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Methods for Detection of Microbial Contaminants

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Methods for Detection of Microbial Contaminants Part I ENVR 421 Mark D. Sobsey Detection of Pathogenic Microbes in Water Three main steps: (1) recovery and ... – PowerPoint PPT presentation

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Title: Methods for Detection of Microbial Contaminants


1
Methods for Detection of Microbial Contaminants
Part I
  • ENVR 421
  • Mark D. Sobsey

2
Detecting Pathogens and Indicators in the
Environment
3
Detection of Pathogenic Microbes in Water
  • Three main steps
  • (1) recovery and concentration,
  • (2) purification and separation, and
  • (3) assay and characterization.

4
Microbial Methods for Pathogen Detection
  • Initial sampling, concentration, or recovery
    methods
  • Efficient recovery of low numbers from waters
  • One of the greatest challenges for environmental
    detection
  • Pathogen detection and isolation methods
  • Modified methods from clinical microbiology
  • Must overcome environmental inhibitors
  • Pathogen confirmation and characterization
  • Where did the fecal waste come from?? (source
    attribution and source tracking)

5
Initial Recovery and Concentration of Pathogens
from Water
  • Sedimentation by Centrifugation
  • Bacteria and Parasites differential
    centrifugation
  • several thousand times gravity for several
    minutes to tens of minutes
  • Blood cell separator continuous flow
    centrifugation and particle accumulation
  • being used for parasites previously used for
    bacteria
  • Viruses ultracentrifugation
  • 50-100,000 x gravity for several hours
  • Recover sedimented microbes in a small volume of
    aqueous solution

6
Filtration Bacteria
  • Membrane and other microporous filters
  • Filter 100s-1000s of ml of water through
    cellulose ester, fiberglass, nylon polycarbonate,
    diatomaceous earth or other filters
  • Apply membrane filters to agar medium and
    incubate to get colonies
  • Place filters in liquid culture medium to culture
    bacteria

7
Filtration Parasites
  • Absolute or nominal pore size filters, 1-several
    micrometer pore size
  • Polypropylene, cotton, et.c. yarn-wound
    cartridge filters
  • Polycarbonate, absolute pore size disk filters
  • Polysulfone, pleated capsule filters
  • Spinning cartridge and hollow fiber ultrafilters
  • Cellulose acetate, absolute pore size, circular
    disks
  • Others
  • Recover retained parasites by elution (washing)
    or recovery of retentate water containing
    particles

8
Filtration Viruses
  • Ultrafiltration 1,000-100,000 MWCO
  • Viruses are retained by size exclusion
  • Hollow fiber, spiral cartridge, multiple sheets,
    flat disks, etc
  • polysulfones, cellulose ester, etc.
  • tangential flow to minimize clogging
  • Recover viruses in retentate facilitate by
    elution of filter medium

9
Filters to Recover and Concentrate Microbes from
Water
10
Filtration Viruses
  • Adsorbent filters pore size of filters larger
    than viruses viruses retained by adsorption
  • electrostatic and hydrophobic interactions
  • negatively charged cellulose esters, fiberglass
  • must acidify water and add multivalent cations
  • Electropositive filters
  • charge-modified fiberglass as disks or pleated
    cartridges
  • fiberglass filter disks one coats with
    precipitated aluminum or iron salts in their own
    laboratory, or
  • positively-charged natural quartz fabricated into
    fiberglass that one packs into a column to make
    an adsorbent filter

11
Cuno 1 MDS Virosorb Electropositive Filter(flat
disk - cartridge)
NOTE course texture
Filter Material
Cartridge filter holder
Flat Disk filter holder
  • Filter material used as double layers
  • Cartridge filter pleated to increase surface area

12
How it works?? Electrostatic and Hydrophobic
Interactions Virus Adsorption
  • Electropositive filter gtgtgt at ambient pH, viruses
    are negatively charged
  • Isoelectric point pH where there is no net
    charge on a particle or surface
  • Hydrophobic interactions gtgtgt hydrophobic areas on
    the filter surface that enhance viral adsorption

