Proceeding Introduction (Monday) and sterilize culture media in accordance with ISO/TS 11133-1 and ISO/TS 11133-2. ... Use - if possible - standardized and certified media. Laboratory

1. 1. Introduction (Monday)Introduction (Monday)

BackgroundBackgroundPrerequisites: LabPrerequisites: LabBasic properties of Basic properties of VibrioVibrio relevant to the analytical laboratoryrelevant to the analytical laboratoryEnrichment protocolsEnrichment protocols

2.2. Specific properties of Specific properties of Vibrio cholerae, Vibrio parahaemolyticus Vibrio cholerae, Vibrio parahaemolyticus and and Vibrio vulnificusVibrio vulnificus (Tuesday)(Tuesday)

SSpecific properties of pecific properties of Vibrio cholerae, Vibrio parahaemolyticusVibrio cholerae, Vibrio parahaemolyticus and and Vibrio Vibrio vulnificusvulnificusIdentification by microbiological, biochemical and immunologicalIdentification by microbiological, biochemical and immunological traits.traits.

3.3. Pathogenicity (Wednesday)Pathogenicity (Wednesday)Genomes and virulence factorsGenomes and virulence factorsPCR MethodsPCR Methods

4.4. SOP'sSOP's, Quantification and uncertainty of results (Thursday), Quantification and uncertainty of results (Thursday)

MPN and plating methodsMPN and plating methodsUncertainty and evaluation of resultsUncertainty and evaluation of results

Proceeding

•• BackgroundBackground

•• Prerequisites: LabPrerequisites: Lab

•• Basic properties of Basic properties of VibrioVibrio relevant to relevant to the analytical laboratorythe analytical laboratory

•• Enrichment protocolsEnrichment protocols

Introduction (Monday)

World freshwater aquaculture

CafeF.vn0

10

20

30

40

19961997

19981999

20002001

20022003

20042005

2006

MT mil.

Others

Thailand

Indonesia

Vietnam

India

China

The world aquaculture production comes from two sources: China and other countries.

Viet Nam ranks thirdViet Nam ranks third. And its aquaculture industry began growing strongly and quickly since 2003, especially shrimp and Pangasius industries.

The freshwater aquaculture

Vietnam Export Market Share2003 - 2008

0%

20%

40%

60%

80%

100%

2003 2004 2005 2006 2007 2008Japan USA EU Russia Korea

ASEAN Ukraina China Others

• The decline in Japan and America markets affects primarily Viet Nam’s shrimp export.

• This might be compensated by opening new markets in EUnew markets in EU and others with high demand for freshwater and low-priced products.

• Some exporters cope with the shrink of Japan and America by diversifying to the new markets outside the 3 big places (EU, America and Japan).

Market trend

Structure of export product 2004-2008 (volume)

0%

20%

40%

60%

80%

100%

2003 2004 2005 2006 2007 T1-6/2008

Pangasius Shrimp Tuna Squid&Octopus Others

• Imbalance in product structure is reflec-ted clearly in exporting.

• Total shrimp and pangasius exporting account for 60%

Structure of export products 2004-2008 (value)

0%

20%

40%

60%

80%

100%

2003 2004 2005 2006 2007 T1-6/2008

Pangasius Shrimp Tuna Squid&Octopus Others

• Until 2003, the main product exported is shrimp.

• After 2003, the share of pangasius industry is rapidly increasing.

Product Structure Economic Aspects: Outbreaks

product already con-sumed / no action taken

border control -screening

Vibrio vulnificus and high number of aerobic plate counts (Pseudomonas dominated) in chilled shrimps (Metapenaeus spp) from India

2008.0583Germany20/05/2008

product (to be) redis-patched or destroyed

Vibrio cholerae NON O:1/NON O:139 (presence in 1/10 samples) in frozen black tiger shrimps from India

2008.AXENorway12/06/2008

distribution restricted to notifying country / public warning public warning --press releasepress release

consumer complaint

Staphylococcal enterotoxin and Vibrio cholerae NON O:1/NON O:139 in frozen pangasius fillets from VietnamVietnam

008.0942Norway01/08/2008

product to be redistri-buted or destroyed

Vibrio cholerae, Vibrio cholerae NON O:1/NON O:139, Vibrio fluvialis, Vibrio parahaemolyticus and Vibrio vulnificus in frozen raw black tiger shrimps (Penaeus monodon) from India

2008.AKBNorway03/03/2008

product (to be) redis-patched or destroyed

distribution restricted to notifying country returned to dispatcher

Status

Vibrio cholerae NON O:1/NON O:139 (in 2 out of 10 samples) in frozen black tiger shrimps from VietnamVietnam

Vibrio cholerae (1 out of 5 samples) in farmed frozen cooked and peeled black tiger shrimps (Penaeusmonodon) from Malaysia

Reason for Notifying

2008.BLTNorway18/09/2008

company's own check

2008.0039Denmark14/01/2008

BasisREFNotified byDATE

Rapid alert system for food and feed (RASFF)

VIBRIO CHOLERAE

VIBRIO VULNIFICUSVIBRIO

PARAHAEMOLYTICUS

The major Culprits






Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological

examinations

Laboratory Standards

Intended for three main Intended for three main usesuses::Implementation of ISO/TC 34 (Food products) subcommittees SC 9 (microbiology) “specific standards”Good laboratory practiceGood laboratory practice for food microbiological laboratoriesGuidance for accreditationGuidance for accreditation of food microbiological laboratories

