Methods for Detection of Microbial Contaminants – Part I

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. 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

6. 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

7. 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

8. 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

9. 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

10. Filters to Recover and Concentrate Microbes from Water

11. 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

12. Electropositive Filter Media(flat disks and cartridges) Cartridge filter holder Filtration medium Cartridge – filter holder Flat Disk – filter holder • Flat filter material used as single or double layers • Cartridge filter pleated to increase surface area

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

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

21. 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

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

23. 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

24. 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

25. 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

26. Waterborne Pathogenic Bacteria For Which Culture Methods Are Underdeveloped • Campylobacter jejuni; other Campylobacters • Yersinia enterocolytica • Helicobacter pylori • Legionella species • Mycobacteriumavium-intracellulare • Shigella Better developed: • Salmonella spp. • Escherichia coli • Clostridium perfringens

27. 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: • Selective media • Dual media plating • Biochemical substrates with colored or fluorescent reaction products

28. 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

29. 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

30. 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 Plaques 10-fold Dilution Series

31. 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 Culture Infected Cell Culture with CPE

32. 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).

33. Detection of Hepatitis A Virus in Cell Culture by Radioimmunoassay

34. 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.

35. 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

36. 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

37. 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

38. Combined Cell Culture and Nucleic Acid Detection and Amplification of Waterborne Pathogens • Inoculate sample into susceptible host cell cultures • incubate 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

39. 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.

40. 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

41. 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

42. 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 But….. Detects cysts and oocysts inactivated by UV and chemical disinfection

43. C. parvum oocysts Dual stain : DAPI (blue) and propidium iodide (red)

44. 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)

45. Detection of total, viable and membrane-perturbed bacteria using triple staining with fluorescent probes • 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 Biochemical Journal www.biochemj.org Biochem. J. (2004) 380, 859-865