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EBOLA AND ANTIBIOTICS: Are they a hazard in sewage and biosolids?
Hannah Sassi, Charles Gerba, and Ian PepperThe University of Arizona
29th Annual BioFest -Ordinary People, Extraordinary Things
September 11-13Blaine, WA
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Implications of Ebola onHuman Health
2014 Outbreak in West Africa: Sierra Leone and Liberia
• 24,797 suspected cases
• ~12,000 laboratory confirmed cases
• 8,764 deaths
Guinea continues to see widespread transmission with 3,351 laboratory confirmed cases and 2,536 deaths (75.7% Case-fatality rate)
Ability to spread to US, UK, Italy, Spain, Nigeria, Mali and Senegal
Funded by CASA
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FATE OF EBOLA IN THE ENVIRONMENT
• Concern over exposure via contaminated sewage
• Is current guidance adequate for waste disposal down the toilet?
• Need to determine fate of Ebola during wastewater/biosolids treatment
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OBJECTIVE
• Survival in human waste that could be flushed down the toilet with and without disinfection
• Survival during mesophilic and thermophylic anaerobic digestion of sewage sludge
Utilize viral surrogates for Ebola to evaluate its fate during toilet disposal and biosolids treatment
APPROACH
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RATIONALE FOR SURROGATE
• Biosafety – level 4 required for Ebola• Surrogates selected based on similarity to
Ebola• In the same viral order • nucleic acid type• contain lipids• replication• Easy to grow in cell culture• Require only a Biosafety Level 2• Related to other emerging viruses
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SURROGATES
MS-2
Phi-6 (lipid containing phage)
Human Coronavirus (229E)
Parainfluenza type 1
Murine norovirus
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Impact of Flushing on Restroom Contamination
MS-2 coliphage
• Inoculum titer: ≈1xE+11 PFU
Collect toilet bowl sample of water
Water samples taken after 1, 2 and 3 flushes
Sample fomites in restroom for aerosolized virus contamination
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Hinge
Top of Toilet
Paper
Dispenser
Trash Bin Door
Wall Wall
Toilet paper
dispenser
Bowl water -all
surfaces
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Surface Contamination of Fomites
Geometric Mean Concentrations, by Sample Site (n=18)
Sample SiteMean SD (Log10 PFU) per surface (100 cm2)
Flush Handle* 1.65 0.91
Toilet Back 2.89 1.04Back Wall 1.63 1.36
Floor 3.44 1.08Toilet Paper Dispens. 1.49 1.41
Toilet Bowl Rim 3.88 1.59
Toilet Seat Top 4.21 1.26Toilet Seat Underside 4.22 1.26
*denotes 90cm2 total surface area
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Percent of Sites in which MS2 was Detected (N=18)
**Only 1/54 flush water samples positive. No positive water samples with ANY treatments.
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Disinfectant Efficacy for Toilet Water
Log-reduction (per mL) by treatment and contact time
Treatment 1 minute 15 minute 30 minuteChlorine Bleach 0.48 1.4 2.83
Hydrogen Peroxide 0.01 0.03 0.06
Quaternary Ammonium 1.99 1.93 2.22
Peracetic Acid 2.26 3.37 3.43
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Log Survival of MS2 by Tested Disinfectants
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TOILET STUDY CONCLUSIONS
• Flushing virus contaminated water leads to significant contamination of fomites within bathroom.
• Efficacy of disinfectants greatly reduced in presence of high organic load within toilet.
