Overview of Microbial Monitoring Technologies

Considered for Use Inside Long Duration Spaceflights and

Planetary Habitats

Monsi C. Roman

NASA ECLSS Chief Microbiologist

C. Mark Ott, PhD

Microbiology Laboratory

NASA Johnson Space Center

4/29/2015 2

Microbial Monitoring in Long Duration Missions

The purpose of this presentation is to start a conversation including the Crew Health, ECLSS, and Planetary Protection communities about the best approach for in-flight microbial monitoring as part of a risk mitigation strategy to prevent forward and back contamination while protecting the crew and vehicle.

Will help set future:

Resource allocations

Monitoring requirements

Minimize duplication of monitoring technologies for use in space

Foster complementary monitoring technologies

Prevention is Important

Regular housekeeping/disinfection

Education of the crew

Minimize conditions that promote growth

Thorough ground disinfection

PreventionDesigned to Meet Current Requirements

•4

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Short-term Effects of Microbial Exposure (days to weeks)

Air/Surfaces:

• Release of volatiles (e.g., odors)

• Allergies (e.g., skin, respiratory)

• Infectious diseases (e.g., Legionnaire’s)

Water:

• Objectionable taste/odor

Long-term Effects of Microbial Exposure (weeks to years)

Air/Surfaces (same as short-term plus):

• Release of toxins (e.g., mycotoxins)

• Sick building syndrome

• Environmental contamination

• Biodegradation of materials

• Systems performance

Water (same as short-term plus):

• System failure

• Clogging, corrosion, pitting, antimicrobial

resistance/regrowth potential (biofilm)

So…Why Are We CurrentlyMonitor Microorganisms?

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Microbial Monitoring Design Considerations*

“Even in high quality water supplies protected by a residual bactericide, viable

organisms can still persist. Therefore, the potential for microbial overgrowth is an

ever-present hazard. Due to the long potential unmanned loiter time contributing

to the duration of flights, routine microbiological monitoring of potable water

coinciding with the re-ocupation by the crew to ensure that it meets the standards

outlined in Table 7.2.3.2-1 and section 5, Natural and Induced Environments, for

microbiological limits may be necessary.”

The document also addresses the potential for BIOFILM formation

*Reference: NASA-STD-3000 Volume VIII- Human-Systems Integration Standards

for the Crew Exploration Vehicle

Current in-flight microbial monitoring

technology is good but it:

Provides only a partial assessment of the microbial

population as it detects the fraction of microorganisms

that will grow in the selected media

Is crew time intensive

Produces a biohazardous waste as microorganisms are

grown in flight

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Microbial Monitoring in Long Duration Missions

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Current US In-flight Microbial Monitoring Capabilities

Water Microbiology Kit (WMK)

Membrane filtration/ 48 hours incubation/ visual analysis

Sample collection/ processing: 122.5 min/ 62.5 min

Water Microbiology Analysis Kit (WMAK)

Presence/absence analysis using Colisure

Final result reported in 24 to 48 hours

Surface Sampler Kit (SSK)

Contact slide or swab/ 48 hohurs incubation/ visual analysis

Sample collection: 100 min; analysis: 220 min

Microbial Air Sampler (MAS kit)

Impaction sampler/ incubation 5 days/ visual analysis

Sample collection: 135 min/ analysis: 220 min

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Current Microbial In-Flight Analysis

No coliformcoliform

ISS Air and Surface MonitoringFungal Isolates

Pierson, et. al. Environmental Monitoring: A Comprehensive Handbook 2012 10

ISS Air and Surface MonitoringBacterial Isolates

Pierson, et. al. Environmental Monitoring: A Comprehensive Handbook 2012 11

U. S. Potable Water Dispenser

Provides “hot” and “ambient” potable water

Processing includes: Catalytic oxidizer

Iodine disinfection

In-line filter (0.2 micron)

Common isolates Ralstonia pickettii

Burkholderia multivorans

Sphingomonas sanguinis

Cupriavidas metallidurans

12

Stakeholders for In-Flight Microbial Monitoring Technology

Crew Health

Life Support Systems-system Health/Environmental

Internal Coolant/Environmental

Experiments/Payloads

Astrobiology and Planetary Protection

Spaceflight Food

Sample Collection

•Use ISS same bags

•Transfer of the sample from the bag to the MMS needs to be addressed

•Detection limit/sample size

•Address microbial viability?

Sample Preparation

•Address microbial viability?

•Extraction of genetic material

•No Crew intervention

PCR

•Processing of genetic material

ANSWER

•WHAT IS THE SAMPLE?

•Address microbial IDENTIFICATIONof VIABLEorganisms

Steps for MMS to meet Medical Requirements

Microbial Monitoring System (MMS)

14

Sample Collection

• Use ISS same bags

• Transfer of the sample from the bag to the MMS needs to be addressed

• Detection limit/sample size

• Address microbial viability?

Sample Preparation

• Extraction of genetic material

• No Crew intervention

PCR

• Processing of genetic material

ANSWER

• HOW MANY ORGANISMS IN THE SAMPLE?

