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Membrane Filtration Technique Sample passed through a membrane filter Filter traps bacteria Filter transferred to media e.g. EMB Total or faecal coliforms may be counted Conditions may be varied to be more or less selective ‘Resuscitation’ step may be included for stressed microorganisms Detection of Coliforms Membrane filter on EMB media showing growth of coliforms Presence-Absence Test Modified MPN procedure Large water sample used (100 ml) ‘Triple-strength’ broth contains indicator Positive test appears yellow (acid production) Colilert Defined Substrate Test Tests for coliforms and faecal coliforms Sample of 100 ml used Medium contains ONPG and MUG Yellow colour after 24 h indicates positive coliform test Fluorescence under UV light indicates positive faecal coliform test Detection of Coliforms The Defined Substrate Test a) Control b) Coliforms c) Faecal coliforms Faecal Enterococci Faecal enterococci have a slower die-out rate than faecal coliforms Particularly applicable to salt water Potential for being more reliable indicators of pollution Detection of Protozoa Lack of correlation between presence of Cryptosporidium oocysts and indicator organisms Can only be removed by filtration and sedimentation Large volumes of water (1-100 L) must be analysed due to low infective dose Concentration of samples Cartridge filtration Membrane filtration Flocculation Detection of Protozoa Purification of samples Density flotation Flow cytometry – probably more sensitive method Fluorescence microscopy Types of Water Drinking water Public supplies Private supplies Water containers Natural mineral water Recreational waters Bathing waters Drinking Water In the UK water for human consumption is covered by EU council legislation Additional legislation covers the detection of Cryptosporidium Testing is required for total coliforms, faecal coliforms and total colony counts Drinking Water Public supplies Samples taken from points before supply and from consumer’s taps Samples tested for coliforms and E.coli 95% of samples must be negative for coliforms when >50 samples analysed from sampling point per annum Continuous monitoring of ‘at risk’ water treatment works for Cryptosporidium oocysts required Requirement: <10 cryptosporidial oocysts /100L Drinking Water Private supplies Supply 1% of UK population Monitoring carried out by local authority Classified into categories Samples tested for coliforms and E.coli 100 ml Samples must be negative Samples tested for colony counts at 22 oC and 37 oC Water quality may change with weather etc. Drinking Water Bottled water and water dispensers Covered by same European directives as public and private supplies Includes spring water and table water Total colony counts apply within 12 h bottling New volume of samples is 250 ml Drinking Water Natural mineral water ‘Microbiologically wholesome water originating in an underground water table or deposit and emerging from a spring tapped at one or more natural or borehole exits’. Water must be from recognised source and not be treated to alter chemical or microbiological composition Similar regulations to those for water in containers Recreational Water Not directly covered by legislation Samples tested for coliforms and E.coli Samples also tested for Pseudomonas aeruginosa Colony counts more important than in drinking water analysis Investigations must be carried out if counts are above guide level and/or E.coli detected Bathing beach water ‘Beaches where large numbers of people are known to bathe (coastal and inland)’ Frequency of illness proportional to indicator organisms and faecal pollution Enterococcus count appearing to become a more accurate indicator 80% of samples should not exceed guide level 95% of samples should not exceed imperative level Incidence of Waterborne Disease Low incidence of waterborne disease in developed countries Incidence of typhoid and cholera declined Chlorination major factor Incidence of Waterborne Disease High incidence of waterborne disease still present in developing countries Cryptosporidiosis may still be a threat in developed countries Outbreak in Milwaukee 1993 400,000 People infected, 85 fatalities Traced to municipal water supply Heavy rain lead to water supply contamination Outbreak in Lancashire 2015 300,000 homes affected Conclusions Coliform standard widely used as indicator for health risk Problems with pathogens resistant to chlorination Enterococci may act as more accurate indicators of health risk Prevention better than cure References Lacey S W.1995. Cholera: calamitous past, ominous future. Clin Infect Dis 20 1409-1419. Gray L.D. Escherichia, Salmonella, Shigella and Yersinia. In Murray PR et al, editors. Manual of clinical microbiology ed 6, Washington DC, 1995, American Society for Microbiology. References MacKenzie W.R., Hoxie J.B., Proctor M.E., Gradus M.S., Blair K.A., Peterson D.E.1994. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 331, 161-167. Vesey G., Slade J.S., Byrne M., Shepherd K. and Fricker C.R. New techniques for the detection of protozoan parasites in water. In New Techniques in Food and Beverage Microbiology. 1993 Society for Applied Microbiology. References Hunter P.R. Water-borne disease: epidemiology and ecology.1997 Chichester:Wiley. Gleeson C., Gray N. The coliform index and waterborne disease. 