• Efficacy of disinfectants:– Peracetic acid > quaternary ammonium > bleach
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RECOMMENDATIONS
oDisinfection of waste should be practiced, when possible
oSurface disinfection still very important after flushing waste to eliminate fomite transmission potential
oPeracetic acid or quaternary ammonium should be used for short contact times (1 min)
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Survival of Viruses during Thermophilic and Mesophilic Anaerobic Digestion:
Assessing Potential for Emerging Viruses
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OBJECTIVEoEvaluate the influence of mesophilic and
thermophilic digestion on the reduction of five
viruses:
o MS2
o Phi 6 (6)
o Murine norovirus
o Poliovirus 1
o Adenovirus 4
oEvaluate using miniature anaerobic digestion
tubes
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Step 1: Addition of substrate
and digestion inoculum
Step 2: Addition of
viral surrogate
Step 3: De-oxygenation
using N2
Step 4: IncubationMesophilic-32ºC
Thermophilic-50ºC(agitation 120 rpm)
METHODS: Assembly of Anaerobic Digestion Tubes
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METHODS: Virus Assayso Cell line types by virus
o Poliovirus 1: BGMK cell line (Buffalo Green Monkey Kidney)
o Adenovirus 4: PLC cell line (Human Liver/Hepatoma)
o Murine norovirus: RAW cell line (Murine Monocyte/Macrophage)
o Adenovirus 4o 24-well cell culture plates; 0.8 mL
sample/wello 2.5 log10/mL detection limit per virus
o Incubated at 37 ºC with 5% CO2atmosphere (6-9 days)
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METHODS: Bacteriophage Assayso MS2: Double agar overlayo Phi 6 (6)o Host: Pseudomonas syringae 10205; 26 ºC
for 24-48 ho Double agar overlay using LB soft agaro 0.2 mL host+1 mL sample
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RESULTS: Mesophilic Digestion
VirusInitial
Concentration (virus/mL)
Concentration after digestion
(virus/mL)
Log10 Reduction (virus/mL) p-value
MS26.38E+08 2.83E+08 152 30 6.6 0.0001
6 1.13E+06 7.69E+05
5.9 0.0029
Adenovirus 4 2.13E+05 2.38E+05 917 574 2 0.0387
Poliovirus 1 3.47E+07 1.20E+07
1.47E+05 1.05E+05 1.8 0.0012
Murine norovirus
4.06E+06 1.20E+06
1.19E+04 3.54E+03 2.2 0.0016
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RESULTS: Thermophilic Digestion
VirusInitial
Concentration (virus/mL)
Concentration after Digestion
(virus/mL)
Log10 Reduction (virus/mL) p-value
MS26.38E+08 2.83E+08 53 17 7.1 0.0001
6 1.13E+06 7.69E+05
5.9 0.0029
Adenovirus 4 2.13E+05 2.38E+05 2.8 0.0399
Poliovirus 1 3.47E+07 1.20E+07 757 237 4.6 0.0013
Murine norovirus
4.06E+06 1.20E+06
4.1 0.0004
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SURVIVAL DURING ANAEROBIC DIGESTION -
CONCLUSIONSo Higher inactivation of bacteriophages than
animal viruseso Phi 6 (6)o Highly sensitive to both digestion typeso Indicative of lipid-containing virus behavior
(Ebola virus)o Lipid-containing viruses likely to be inactivated
at a greater level than non-lipid containing enteric viruses
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OVERALL CONCLUSIONSo Inactivation of Ebola virus in human wastes is difficult
due to high organic content
o Isolation and separation of human wastes may be necessary on a case-by-case basis
oControl spread to protect healthcare worker health in hospitals and wastewater treatment works in downstream processes
o Stronger disinfectants may need to be implemented in large-scale outbreak situations
o Surrogates for Ebola did not survive well during mesophilic or thermophilic digestion
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THE THREAT OFANTIBIOTIC RESISTANT BACTERIA:
A FRESH PERSPECTIVEIan Pepper1, John Brooks2, and Chuck Gerba1
1Water & Energy Sustainable Technology Center (WEST)2Genetics & Sustainable Agriculture Research Unit, USDA ARS
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BACKGROUNDAntibiotics:• Natural compounds produced by soil microorganisms that kill
or inhibit the growth of other competing microorganisms
History:• 1929 Alexander Fleming discovers penicillin isolated from soil
fungus Penicillium• 1943 Selman Waksman discovers streptomycin isolated from
the soil actinomycete Streptomyces
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Zone of inhibition of bacterial growth on aspread plate.