Virus Isoelectric Point
Poliovirus 7.0
Coliphage MS2 3.9
Coliphage PRD1 4.2
Coliphage Q? 5.3
Coliphage ?X174 6.6
Norovirus 5 6 (depending on strain)
Adenovirus 4.55 (hexon) 4.69 (penton) 7.07 (fiber)
13
How it works?? Electrostatic and Hydrophobic
Interactions Virus Elution
  • Eluting solutions at higher pH (typically pH 9.5)
    surpass the isoelectric point of the filter gt
    both filter and virus have net negative (-)
    charge
  • Virus and filter repels each other releasing
    viruses into solution
  • Negatively charged constituents within the
    eluting solution that may compete for adsorption
    sites on the filter surface
  • enhanced by particles with high isoelectric
    points that remain negatively charged if the pH
    of the eluting solution is raised
  • a contributing factor for elution with beef
    extract/glycine
  • positively charged eluting solutions may compete
    with the filter surface for the negatively
    charged virus particles
  • a contributing factor for elution with
    alternative eluting solutions

14
Virus Elution from Adsorbent Filters
  • Elute adsorbed viruses with alkaline organic
    buffer solutions
  • Beef extract
  • Amino acids
  • Others
  • Beef extract less compatible with nucleic acid
    detection methods

15
Initial Recovery and Concentration of Pathogens
from Water by Chemical Precipitation Methods
  • Viruses precipitate with polyethylene glycol or
    aluminum hydroxide
  • resuspend PEG precipitate in aqueous buffer
  • dissolve aluminum floc in dilute acid solution
  • both have been used as second-step concentration
    and purification methods
  • Parasites precipitate with calcium carbonate
  • dissolve precipitate in dilute sulfamic acid

16
Secondary Concentration PEG Precipitation
  • Polyethylene glycol (C2H6O2)
  • General mode of action of a precipitation reagent
    is the binding of water
  • Used along with NaCl gt essentially salting out
    protein particles
  • Rapid, inexpensive, non-destructive to viruses
  • Gentle precipitation at neutral pH

17
Other Primary Recovery and Concentration Methods
  • Minerals, such as iron oxide and talc used to
    adsorb viruses
  • Synthetic resins ion exchange and adsorbent
  • Other granular media glass beads and sand
  • Less widely used less reliable, cumbersome
    uncertain elution, desorption, exchange
    efficiencies

18
Separation and Purification Methods
  • Purification, separation and concentration of
    target microbes in primary sample or sample
    concentrate
  • Separate target microbes from other particles and
    from solutes
  • Reduce sample size (further concentrate)
  • Variety of physical, chemical and immunochemical
    methods
  • Sedimentation and flotation (primarily parasites)
  • Precipitation (viruses)
  • Filtration (all classes)
  • Immunomagnetic separation or IMS (all classes)
  • Flow cytometry (bacteria and parasites) an
    analytical method, too

19
Assay Methods for Waterborne Pathogens
  • culture or infectivity
  • viability or activity measurements
  • immunoassays
  • nucleic acid assays
  • microscopic examinations

20
Culturing Waterborne Microbes
  • Detection by culture or infectivity assays is
    preferred
  • demonstrates that the target microbes are alive
    and capable of multiplication or replication.
  • From a public health and risk assessment
    standpoint, microbial pathogen assays based on
    infectious units are the most relevant and
    interpretable ones

21
Traditional Approach Culture or Infectivity
Assays for Bacteria
  1. pre-enrich and/or enrich using non-selective and
    then selective broth media, or
  2. grow colonies on membrane filters
  3. Transfer to differential and selective agars
  4. Recover presumptive positive colonies
  5. Biochemical, metabolic and other physiological
    testing
  6. Serological or other immunochemical typing and
    identification (agglutination, enzyme
    immunoassay, etc.)
  7. Other characterization phage typing, nucleic
    acid analyses, virulence tests (cell cultures and
    animal ileal loop assays for pathophysiologic
    response, animal infection, etc.)

22
Enrichment Cultures
  • Cultures may have characteristic appearance
  • Color change
  • Other phenomena
  • E.g., stormy fermentation
  • Cultures may require additional measurement to
    confirm a positive result
  • Subculture
  • Apply other analytical measurement
  • Immunoassay
  • Nucleic acid assay

23
Culturing Waterborne Bacteria Pathogens
  • Continued interest and use because of newly
    recognized, newly appreciated and evolving
    agents.
  • Ability to culture some bacterial pathogens goes
    back more than a century,
  • Culturing bacterial pathogens from water remains
    technologically underdeveloped
  • Has not advanced greatly beyond the adaptation
    of methods used in clinical diagnostic and/or
    food bacteriology

24
Culturing Waterborne Bacteria Pathogens
  • Salmonella, Shigella, Campylobacters and Vibrios
    culture methods little changed beyond efforts to
    improve recoveries using modified pre-enrichment
    and enrichment broths and differential and
    selective agars
  • For some other bacterial pathogens e.g.,
    enterohemorrhagic strains of Escherichia coli
    (O157 H7), culturing from water is a challenge
    due to relative abundance of other,
    non-pathogenic strains of E. coli.
  • select for their growth based on unique
    biochemical or other properties to facilitate
    their separation from the other, non-target
    strains
  • e.g., sorbitol-MacConkey Agar for E. coli O157H7