1996. London:E and FN Spon. Baker K.H. 1995. Detection and occurrence of indicator organisms and pathogens. Water Environ. Res.67 406-10. References The Water Supply (Water Quality) Regulations 1989 (Statutory Instrument 1989 No. 1147 as amended by Statutory Instrument 1989 No.1384 and Statutory Instrument 1991 No.1837.) London:HMSO, 1991. The Private Water Supply (Water Quality) (Amendment) Regulations 1999. Statutory Instrument 1999 No. 1524. London:Stationery Office, 1999. Water and Environmental Microbiology Dr Sarah Atchia Microbial flora of aquatic environments Most environments contain large varieties of microbial species Most species are adapted for relatively nutrient poor environment Factors Influencing Microbial Flora Temperature pH Nutrient content Salt content Extreme environments Pollution Sewage contamination Litter Chemicals e.g. Fertilisers Point source of pollution Release of wastes into watercourses Nonpoint source of pollution Run off from fields etc Eutrophication (nutrient enrichment) Water Treatment Precipitate removal Sedimentation basin Coagulation and flocculation Settling basin Filtration Rapid sand filters Disinfection Chlorination Ozonation Waterborne Disease Pathogens may be transmitted via a variety of types of water Growth occurs in the intestines Microorganisms are shed in the faeces Infections are transmitted by the faecal-oral route Involves survival of acidic environment of the stomach Historical Perspective First documented water-borne disease: 1854 cholera outbreak traced to Broad Street pump Cholera in London - 1854 Cholera fatalities in Soho Location of water pumps Dr John Snow identifies the contaminated pump Pathogens Viruses Bacteria Protozoa Algae Viruses Enteroviruses e.g. poliovirus Hepatitis A Bacteria Vibrio species Gram negative curved rods Vibrio cholerae Enterobacteriaceae species Enteric bacteria which are members of the intestinal microbiota of humans and animals. Can be divided into coliforms and noncoliforms Found in water, soil and on vegetation Salmonella typhi Escherichia coli Vibrio cholerae Gram-negative curved rod causes cholera Endemic in India, Pakistan and Central and South America Epidemics Pandemics Vibrio cholerae Vibrio cholerae: Electronmicrograph x12,000 Cholera Cholera produced by enterotoxin Vibrios attach to small intestine Choleragen released resulting in diarrhea and massive fluid loss Ingestion of large inoculum (108 – 109 vibrios) causes disease Cholera Treatment involves replacement of fluid and electrolytes Antibiotic therapy not normally recommended but may increase speed of bacterial elimination Vaccine available for protection against some strains Salmonella typhi Gram-negative rod causes typhoid Children, the elderly and travellers most at risk Most common in warmer months Ingestion of large inoculum (106 – 108 bacteria) causes disease Bacteria invade cells in small intestine Strains causing enteric fever pass through intestines and to other organs Typhoid Fever Enteric fever (typhoid fever) Bacteremic phase- systemic illness (fever, headache, myalgias) Gastrointestinal phase Paratyphoid fever (S.paratyphi) Milder disease Typhoid Fever Asymptomatic carriage Chronic carriage after paratyphoid or typhoid fever E.g. ‘Typhoid Mary’ Enteritis Nausea, vomiting, diarrhea Septicaemia Protozoa Cryptosporidium parvum Giardia lamblia Entamoeba histolytica Cyclospora Dracunculus medinensis Cryptosporidium parvum Causative agent of cryptosporidiosis Cells attach to intestinal epithelium Life cycle includes production of oocysts Oocysts and cysts are highly resistant to chlorine (Cryptosporidium up to x14 than Giardia) Protozoan parasites may infect wide range of animal hosts Cryptosporidium parvum Cryptosporidium oocysts: acid-fast stain Cryptosporidiosis Infection occurs with ingestion of oocysts produced in faeces Causes enterocolitis Watery diarrhea, abdominal pain Risk to immunocompromised individuals exposed to contaminated water May cause chronic disease Treatment involves replacement of fluid Microbiological Analysis Water cannot be examined for presence of all pathogens Waterborne disease is largely due to faecal pollution Therefore specific indicator organisms are utilised as markers of risk Indicator Organisms Criteria required: Suitable for analysis of all water types Indicator must reflect presence of enteric pathogens Indicator should survive longer than enteric pathogens Indicator should not grow in contaminated water giving inflated counts Testing procedure should be specific for the indicator Testing procedure should be easy to perform Indicator should be harmless Indicator level must directly reflect the degree of faecal pollution Coliforms Facultatively anaerobic Gram-negative Non-sporing Rod-shaped Ferment lactose and produce gas within 48 h at 35 oC Coliforms Mostly of intestinal origin Members of Enterobacteriaceae and make up 10% of intestinal flora Slow die-out rate in comparison to other intestinal pathogens Contain Escherichia coli, Klebsiella pneumoniae (intestinal inhabitant) Enterobacter aerogenes (non-intestinal inhabitant) Coliform Test Most probable number procedure Utilises liquid media Presumptive test Lactose broth inoculated with three different sample volumes (differ by powers of 10) Tubes examined for gas production after 24 h Confirmed test Positive tubes inoculated into brilliant green lactose bile broth Tubes examined for gas production after 48 h MPN values determined Most probable number method Principle: Probability of viable cells being present in a diluted sample follows Poisson distribution Data are analysed using MPN tables * Coliform Test Most probable number procedure Completed test Positive tubes used to inoculate eosin-methylene blue (EMB) agar Media is selective and differential After 24 h colonies retested in brilliant green lactose bile broth Colonies subjected to further tests for coliforms Detection of Coliforms EMB agar showing growth of E.coli, S.typhimurium and E.aerogenes Faecal Coliforms Narrower range of microorganisms allowing for more specific testing Intestinal coliforms from warm-blooded animals Involves detection of E.coli Can grow at 44.5 oC Pakistan and Central and South America Epidemics Pandemics Vibrio cholerae Vibrio cholerae: Electronmicrograph x12,000 Cholera Cholera produced by enterotoxin Vibrios attach to small intestine Choleragen released resulting in diarrhea and massive fluid loss Ingestion of large inoculum (108 – 109 vibrios) causes disease Cholera Treatment involves replacement of fluid and electrolytes Antibiotic therapy not normally recommended but may increase speed of bacterial elimination Vaccine available for protection against some strains Salmonella typhi Gram-negative rod causes typhoid C