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BACKGROUND
Antibiotic Resistant Bacteria (ARB) with Antibiotic Resistance Genes (ARGs)
Antibiotic Bacterial cell
genetic or
mutational change
ARB ARB
ARB ARB
ARB ARB
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BACKGROUND:The Concern
• The more an antibiotic is used the greater the likelihood of antibiotic resistant strains
• The more the antibiotic is used to fight infectious disease, the less effective it becomes
• Of particular concern, are bacteria resistant to multiple antibiotics
e.g. Methicillin-resistantStaphylococcus aureus (MRSA)
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MICROBIAL PRODUCTION OF ANTIBIOTICS
• Vast majority of antibiotics synthesized by SOIL MICROORGANISMS‒ Penicillin effective on Gm +ve bacteria‒ Polymixin effective on Gm –ve‒ Chloramphenicol effective against Gm +ve and -ve
• Antibiotics produced by bacteria, actinomycetes and fungi• Utilized as major form of self-defense against other
indigenous soil microbes
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SOURCE OF ANTIBIOTICS ARBs and ARGs
Anthropogenic:• Sewage effluents and biosolids• CAFO effluent and animal manures• Hospital wastes discharged into sewers
Natural:• Soils• Water
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ENVIRONMENTAL ANTIBIOTIC RESISTANCE
• New term introduced in 2013• Caused by anthropogenic activity• Wastewater treatment plants blamed for increasing
“environmental antibiotic resistance”
Rizzo et al., 2013:“Urban Wastewater Treatment Plants as Hotspots for Antibiotic Resistant Bacteria and Genes Spread into the Environment: A Review”
IS THIS TRUE: a fresh perspective?
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NATURAL INCIDENCE OF ANTIBIOTICS IN SOIL
• Present in soil for over 3 billion years• Even pristine soils contain ARBs• ARBs within soil resistant to tetracycline,
ciprofloxacin, cephalothin and ampicillin ≃ 107
CFUs/g soil • Total numbers including non-culturable ARBs ≃
109 – 1010/g soil
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WE HAVE DATA ON THE INCIDENCE OF ANTIBIOTIC RESISTANT BACTERIA
(ARB)● soil ● groundwater ● biosolids ● chicken● compost ● hamburger meat●manure ● lettuce● household dust ● tomatoes
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BACTERIAL ANTIBIOTIC RESISTANCE
0 10
20
30
40
50
60
70
80
90
Biosolid
Compost
Dust
Manure
Soil Biosolid Never Appl
Soil Biosolid Appl
Well Biosolid App
Irrigation Well
Fresh Cow Manure
Ready to eat lettuce
Ground Beef
Chicken (Whole)
Tomatoes (Cherry)
Lettuce (Head)
Tap Water/mL
Antibiotic Resistant Bacteria (%)
Tetracycline %
Ciprofloxacin %
Cephalothin %
Ampicillin %
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HETEROTROPHIC PLATE COUNT BACTERIA (HPC) SAMPLE
CONCENTRATIONS
1.0
0E
+0
0
1.0
0E
+0
1
1.0
0E
+0
2
1.0
0E
+0
3
1.0
0E
+0
4
1.0
0E
+0
5
1.0
0E
+0
6
1.0
0E
+0
7
1.0
0E
+0
8
1.0
0E
+0
9
Biosolid Compost
Dust Manure
Soil Biosolid Never ApplSoil Biosolid ApplWell Biosolid App
Irrigation WellFresh Cow ManureReady to eat lettuce
Ground BeefChicken (Whole)
Tomatoes (Cherry)Lettuce (Head)Tap Water/mL
Log10 CFU/g or ml
HPC
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THE GREATEST CONCERN: LETTUCE
•Highest # ARBs/g•Eaten raw
•We eat more lettuce than biosolids
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HOMEOWNER ON A 1-ACRE LOT
Total bacteria:• One acre furrow slice = 2 x 106 lbs soil = 9 x 108 g soil• Assume 109 bacterial cells/g soil 109 x 9 x 108 bacteria/g soil≃ 1018 bacteria
ARBs:• 9 x 108 g soil in the acre furrow slice• Assume 107 ARB/g soil resistant to any individual
antibiotic≃ 1016 ARB resistant to any antibiotic
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Environmental Sample ARBs
(CFU/g or
CFU/ml)
ARGs
(# gene copies/g or
ml)*
Soil 106 – 107 108 – 109
Class B Biosolids 104 – 109 106 - 109
Wastewater Final
Effluents
101 – 103 0 – 104
Poultry Manure 103 - 104
Bovine Manure 106 - 108 103 – 109
Swine Manure 104 - 107
Example concentrations of ARBs and ARGs in environmental samples.