• Address microbial ENUMERATION

Steps for MMS to meet Engineering Requirements

Microbial Monitoring System (MMS)

15

Sample Collection

•Use ISS same bags

•Transfer of the sample from the bag to the MMS needs to be addressed

•Detection limit/sample size

•Address microbial viability?

Sample Preparation

•Address microbial viability?

•Extraction of genetic material

•No Crew intervention

PCR

•Processing of genetic material

ANSWER

•WHAT IS THE SAMPLE?

•Address microbial IDENTIFICATIONof VIABLEorganisms

Microbial Monitoring System (MMS)

16

Sample Collection

• Use ISS same bags

• Transfer of the sample from the bag to the MMS needs to be addressed

• Detection limit/sample size

• Address microbial viability?

Sample Preparation

• Extraction of genetic material

• No Crew intervention

PCR

• Processing of genetic material

ANSWER

• HOW MANY ORGANISMS IN THE SAMPLE?

• Address microbial ENUMERATION

QPCR can support both

MED

REQ

ENG

REQ

WHAT ARE THE DIFFERENCES?

Current Hardware Efforts

Two DNA based microbiological monitoring

systems are being evaluated under the ISS 2 x

2015 technology demonstration initiative

One effort is evaluating the RAZOR QPCR system

developed by Biofire Diagnostics

One effort is evaluating the MinION system

developed by Oxford Nanopore

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2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026

Microbial Monitoring (Air, Water, Surface)

18

Fiscal Year

• Objectives/FOMs: Non-culture based in-flight microbial monitor (enabling), in-flight species id (enabling), minimal crew time <1 hr/sample (enhancing), minimal consumables (enhancing), fast response time <6 hrs(enabling), distinguish viable from non-viable species (enabling for ECLSS); consumables shelf life >3 yrs

Assmnt of Microbial Viability Tech (for

Medical Req)

Flight Operations

Microbiology Requirements for Exploration (Water, Air, Surfaces)

MiDASS(ESA)

Razor (QPCR)Meets

Medical ID Req?

Trad

e,

do

wn

-se

lect

Other MicrobialID Systems (test strip, Mini sequencers, etc)

Ground Test

WetLab IIPCR Flight

UnitMeets

ECLSS/Med Req?

Missions Enabled:- ISS operations- Extending Reach Beyond LEO

- Initial Orion missions- Into the Solar System

- NEA < 1 mo- NEA > 1 mo

- Exploring Other Worlds- Lunar < 1 mo- Lunar > 1 mo- Phobos/Deimos

- Planetary Exploration

System for Exploration

Yes

Ground Test

Flight Demo

iATPMeets

ISS ECLSS Quant

Needs?

Ground Test

Yes?

Assmnt of Microbial Concentrator Technologies

Automate?

Trad

e, d

ow

n-s

elec

t fo

r Ex

plo

rati

on

Flight Demo (COTS)

Yes?

NO

ViabilityDetection Limits

Quantify

Combination of technologies for

Exploration (PCR + ATP) Other?

Work needs to be continued?

NO

ISS Microbial Kits (based on culture) will be used as back-ups until they can be completely replaced

Flight Demo (COTS)

Yes?

Yes?

Mini DNA Sequencer

Flight Demo

Ground Test

Lessons Learned

No single technology may provide the needed data (“a silver bullet solution”); combination of multiple technologies may provide the best approach

• Assessment of viability important for crew health• Enumeration is important to assess hardware performance• Science community wants/needs are different to operational

needs• Hardware for “day to day” operations needs to be simple

sample to answer equipment

Defining the requirements of all stakeholders is essential. For example, crew health requirements using non-culture based methodologies do not exist.

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Lessons Learned

• Changes in mission architecture can cause changes in monitoring requirements.

• In the search for new technologies, in-flight sample collection and processing are often under emphasized.

• Detection limits can be a challenge (Sample size, etc)

• Chosen technologies need to be extensively validated in the proper environment with appropriate samples prior to use in long duration missions.

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Back-up Material

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Issues that Need to be Addressed

Microbial count (quantification) Viable vs non-viable

How will it compare with culture methods?

Real-time identification Bacteria, Fungi, Viruses

Flexible Integrated to systems (in-line)

Hand-held (for clinical applications)

Robustness Will the hardware survive qual/acceptance testing?

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Issues that Need to be Addressed (cont’d)

If gene-base technology will be used what challenges, like damage to genetic material due to radiation, will need to be addressed?

Expendables (how much waste will be generated)

Consumables (reusable is preferred)

Low power consumption

Equipment size

Non-hazardous reagents

Non-generation of hazardous waste

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Issues that Need to be Addressed (cont’d)

Calibration (positive/negative controls?)

Cleaning/disinfection of the sample collection areas How to avoid cross contamination?

What chemicals/conditions(temp, humidity, etc) could cause a problem (void the reaction)?

Maintenance/repair (ORU’s?)

Construction materials Are the materials acceptable in a close environment?

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Issues that Need to be Addressed (cont’d)

Sample size

Detection limit (currently <300 CFU/100 mL)

Microgravity sensitivity

Sensitivity to particles/precipitates in the fluid

A system that can be upgraded as needed is preferable (as “target” organisms are identified)

Will the crew be able to “read” the results on-orbit; can the results be sent to the ground?

Sample archival for later analyses