Methods for Detection of Microbial Contaminants – Part I

ENVR 421

Mark D. Sobsey

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Detecting Pathogens andIndicators in the Environment

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Detection of Pathogenic Microbes in Water

• 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)

Direct Plating for Culture• Used for bacteria

and bacteriophages– Combine sample

with medium– Liquid broth

culture • quantal; MPN)

– Agar (gel) medium culture for colony count

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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, etc.. 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• Compressed sponge filter medium

Recover retained parasites by elution (washing) or recovery of retentate water containing particles

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Filtration: Viruses(Used for cellular microbes, too)

• 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: VirusesAdsorbent 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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Electropositive Filter Media(flat disks and cartridges)

Flat Disk – filter holder Cartridge – filter holder

•Flat filter material used as single or double layers•Cartridge filter pleated to increase surface area

Cartridge filter holder

Filtration medium

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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

• Hydrophobic interactions >>> 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)

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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 so: – Both filter and virus have net negative (-) charge– Virus and filter repel, releasing viruses into solution

• Negatively charged constituents in eluting solution to compete for adsorption sites on the filter surface– enhanced by constituents with high isoelectric points

that remain negatively charged when pH is raised– a contributing factor for elution with beef

extract/glycine• Positively charged eluting solutions to compete as

competing surface for the negatively charged viruses– a contributing factor for elution by some 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

analytical method, too

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Assay Methods for Waterborne Pathogens

• 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 filters3. Transfer to differential and selective agars4. 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.)

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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

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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– Has not advanced greatly beyond the

adaptation of methods used in clinical diagnostic and/or food bacteriology

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Culturing Waterborne Bacteria Pathogens

• Salmonella, Shigella, Campylobacter &Vibrio spp.:• 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 O157:H7

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Waterborne Pathogenic Bacteria For Which Culture Methods Are Underdeveloped

• Campylobacter jejuni; other Campylobacters• Yersinia enterocolytica • Helicobacter pylori• Legionella species • Mycobacterium avium-intracellulare• Shigella

Better developed:• Salmonella spp.• Escherichia coli• Clostridium perfringens

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Problems in Culture Methods for Bacterial Pathogens in Water

1. Inefficient growth (low plating efficiency) 2. Slow growth rates3. 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:

– Selective media– Dual media plating– Biochemical substrates with colored or fluorescent

reaction products

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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

– Role of environmental change in selection or emergence of new pathogenic strains

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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

Detection Of Viral Pathogens by Culture Infectivity

• Viruses: 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)

Virus Plaques10-fold Dilution Series

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Enteric Virus Detection in Cell Culture

• Some viruses propagate in susceptible host cell cultures and produce morphologically distinct cytopathogenic effects (CPE):

• Enteroviruses, reoviruses, adenoviruses and astroviruses

Uninfected Cell Uninfected Cell CultureCulture

Infected Cell Culture with Infected Cell Culture with CPECPE

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Enteric Virus Detection in Cell Culture

• Other viruses (some enteroviruses, enteric adenoviruses, rotaviruses, astroviruses and hepatitis A virus) grow poorly or slowly in cell cultures and produce little or no CPE.– Detection 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

• Some important human enteric viruses can not be propagated in any known cell cultures– Human noroviruses– Hepatitis E Virus

• Not detectable in water unless an alternative analytical method, such as direct 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:

• 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 cultures2. incubate to allow the viruses or parasites to infect

the cells and proliferate. 3. 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

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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]-5-phenyltetrazolium 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 PI-negative

• Non-viable oocysts are DAPI-negative and PI-positive

Alternative stains may be more reliableViability staining is often poorly associated with infectivity;

detects inactivated cysts and oocysts

But…..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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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; next lecture)

Detection of total, viable and membrane-perturbed bacteria using triple staining with fluorescent probes

Biochemical Journal www.biochemj.org Biochem. J. (2004) 380, 859-865 Biochemical Journal www.biochemj.org Biochem. J. (2004) 380, 859-865

• E. coli cells immobilized on poly(L-lysine)-coated glass slides, and incubated with CTC, DAPI and FITC

• DAPI: double-stranded DNA-binding dye stains all cells• FITC: green fluorescent probe unable to traverse the cytoplasmic membrane of cells unless permeabilized by a peptide.• CTC: vital dye CTC stains viable cells