New Testing Methods for Microorganisms and Pathogens

Dr. Joan B. Rose Homer Nowlin Chair in Water Research

Michigan State Universityrosejo@msu.edu

I am a Water Pollution & Public Health Microbiologist

I study the fecal pollution of water and the spread of waterborne pathogens to humans due primarily to recreational and drinking water contamination.

Began working in partnership with public health laboratories early in my career.

In 2002 I moved to Michigan beganworking in collaboration with County Health Laboratories, DEQ and Great Lakes Beachnet.

THREATS TO THE COASTAL GL Sewage; Combine sewer overflowsStorm water; non-point source pollutionInadequate InfrastructureInvasive speciesAlgal bloomsClimate change

Waterborne pathogens threaten human health in the Great Lakes region

Campylobacter, Arcobacter,

GiardiaLegionella

Cryptosporidium

Toxic Algal blooms

Norovirus

E.Coli 0157H7 and

Campylobacter

A sample glass of Lake Erie water is photographed near the Toledo water intake crib in Lake Erie. (Haraz

N. Ghanbari/Associated Press)

Improving Water DiagnosticsMoving from Escherichia coli (E. coli) and Enterococci to qPCRGeneral indicators of fecal contamination using Membrane filtration, IDEXX Colilert® Quanti-Tray 2000®Specific targets for pathogens and microbial source tracking markers using PCR

Building qPCR capacity in MI

2014Michigan funds new labs

2015Train-the-trainer

2016 Lab trainingqPCR validation study

2017Colilert + qPCR analysis during beach season

Beach samples frozen for later analysis

The qPCR multi-lab validation studyDr. Shannon Briggs, DEQ and Dr. Erin Dreelin, MSU

Our Network ofMichigan qPCR LabsMarquette Area Wastewater Treatment PlantLake Superior State UniversityNorthwest Michigan Regional LabNPS- Sleeping Bear DunesCentral Michigan Health DistrictFerris State UniversitySaginaw County Dept of Public HealthSaginaw Valley State UniversityGrand Valley State UniversityHope CollegeKalamazoo County Health & Community ServicesMichigan State UniversityUSGS- LansingOakland County Health DepartmentOakland University

Our Network ofCollaborators

Training Labs: Feb 2016 @ MSU; Developing publications on the efforts.

Sivaganesan, M., R. Haugland, T.G. Aw, K. Oshima, E.A. Dreelin, S. Briggs, et al. Global data quality acceptance criteria for a draft U.S. EPA E. coli qPCR method from multiple laboratory analyses of standardized reference and control materials.

Aw. T.G, R. Haugland, M.Sivaganesan, E.A. Dreelin, S. Briggs, K. Oshima, et al. Evaluation of multiple laboratory performance and variability in analysis of Michigan recreational waters by an E. coli qPCR method.

How would you rate your confidence in your skills for completing each step of qPCR?

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Pipetting Preparingreagents

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E. coli Calibrator Ct’s and acceptance range

• Y-axis: laboratories• X-Axis: CT VALUEs• Horizontal lines: 95%

Bayesian Credible intervals

• Hash Marks: Mean Ct values

• Red Highlighted Results excluded from Determination of acceptance range

• Acceptance Range: 26.49 - 29.63

Phase 2 Results: total method variability

Color key for quantification models:Blue = ΔΔCtBrown = master standard curve

Dots = mean log10copies/reactionLines = 95% BCI*

*Bayesian credible interval

Sources of E.coli and Pathogens

Agricultural run-off

Animal farming operations

Waste water/Sewage treatment Septic systems

Combined Sewer OverflowWildlife

What’s going on with Microbial Source Tracking

(State of Michigan MST capacity building 2018)Dr. Jean Pierre Nshimyimana, MSU

State of Michigan provided $230,000 for: • Providing MST training for lab staff performing molecular methods (e.g. Method

C) for local health departments in Michigan.

• Conducting a validation study to test proficiency of MST trained laboratories.

• Using the validation study to test effectiveness of the training provided to labs.

• Conducting a study to validate the performance of MST assays for animal source tracking in Michigan (cattle, pig, dog, chicken, deer, goose, and gull)

How Do We Apply Bacteroidales-associated Genetic Markers

for Microbial Source Tracking?

Source: MSU-MST-Fact Sheet & Nshimyimana et al. 2017

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Water Diagnostics using digital droplet polymerase chain reaction

ddPCR for Source Tracking

• B.theta for human sewage

• M2 bovine marker

• > 10,000 tests (droplets) per well

Sewage Positive

Cow manure Positive

Positive for both genes

NegativeNazarian, E.J., et al. (2008). Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples. Diagn. Microbiol. Infect. Dis. 62:125-132.

The distribution of the human sewage marker

Bacteroides

• Increasing B. theta related to more septic tanks

• More E.coli related to more total phosphorous and increasing stream temperature

Significant Knowledge Gaps Exist for Septics

a) Base flow

b) Summer rain

Transport of animal feacal source tracking markers during summer rains 64 watersheds

Sources

Microbiota

Community DNA

Metagenome library & sequence analysis

ALL ENVIRONMENTS INCLUDING WATER HAVE A MICROBIOME

Basic features and performances of NGS platforms

Modified from Kulski, 2015

NGS platform (company)

# Reads/run

Read length (bp)/run

Cost (USD)/106

bases

Raw error rate (%)

Platform cost (USD)

1st generationSanger (Life technologies) 1 800 2,400 0.3 95,000

2nd generation454 GS FLX (Roche) 1 x 106 700 10 1 500,000

HiSeq(Illumina) 5 x 109 2 x 150 0.1 0.8 750,000

MiSeq(Illumina) 3 x 108 2 x 300 0.13 0.8 125,000

…..3rd generationSMRT (Pac Bio) 1 x 106 >10,000 2 12.9 750,000

Nanopore(Oxford) 6 x 104 >5,000 <1 34 1,000

…..

examples of 2nd

generation sequencers

• Illumina- Amplified single-molecule sequencing platform

Images from Illumina

An example of 3rd generation sequencer

• Oxford Nanopore- Single-molecule sequencing platform- Powered and operated by a laptop via a USB- Low cost for reagents and instrumentation

(USD 1,000)- Longer reads (average 5 kb)- Real-time sequence analysis

Images from University of Oxford

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Dr. Pan JI

• Portable device and fast library prep available (can be less than 10 min);

• ‘Real-time’ data analysis- does not have a fixed run time;

• Ultra-long reads (longest read reported by MinION user is 950 kb);

• Other advantages (low starting cost etc.).

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MS2 ssRNAvirus

MinIONTM by OxfordNanopore Technology30 min run, max at 120 min

Recreational

Irrigation

Drinking

FRESH WATER RESOURCES ARE DEGRADING

Ecosystems

By Todd C. Frankel August 11 2014Follow @tcfrankel The Washington Post

A sample glass of Lake Erie water is photographed near the Toledo water intake crib in Lake Erie. (Haraz N.

Ghanbari/Associated Press)

AcknowledgementsErin DreelinBecca IvesJP NshimyimanaTiong AwRose GANG

THANK YOU