25
Waterborne Pathogenic Bacteria For Which Culture
Methods Are Underdeveloped
  • Campylobacter jejuni other Campylobacters
  • Yersinia enterocolytica,
  • Aeromonas hydrophila,
  • Helicobacter pylori,
  • Legionella species
  • Mycobacterium avium-intracellulare
  • Shigella
  • Better developed
  • Salmonella

26
Problems in Culture Methods for Bacterial
Pathogens in Water
  • Inefficient growth (low plating efficiency),
  • Slow growth rates
  • Overgrowth by other non-target bacteria.
  • Efforts to improve culture and reduce or
    eliminate non-target bacteria
  • antibiotics
  • physical (heat) treatments
  • chemical (acid) treatments
  • specialized plating
  • Dual media plating
  • Biochemicals with chromogenic reaction products

27
Problems in Culturing Bacterial Pathogens in Water
  • Inability of typical culture methods now in use
    to detect or distinguish
  • pathogenic from non-pathogenic strains
  • the sources of pathogens
  • newly emerging pathogenic strains
  • evolutionary processes and mechanisms
  • the role of environmental change in selection for
    or emergence of new pathogenic strains

28
Detection of Stressed, Injured and
Viable-But-Nonculturable (VBNC) Bacteria
  • Waterborne bacterial pathogens and indicators are
    often physiologically altered/stressed and not
    efficiently cultured using standard selective and
    differential media
  • Causes great underestimation of true
    concentrations in water and other samples
  • Underestimation of their risks to human health
  • Stressed, injured and VBNC bacteria may still be
    fully infectious for humans and other animal
    hosts (there is disagreement on this point!)
  • Repair and resuscitation methods to improve the
    detection of viable and potentially cultural
    bacteria
  • Such methods are rarely used to detect pathogens
    in drinking water more so in foods

29
Detection Of Viral Pathogens by Culture
  • Viruses are obligate intracellular parasites,
  • many enteric viruses can be propagated or
    cultured in susceptible hosts
  • whole animals
  • mammalian cells grown in culture
  • Quantify viruses in animals and cells using
    quantal methods (e.g., Most Probable Number or
    MPN)
  • Virus assays in cell cultures by quantal (e.g.,
    MPN) or enumerative methods (plaque or local
    lesion assays)

30
Enteric Virus Detection in Cell Culture
  • Some viruses (enteroviruses, reoviruses,
    adenoviruses and astroviruses) propagate in
    susceptible host cell cultures and produce
    morphologically distinct cytopathogenic effects
    (CPE).

Uninfected Cell Culture
Infected Cell Culture with CPE
31
Enteric Virus Detection in Cell Culture
  • Other viruses (some enteroviruses, adenoviruses,
    rotaviruses, astroviruses and hepatitis A virus)
    grow poorly or slowly in cell cultures and
    produce little or no CPE.
  • Detection of these viruses requires the used of
    additional analytical techniques directed at
    detecting viral antigens (immunofluorescence
    assay, enzyme immunoassays and radioimmunoassays)
    and nucleic acid (nucleic acid hybridization or
    amplification assays).

32
Detection of Hepatitis A Virus in Cell Culture by
Radioimmunoassay
33
Viruses Not Detected in Cell Culture
  • Enteroviruses, caliciviruses (noroviruses)
    parvoviruses, coronaviruses (some),
    picobirnaviruses and hepatitis E virus can not be
    propagated in any known cell cultures.
  • Not detectable in water unless an alternative
    analytical method, such as nucleic acid
    amplification by PCR or RT-PCR, is applied
    directly to concentrated samples.

