Chapter outline: Sampling of Wastewater and Biosolids for Bacteria and Viruses

Proposed title:  Sampling of Wastewater and Biosolids for Bacteria and Viruses

Chapter no.: _________

Author(s):       John Scott Meschke

                        Department of Environmental and Occupational Health Sciences

University of Washington

                        4225 Roosevelt Way NE, suite 2338

                        Seattle, WA 98105-6099

Notes:

·          Material in this chapter may overlap with other planned chapters on Detection of Pathogens in Sludges, etc. (Judy Blackbeard) and Assessing the Efficacy of Wastewater Treatment (Wesley Pipes). 

·          Content covered in this chapter will include microbiological sampling considerations, primary and secondary concentration techniques, and separation/purification processes for target microbes in raw waste water, primary, secondary, and final effluent, and biosolids.

·          Techniques relevant to viruses, bacteria, and eukaryotic microbes will be covered. Application to both pathogenic and non-pathogenic targets will be discussed.

·          Culture and other detection techniques will not be covered in detail (rather they will only be referred to as they related to the upstream sampling, concentration, and purification techniques).

Proposed topics

1)     Introduction: overview of wastewater treatment (purpose, processes, communities involved, etc.), matrix description/characterization (raw wastewater, primary effluent, secondary effluent, final/tertiary effluents, biosolids), etc.

2)     Relevant sampling schemes:  purpose/rationale for sampling, types of sampling schemes and appropriate uses, relevant regulation, qualitative and quantitative approaches, etc.

3)     Methods for Sampling of Viruses

a.      Viral Targets: pathogens and phage

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Sample Collection: sample volumes, sample preservation, time to processing, etc.

                                                  ii.     Concentration Methods:  filtration, precipitation, acid adsorption/elution and centrifugal methods

                                                iii.     Separation/Purification Techniques: phase separation, organic extraction, serial filtration, gradient centrifugation, etc.

c.      Biosolids

                                                    i.     Sample Collection: sample mass, preservation, time to processing, etc.

                                                  ii.     Elution/Extraction Techniques

                                                iii.     Separation/Purification Techniques: phase separation, organic extraction, serial filtration, gradient centrifugation, etc.

d.     Nucleic Acid Purification

4)     Methods for Sampling of Bacteria

a.      Bacterial Targets: indicator bacteria, pathogens, process bacteria (active and nuisance)

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Concentration and Enrichment Methods

                                                  ii.     Separation Methods

c.      Biosolids

                                                    i.     Elution/Enrichment Methods

                                                  ii.     Separation Methods

d.     Nucleic Acid Purification

5)     Methods for Sampling of Eukaryotic Microbes

a.      Eukaryotic Targets: algae, fungi, protozoa, helminth ova

b.     Raw Wastewater; Primary, Secondary, Advanced, and Final Effluents

                                                    i.     Concentration Methods

                                                  ii.     Separation Methods

c.      Biosolids

                                                    i.     Elution Methods

                                                  ii.     Separation Methods

d.     Nucleic Acid Purification

6)     Use of Sampling and Recovery Controls

Chapter Highlights

The following concepts will be conveyed in this chapter:

1.  Matrix effects of sampling methods

2.  Impact of target on sampling methods

3.  Sampling for whole cell or culture based detection versus nucleic acid based detection.

Chapter outline: Animal Gut Microbiomes

Animal Gut Microbiomes--Marchesi

Focus on livestock and companion animals

Introduction

Focus and benefits of studying animal gut microbiota:

·     Comparison with other animals and humans – relating to phylogeny and dietary differences

·     Understanding and improving feed conversion and nutrition (diet)

·     Controlling methane production in ruminants

·     Understanding enteric disease (infection > imbalance)

·     Public Health Microbiology (carriage of zoonotic agents)

·     Novel pathogen discovery (bacterial and viral?)

·     Impact of antibiotics, vaccines, etc

·     Sample sizes (number of replicates) can be much larger than human studies

Sampling considerations

·     Faecal samples are readily available but exposure to air means they may not be completely representative of communities in the gut.

·     Individual gut sections and/or luminal contents can be sampled during post-mortem examinations or via surgical fistulations – different regions will harbour different microbial communities and will have different physicochemical conditions.

·     Methods for selective recovery of planktonic and adherent microbial communities from digesta.

