Download Microbial Detection in the Environment, Part 1

ENVR 133
Methods for Detection of Microbial
Contaminants – Part I
Mark D. Sobsey
University of North Carolina
at Chapel Hill
Detecting Pathogens and
Indicators in the
Environment
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Detection of Pathogenic Microbes in
Water
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Three main steps:
(1) recovery and concentration,
(2) purification and separation, and
(3) assay and characterization.
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Microbial Methods for Pathogen Detection
1. Initial sampling, concentration, or recovery
methods
– Efficient recovery of low numbers from waters
– One of the greatest challenges for environmental
detection
2. Pathogen detection and isolation methods
– Modified methods from clinical microbiology
– Must overcome environmental inhibitors
3. Pathogen confirmation and characterization
– Where did the fecal waste come from?? (source
attribution and source tracking)
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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
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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
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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
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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
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Filters to Recover and Concentrate Microbes from Water
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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
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Cuno 1 MDS Virosorb Electropositive Filter
(flat disk - cartridge)
NOTE: course texture
Flat Disk – filter holder
Filter Material
Cartridge – filter holder
• Filter material used as double layers
• Cartridge filter pleated to increase surface area
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How it works?? – Electrostatic and
Hydrophobic Interactions: Virus Adsorption
• Electropositive filter >>> at ambient pH, viruses
are negatively charged
• Isoelectric point – pH where there is no net
charge on a particle or surface
Virus
Isoelectric Point
Poliovirus
7.0
Coliphage MS2
3.9
Coliphage PRD1
4.2
Coliphage Q
5.3
Coliphage X174
6.6
Norovirus
Adenovirus
5 – 6 (depending on strain)
4.55 (hexon)
4.69 (penton)
7.07 (fiber)
• Hydrophobic interactions >>> hydrophobic areas
on the filter surface that enhance viral adsorption
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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 > 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
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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
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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
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Secondary Concentration:
PEG Precipitation
• Polyethylene glycol (C2H6O2)
• General mode of action of a precipitation reagent
is the binding of water
• Used along with NaCl > essentially “salting out”
protein particles
• Rapid, inexpensive, non-destructive to viruses
• Gentle precipitation at neutral pH
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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
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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
analysis, too
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Assay Methods for Waterborne
Pathogens
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culture or infectivity
viability or activity measurements
immunoassays
nucleic acid assays
microscopic examinations
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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
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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, animal ileal
loops, animal infection, etc.)
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Enrichment Cultures
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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
and has not advanced greatly beyond the
application of methods used in clinical
diagnostic and/or food bacteriology
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Culturing Waterborne Bacteria
Pathogens
• Salmonella, Shigella, Campylobacters and
Vibrios: culture methods little changed beyond
efforts to improve recoveries using modified preenrichment 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 continues to
be a challenge because of the relative abundance
of other, non-pathogenic strains of E. coli.
– select for their growth based on distinctive
biochemical or other properties to facilitate
their separation from the other, non-target
strains
• e.g., sorbitol-MacConkey Agar for E. coli
O157:H7
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Waterborne Pathogenic Bacteria For
Which Culture Methods Are Most
Underdeveloped
Campylobacter jejuni; other Campylobacters
Yersinia enterocolytica,
Aeromonas hydrophila,
Helicobacter pylori,
Legionella species
Mycobacterium avium-intracellulare
Shigella
Better developed:
• Salmonella
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Problems in Culture Methods for Bacterial
Pathogens in Water
1. Inefficient growth (low plating efficiency),
2. Slow growth rates
3. Overgrowth by other non-target bacteria.
Efforts to improve culture and reduce or eliminate
non-target bacteria:
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antibiotics
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physical (heat) treatments
• chemical (acid) treatments
• specialized plating: e.g., dual media plating
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Problems in Culturing Bacterial
Pathogens in Water
Inability of typical culture methods now in
use to detect or distinguish:
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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
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Detection of Stressed, Injured and ViableBut-Nonculturable (VBNC) Bacteria
• Waterborne bacterial pathogens and indicators
are often physiologically altered/stressed and not
efficiently cultured using standard selective and
differential media
• Causes considerable underestimation of the
concentrations of these bacteria in water and
therefore, 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 improve the
detection of viable and potentially cultural
bacteria, but, these methods are rarely used to
detect pathogens in drinking water.
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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)
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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
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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).
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Detection of Hepatitis A Virus in Cell
Culture by Radioimmunoassay
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Viruses Not Detected in Cell Culture
• Other viruses (some enteroviruses,
caliciviruses, parvoviruses,
coronaviruses, picobirnaviruses and
hepatitis E virus) can not be propagated in
any known cell cultures.
– They will not be detected in water unless an
alternative analytical method, such as nucleic
acid amplification by PCR or RT-PCR, is
applied directly to concentrated samples.
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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
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Detection of Protozoan Parasites by Culture:
Some New Developments
• 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
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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
Stages:
in MDCK Cells with
C3C3-FITC Antibody
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Combined Cell Culture and Nucleic Acid Detection
and Amplification of Waterborne Pathogens
1. Inoculate sample into susceptible host cell cultures
2. incubated to allow the viruses or parasites to infect
the cells and proliferate.
3. After producing enough pathogen nucleic acid,
extract and either hybridized directly with a gene
probe or further amplify by PCR or RT-PCR.
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Facilitates detection of infectious but noncytopathogenic 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
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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]-5phenyltetrazolium 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.
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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
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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
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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 PInegative
• 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
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C. parvum oocysts
Dual stain : DAPI (blue) and propidium iodide (red)
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Detection of Protozoan Parasites by Cell Culture: C. parvum
Immunofocus of C. parvum Living Stages:
in MDCK Cells with C3C3-FITC Antibody
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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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