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

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

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

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

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

SURROGATES

MS-2

Phi-6 (lipid containing phage)

Human Coronavirus (229E)

Parainfluenza type 1

Murine norovirus

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

Hinge

Top of Toilet

Paper

Dispenser

Trash Bin Door

Wall Wall

Toilet paper

dispenser

Bowl water -all

surfaces

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

Percent of Sites in which MS2 was Detected (N=18)

**Only 1/54 flush water samples positive. No positive water samples with ANY treatments.

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

Log Survival of MS2 by Tested Disinfectants

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

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)

Survival of Viruses during Thermophilic and Mesophilic Anaerobic Digestion:

Assessing Potential for Emerging Viruses

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

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

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)

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

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

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

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

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

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

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

Zone of inhibition of bacterial growth on aspread plate.

BACKGROUND

Antibiotic Resistant Bacteria (ARB) with Antibiotic Resistance Genes (ARGs)

Antibiotic Bacterial cell

genetic or

mutational change

ARB ARB

ARB ARB

ARB ARB

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)

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

SOURCE OF ANTIBIOTICS ARBs and ARGs

Anthropogenic:• Sewage effluents and biosolids• CAFO effluent and animal manures• Hospital wastes discharged into sewers

Natural:• Soils• Water

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?

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

WE HAVE DATA ON THE INCIDENCE OF ANTIBIOTIC RESISTANT BACTERIA

(ARB)● soil ● groundwater ● biosolids ● chicken● compost ● hamburger meat●manure ● lettuce● household dust ● tomatoes

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 %

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

THE GREATEST CONCERN: LETTUCE

•Highest # ARBs/g•Eaten raw

•We eat more lettuce than biosolids

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

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

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)

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

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

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

OTHER MICROBIAL ISSUES

Staphylococcus aureus

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

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

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)

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.