Detection of specific microorganisms of interest (e.g. enteric pathogens)

·     Bacteria (e.g. Salmonella, Campylobacter, etc)

·     Viruses and bacteriophage

·     Protozoa

·     Archaea

·     Yeasts and Fungi

Microbial community analysis

·     Nucleic acid extraction methods

·     Metagenome shotgun sequencing (including library construction and screening)

·     Targeted amplicon sequencing (e.g. 16S, Fungal ITS, AMR genes, etc)

·     RNA extraction, enrichment and analysis methods

·     Data analysis and interpretation

Metabolism of the gut microbiome

·     Methanogenesis and other hydrogen sinks in the GI tract

·     Role of hydrogen consuming organisms in the rumen

·     Microbial groups which utilise hydrogen

·     Approaches and methods for characterisation of the different functional groups

·     Biotransformation and biodegradation of phytochemicals (that toxify and detoxify)

·     Significance of microorganisms that metabolise phytochemicals.

Metaproteomics

·     Extraction methods

    Analysis

Chapter outline: Surface Sampling

Proposed topics for “Surface Sampling” chapter                   L. J. Rose, M.J. Arduino, J. Noble-Wang

Proposed as an overview; not a “how to,” but instead a “principles of surface sampling” approach.

Objectives:      1) present general methods, tools and approaches to surface sampling for a variety of purposes, and    2) discuss relevant caveats to be considered before, during and after sampling.

1.     Who, what, where and when of surface sampling : food industry, healthcare, aerospace, biothreat investigation, etc., and current industry standards for surface sampling

2.     Why surface sampling is needed: monitor, quantitate, identify

3.     The How of sampling:

a.     Preparing to sample: Define goal/approach (sampling strategy)

b.     Qualitative sampling: Sensitivity, specificity

c.      Quantitative sampling: Efficiency, level of detection, strategy (number of replicates, field blanks  controls , consistency of technique)

d.     Considerations: Surface Area, roughness, porosity, composite samples, efficiency of device for target organism(s), use of neutralizers, viability of organism on surface

4.     Tools and general steps for sampling; advantages, disadvantages and  limitations of each:

a.     Non porous surfaces: swabs, wipes, sponges, contact plates

b.     Porous surfaces: vacuum socks, filter cassettes, forensic filter devices, wet vacuum

5.     Transport to laboratory: dry spores, vegetative cells, viruses

6.     Elution: shaking, vortexing, sonicating, stomaching

7.     Analysis: Considerations for choosing   

a.     Culture: Competition from other organisms, selective media, broth enrichment, neutralizers, ability of organism to be cultured.

b.     Molecular detection: Inhibition by environmental matrices (humic acid, metals, etc.)

8.     Interpretation of data: limitations   

 

 

 

 

Chapter outline: Sampling of Airborne Microorganisms

Sampling of Airborne Microorganisms--Mainelis

 

Introduction

Interest and need to sample in many and diverse areas:

agricultural and industrial settings, medicine, home and office environments, and military research; surgeries; role of microorganisms in air processes; hospitals; food industry

Define "bioaerosol"

No single method to collect all; no single method to analyze; No standardized protocol.

Human exposure limits have not been established

The purpose of this chapter

Present common and known samplers as well as recently introduced samplers and techniques. Active field; Describe advantages and disadvantages of common techniques; present some comparative studies

Physical and biological components of sampling

Efficient removal from the air stream while preserving the characteristics critical for enumeration and identification

Bioaerosol motion and behavior the same as for non-biological particles

                           Bioaerosol characteristics: single, aggregates, attached to other particles

Viable and non-viable; sampling technique has to be suited for intended purpose and analysis technique

Common notes

Purpose of sampling

Common concentrations

Standards do not exist; no single method can be applied; differences in sampling flow rate, recommended sampling time, media used.

Sampling methods (description of methods)

Impaction: how it works. Depends on inertial properties and physical parameters of the impactor

Impingement (liquid techniques); Usually high inlet velocities and agglomerates can be broken up.

Filtration; can collect smaller particles than the pore of a filter

Gravitational

Pollen traps

Electrostatic collection

Sampler types

Intro: wide variety of samplers is available. Selection depends on sampling method, expected concentration, sampling media and analysis method. Guidelines published to help with selection

Impaction samplers: most commonly used and a variety are commercially available.

Stationary

Andersen 6, 2 and 1. Andersen-type by other companies (Tisch,SKC Inc., others); Single-use agar impactor.

Portable; single stage only;

Use of Andersen N6 as portable

Slit impactors depositing on agar surface

Spore-traps

Impingers (liquid techniques); sample can be diluted and analyzed by several different techniques.