34
Detection of Protozoan Parasites by Culture
  • Environmental forms of some protozoan parasites,
    such as spores and oocysts, are culturable in
    susceptible host cells
  • Culture free-living amoebas (Naegleria spp. and
    Acanthamoeba spp.) on lawns of host bacteria,
    such as E. coli, on nonnutrient agar they form
    local lesions.
  • For other waterborne parasites, such as Giardia
    lamblia and Cyclospora cayatenensis, culture from
    the environmental stage (the cyst or oocyst)
    recovered from water is still not possible

35
Detection of Protozoan Parasites by Culture
  • Spores of some microsporidia (Encephalitozoon
    intestinalis) and the oocysts of Cryptosporidium
    parvum can be cultured in mammalian host cells
    where spores germinate or oocysts excyst and
    active stages of the organisms can proliferate.
  • Living stages detected (after immunofluorescent
    or other staining) and quantified score positive
    and negative microscope fields or cell areas
    (slide wells), or count numbers of foci of living
    stages or discrete living stages.
  • Express concentrations MPNs or other units, such
    as numbers of live stages.
  • Detection also possible by PCR or immunoblotting
  • Facilitates molecular characterization

36
Progress in Detection of Protozoan Parasites by
Culture
  • Oocysts of Cryptosporidium parvum and spores of
    some microsporidia (Encephalitozoon intestinalis)
    infect mammalian host cells
  • Spores germinate and oocysts excyst
  • Active stages of the organisms proliferate
  • Detect and quantify (after immunofluorescent or
    other staining)
  • Score positive and negative microscope fields or
    cell areas (slide wells), or count numbers of
    foci of living stages or discrete living stages.
  • Express concentrations as MPNs or other units
    based numbers of live stages, numbers of
    infectious foci or number of positive microscope
    fields
  • Detect by NA methods (PCR, FISH, etc.)
  • Facilitates molecular characterization

Immunofocus of C. parvum Living Stagesin MDCK
Cells with C3C3-FITC Antibody
37
Combined Cell Culture and Nucleic Acid Detection
and Amplification of Waterborne Pathogens
  • Inoculate sample into susceptible host cell
    cultures
  • incubated to allow the viruses or parasites to
    infect the cells and proliferate.
  • After producing enough nucleic acid, extract and
    either hybridize directly with a gene probe or
    further amplify by PCR or RT-PCR
  • Facilitates detection of infectious but
    non-cytopathogenic viral and protozoan pathogens
    able to proliferate in cell cultures.
  • Reduces incubation time to detect pathogen
    nucleic acid.
  • Facilitates molecular or other methods of
    characterization

38
Detection of Waterborne Pathogens by Viability or
Activity Assays
  • Assay bacteria for viability or activity by
    combining microscopic examination with chemical
    treatments to detect activity or "viability".
  • measure enzymatic activities, such as
    dehydrogenase, esterase, protease, lipase,
    amylase, etc.
  • Example tetrazolium dye (INT) reduction
  • 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetraz
    olium Cl (measures dehydrogenase activity).
  • Reduction of tetrazolium dye leads to
    precipitation of reduced products in the
    bacterial cells that are seen microscopically as
    dark crystals.

39
Progress in Detection of Waterborne Bacteria by
Viability or Activity Assays
  • Combine activity measurement and immunochemical
    assay (for specific bacteria).
  • Combine fluorescent antibody (FA) (for detection
    of specific bacterium or group) with enzymatic or
    other activity measurement
  • Use image analysis tools to improve detection and
    quantitation
  • Flow cytometry
  • Computer-aided laser scanning of cells or
    colonies on filters

40
Detection of Waterborne Bacterial Pathogens by
Viability or Activity Assays
  • Combine methods for bacterial detection in water,
    such as activity measurement and immunochemical
    assay (for specific bacteria).
  • Example "FAINT combines fluorescent antibody
    (FA) (for detection of specific bacterium or
    group) with tetrazolium dye reduction (INT)
  • Look for INT crystals in cells specifically
    stained with fluorescent antibodies

41
Viability or Activity Assays for Protozoan Cysts
and Oocysts
  • Example Stain with DAPI (the fluorogenic stain
    4',6-diamidino-2-phenylindole taken up by live
    oocysts and propidium iodide (PI taken up by
    dead oocysts).
  • Viable Cryptosporidium oocysts are DAPI-positive
    and PI-negative
  • Non-viable oocysts are DAPI-negative and
    PI-positive
  • Alternative stains may be more reliable
  • Viability staining is often poorly associated
    with infectivity detects inactivated cysts and
    oocysts

Detects cysts and oocysts inactivated by UV and
chemical disinfection
42
C. parvum oocysts Dual stain DAPI (blue) and
propidium iodide (red)
43
Detecting Active or Viable Pathogens Using
Nucleic Acid Targets
  • Detect short-lived nucleic acids present in only
    viable/infectious microbes
  • ribosomal RNA
  • messenger RNA
  • genomic RNA of viruses (large amplicons)
  • Detect pathogen nucleic acid by fluorescent
    in-situ hybridization (FISH)
  • applied to bacteria, protozoan cysts and oocysts,
    as well as viruses in infected cell cultures
  • (see pictures in later slides)
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