*Assumes 99% of ARB population non-culturable and 1 ARG per bacterial cell
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Sample ARBs
(CFU per acre furrow
slice)1
ARGs
(gene copies per acre
furrow slice)
Soil 1016 1018
Biosolids applied, 2 Tons Per Acre 2 x 1012 2 x 1013
1 Acre Foot Effluent 1 x 1011 1 x 1013
2 Tons Per Acre Solid Manure 2 x 1013 2 x 1015
1 Acre Foot Manure Effluent 2 x 1013
Land Application (% increase) ARBs ARGs
to Biosolids 0.02% 0.002%
to Effluent 0.001% 0.001%
to Manure 0.2% 0.2%
Impact of land application of sewage effluents and Class B biosolids on ARBs and ARGs in soil.
1Assumes one furrow slice = 2 million lbs (~9 x 108 g) soil and 107 ARBs per g soil
a)
b)
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Indigenous Pathogens Number
(per gram soil)
Number
(per acre furrow slice)
Bacillus anthracis 104 1013
Clostridium perfringens 900 1012
Pathogens Number
(per gram)
Number
(per 2 tons biosolid)
Thermotolerant coliforms (including E. coli) 106 1012
Fecal Streptococcus 106 9 x 1012
Salmonella 40 4 x 107
Effluent Number
(per L)
Number
(per acre foot)
Salmonella 3 x 102 3 x 108
Shigella 1 x 103 1 x 109
E.coli 3 x 105 3 x 1011
Manure Number
(per gram)
Number
(per 2 tons manure)
Thermotolerant coliforms (including E. coli) 102 1 x 108
Campylobacter jejuni 2 x 103 2 x 109
Listeria monocytogenes 6 x 102 6 x 108
Salmonella 2 x 103 2 x 109
Example concentrations of pathogens in soil, Class B biosolids and treated wastewater effluent.A) Soil
B) Class B Biosolids
C) Effluent
D) Fresh Manure
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Percent antibiotic resistant bacteria in land applied biosolids.
0
2
4
6
8
10
12
14
16
Prior toApplication
Day 0 Day 7 Day 14 Month 1 Month 2 Month 3 Month 4 Month 5 Month 6
% R
esis
tan
ce/g
Ampicillin
Cephalothin
Ciprofloxacin
Tetracycline
EFFECTS OF LAND APPLICATION OF BIOSOLIDS ON ARBs IN SOIL
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IMPACT OF SEWAGE EFFLUENTS AND CLASS B BIOSOLIDS ON PATHOGENIC ARBs IN SOIL
• Minimal ARBs added relative to what is already in soil• Number of enteric pathogens (e.g. E.coli) introduced via
effluent and biosolids is less than pathogens indigenous to soil (e.g. Bacillus anthracis or Clostridium perfringens)
• Enteric pathogens and ARBs introduced into soil normally die-off quickly
• When E.coli adapt to soil environment, pathogenicity lost
• Horizontal gene transfer in soil limited due to spatial separation of cells
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OTHER MICROBIAL ISSUES
Staphylococcus aureus
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THE STAPHYLOCOCCUSSTORY: FACTS
Staphylococcus aureus
• Gram positive coccus
• Commonly found within nose of healthy people
• Can result in minor or major skin infections
• To date, no scientific data or epidemiological study has been published linking S. aureus to land application of biosolids
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THE STAPHYLOCOCCUSSTORY: ALLEGATIONS
• S. aureus is found in biosolids
• S. aureus from biosolids results in adverse public health affects
• S. aureus from land applied biosolids has resulted in deaths
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S. AUREUS RESEARCH AT THE UNIVERSITY OF
ARIZONA
• S. aureus found in 3 of 5 sewage samples (60% incidence)
• S. aureus never detected in 23 biosolid samples (8 Class A and 15 Class B) (0% incidence)
• S. aureus never detected in bioaerosol samples (0% incidence)
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S. AUREUS RESEARCH ATTHE UNIVERSITY OF
ARIZONA—Discussion
• This study provides scientific evidence for the absence of S. aureus in land applied biosolids
• It shows that biosolids are not a source of S. aureus human exposure
• Rusin, P., S. Maxwell, J. Brooks, C. Gerba, and I. Pepper. (2003) Evidence for the Absence of Staphylococcus aureus in Land Applied Biosolids. Environ. Sci. Technol. 37:4027–4030.