat fungal infections, topical
  Membranes                                                     application for Eczema
Antagonize Metabolic           Sulfonamides,                    Humans, livestock,
  Processes                    trimethoprim (TMP)               aquaculture; TMP and
                                                                sulfamethoxazole often
                                                                prescribed together
Antibiotics in the Environment
                                                     Antibiotic
                                                    Manufacturers




Livestock Feed
                                  Veterinarians                                        Phamacies
Manufacturers



                 Livestock Use                                                         Human Use



                  Injestion/                          Injestion/                                                         Topical
                                                                                     Flushing unused
                  Excretion                           Excretion                                                        application




                 Treatment of                                                                          Individual WW
                                                                    Municipal WW
                   Manure                                                                                (ie septic)



   Treated                       Land Application                                                         Treated
                                                                       Runoff
  Discharge                         of Solids                                                            Discharge



                                                     Leaching to                     Surface Water
                                                    Groundwater                      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     b-lacatams, cephalosporins,        Human therapeutic and animal
  Biosynthesis                  glycopeptides, everninomycins      therapeutic/ subtherapeutic
Block 30S or 50S Ribosomes      Macrolides, streptogramins,        Human therapeutic and animal
  (inhibit protein synthesis)   chloramphenicol, fusidic acid,     therapeutic/ subtherapeutic
                                tetracyclines, lincosamides,
                                aminoglycosides, oxazolidinones,

Block Replication of DNA        Quinolines, rifamycines            Treat urinary tract, systemic, and
                                                                   respiratory infections
Disrupt Integrity of Cell       Antifungal azoles, polyenes        Treat fungal infections, topical
  Membranes                                                        application for Eczema
Antagonize Metabolic            Sulfonamides,                      Humans, livestock, aquaculture;
  Processes                     trimethoprim (TMP)                 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                   Yellow Yellow with 50/50   Purple with   Purple
                               some purple         some yellow
Antibiotic effect (%)     0        25        50         75         100
Comparative              <25       40       50.7        63         >100
erythromycin
potency (mg/L)
Comparative              <40       40        100       155         >250
sulfamethoxazole
potency (mg/L)
       Smith et al, 2007 (cont.)
• Calibration curves
• Surface Water Samples
Other similar antibiotic activity assays

 Antibiotic   Manufacturer Time per test   Antibiotic   Reaction
 Detection                                 Tested       Type
 Assay
 Delvotest SP DSM          2.5 hours       Broad        Color Change
 Kit                                       Spectrum
 Premitest    DSM          3.5 hours       Broad        Color Change
                                           Spectrum
 Copan test  Copan Italia 2.5 – 3 hours    Broad        Color Change
             SpA                           Spectrum
 Charm Farm Charm         2.5 – 3 hours    Broad        Color Change/
 Test        Sciences                      Spectrum     pH Change
 Charm AIM – Charm        4 hours          Broad        Color Change
 96          Sciences                      Spectrum
                 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
                               Positive
                       Seg 2   Control

               Seg 1
                       Data
                       Point
    Negative
    Control
                                 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
                       0.7                                    R² = 0.947
 Fractional Response




                                                                           y = 3.648x
                       0.6                                                 R² = 0.9935

                       0.5
                                                                                                          Very minimal
                       0.4
                                                                                                          interference in
                                                                     Penicillin in DI
                                                                     Penicillin in Natural Water
                                                                                                          “pristine, natural
                       0.3
                                                                                                          water”
                       0.2

                       0.1

                        0
                             0   0.05    0.1                 0.15                  0.2             0.25
                                        Disolved Penicillin (mg/L)
              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??

								
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