Traditional impingers; some examples; high velocity; impact and violent motion of liquid

BioSampler; combines impingement with centrifugal motion. Can be used with viscous fluids

Agranowski's sampler

Cyclones with liquid(Bioguardian, SpinCon, OMNI, Coriolis)

BioCapture 650

Wetted-wall cyclones (SASS 2300 and 2400; XMC; McFarland's sampler)

CIP-10

Filter samplers

Filter cassettes

 Could be used as personal samplers due to small size

  Filter types and cassette types;

Gelatin filter; MD8 is designed for gelatin. 25 mm gelatin filter has been used with filter cassettes of that size.

Use of PUF

InnovaPrep filter sampler

Electrostatic samplers and techniques

Brief history

Main developments. Low flow rate and high flow rate samplers. EPSS (collector into small amount of liquid)

Integrated and autonomous samplers; Developed for biodefense purpose.

APDS;

Joint BPDS

Others?

Sampler performance

Many studies have been performed

Different samplers, different organisms, different references; sampling time and volume

When several different samplers are operated simultaneously  and the same method is used for sample analysis, we can make comparisons about their performance.

Laboratory studies

Field comparisons

Physical and biological components

Physical: inlet efficiency and collection efficiency. Inlet efficiency: moving air vs calm air; Isokinetic sampling is desired, but not always achievable. 

Biological efficiency:

effect of sampling on culturability.

Effect on sample integrity

What d50 represents. How it should relate to size of the microorganism to be collected. Theoretical and experimental d50  for inertia-based samplers; Theoretical d50 has been calculated for many samplers.

Typical collection efficiencies.  Agar parameters affecting the efficiencies

Marple's design criteria. Impactors with very low S/W

Biological efficiency and sampling stress.

Stress with filters. Loss of culturability, but recovery can be improved by using certain culture media.

The length of collection time.

Effect of sampling method on microorganism diversity

Advantages and disadvantages of impactors;

Direct collection

Culturable only. Non culturable could be a substantial component

Bounce;  particle bounce from agar? If agar dries up.

Clumping

Electrostatic forces

Overloading

Spore traps. Particle bounce. Contributes to non-uniformity of spore distribution and difficulties in enumeration by microscopy.

Advantages and disadvantages of liquid samplers

Can handle high concentrations. Can be diluted for analysis with multiple and different methods.

Impingement (amount of liquid) and loss of liquid over time; mineral oil;

Reaerosolization; internal losses.

Concentration rate and factor for liquid samplers; traditional impingers have low d50, but also low concentration rate; high-flow impingers and high concentration rate

Filter samplers

Extraction difficulties; Accuracy; filter types and extraction techniques.

Possible overloading too. Loss of viability due to desiccation; Gelatin filters offer some improvement.

Advantages and disadvantages of electrostatic collectors

        Effect on viability. Low velocity

        Effect of charge and exposure to electrostatic fields

                              Low sample volume and high concentration rate possible

                              Low footprint and power consumption

Comparative studies (Table). Brief description based on table

Other factors affecting sampler performance:

Collection time

The importance of having the right collection time; concentrations vary greatly with time. One longer sample (if possible) or multiple short samples. A longer sample integrates the concentrations over time and will give an average value. An option for liquid-based samplers.

Doubling sampling time may not double the count due to desiccation. Several consecutive samples preferred over a few long-time samples.

Positive hole correction factor (factor of time and volume)

Sampler calibration

Surface sampling?

Introduction why it is used

Methods

Swab sampling

Surface wipes. Cloth types

Tape sampling. Transparent tape. Microscopy and stain

Agar contact. Limited to smooth surfaces; microorganisms may not grow on a particular agar

Vacuum sampling

Effectiveness of surface sampling

??? Sample analysis methods

Short description

??? Method s to collect airborne viruses

??? Method s to collect airborne endotoxin

??? Method s to collect airborne allergens

Table 1. General characteristics of several bioaerosol samplers

Sampler/ Prototype	Manufacturer/ Representative/ Developer	Collection medium or sampling volume if applicable	Collection flow rate	Analysis method	Comments

Table 2. Calculated and reported d50 (cut-off) sizes of several inertia-based bioaerosol samplers

Sampler

Calculated d50

Reported d50

Reference s

 

Table 3. Sampler performance studies

Samplers investigated

Microorganisms used

Sample analysis method

Reference sampler or method

Reference s

 

Figure 1. Sampling mechanisms