PurposePurpose of this International Standardof this International StandardTo ensure the validityensure the validity of food microbiology examinationsTo assist in ensuring that the general techniques usedtechniques used for the samethe samein all laboratoriesin all laboratoriesTo help achieve homogeneous resultshomogeneous results in different laboratoriesFor safetysafety of the laboratory personnel

Laboratory StandardISO 7218:2007


Laboratory design shall comply with safety requirementsMicroorganisms are usually classified in four risk categories. Depending on the country and/or organization, this classification system might take the following factors into consideration:

Pathogenicity of the organism Mode of transmission and host range Availability of effective preventive measures (e.g., vaccines) Availability of effective treatment (e.g., antibiotics) Other factors


WHO Classification of Infective Microorganisms by Risk Group (2004)Laboratory Biosafety Manual". 3rd Editionhttp://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/index.html

Australian/New Zealand Standard (2002) Standard AS/NZS 2243.3:2002. Safety in laboratories Part 3: Microbiological aspects and containment facilities.(www.standards.com.au and www.standards.com.nz )

Canadian Laboratory Safety Guidelines (2004)http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

European Economic Community (2000)Directives 2000/54/EC and 90/679/EEC http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32000L0054:EN:HTML

NIH Recombinant DNA Guidelines (USA, 2002)April 2002. Appendix B. http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html

Biosafety levels as defined by various authorities

European Community classification: Risk Group 2

NHI Biosafety guidelines Risk Group 2

ATCC Biosafety level 2

Vibrio cholerae, V. parahaemolyticus, V. vulnificus Vibrio cholerae, V. parahaemolyticus, V. vulnificus

Recommended Precautions:Biosafety Level 2 practices, containment equipment, and facilities are recommended for activities with cultures or potentially infectious clinical materials. Animal Biosafety Level 2 practices, containment equipment, and facilities are re com mended for activities with naturally or experimentally infected animals. Although cholera vaccines exist, their routine use by laboratory staff has not been recommended.


4 Staff4.1 GeneralGeneral requirements on the competence of staff competence of staff can be found in ISO/IEC 17025.ISO/IEC 17025.4.2 Competence

For each method or technique, objective criteria shall be definedcriteria shall be defined for assessment of appropriate competence, both initially and on an ongoing basis.The competence may be established within the laboratorywithin the laboratory by internal quality control quality control (see 15.1.2).

NOTE:NOTE: One of the means of investigating the cause of poor performance (pipetting, poor homo-geneity of the initial suspension, counting, etc.) in the case of enumerations by counting colonies is given in ISO 14461-1.

4.3 Verification of on-going staff competenceVerification of on-going staff competence should be evaluated regularly against objective parameters. This includes participation in internal quality assurance programmesparticipation in internal quality assurance programmes, proficiency testsproficiency tests (see ISO/IEC Guide 43-1), the use of reference materialsreference materials or by selfself--assessment testsassessment tests for enumeration of microorganisms as described in ISO 14461-2.


5 Apparatus and equipment 6 Preparation6.1 Preparation of glassware and other laboratory materials6.2 Sterilization/decontamination 6.3 Disposable equipment and materials 6.4 Storage of clean glassware and materials6.5 Management of sterile glassware and materials6.6 Use of decontamination and disinfection6.7 Waste management 6.8 Washing


Avoid cross-contaminationUse different chambers, or at least different containersdifferent chambers, or at least different containers, for the storage of :

Uninoculated culture media and reagent.Test samples.Microorganism cultures and incubated media.Allow appropriate air circulation.Minimize cross-contamination.

5.9 Refrigerator, cold-storage room5.9.2 Use


7 Preparation and sterilization of culture mediaPrepare and sterilize culture media in accordance with ISO/TS 11133-1 and

ISO/TS 11133-2.

ENV ISO 11133ENV ISO 11133--1:20001:2000 Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media - Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory (ISO/TS 11133-1:2000)

CEN ISO/TS 11133CEN ISO/TS 11133--2:20032:2003 Microbiology of food and animal feeding stuffs - Guidelines on preparation and production of culture media - Part 2: Practical guidelines on performance testing of culture media (ISO/TS 11133-2:2003)

Use Use -- if possible if possible -- standardized and certified media.standardized and certified media.


Prepare media strictly according established and documented procedures established and documented procedures (e.g. ISO 11133, manufacturers specifications).

Use glass distilled waterdistilled water. (Ion-exchange-) Desalted water might contain considerable numbers of microorganisms, particularly if connected with plastic tubes. The conductivity of distilled water should not exceed 25 µS/cm at 25 °C (ISO 7218-rev:2002).

Check pH pH carefully, in particular if alkaline pH values are desired (Vibrio). Remember that during autoclaving the pH might drop by 0.2-0.4 units.

Pour autoclaved media into plates only after sufficient coolingcooling (47 ± 2 °C).

Store and maintainStore and maintain media according to manufacturers instructions and good laboratory practice.

Lot identificationLot identification: Give each preparation an identification number for tracking purchase or ingredients, preparation protocol, person and date.

Label Label each medium (particularly plates) unambiguously (type, lot).

Control functionalityControl functionality of media in regular intervals with reference strains. This holds true particularly for chromogenic or other selective media which must be tested with the species the medium should be selective for as well as with species presumed to be suppressed. Document results (see e.g. Corry, J.E.L., Curtis, G.D.W., Baird, R.M.: Handbook of culture media for food microbiology, Elsevier, Amsterdam, 2003

Observe maximal storage life. Check storage life. Check regularly exsiccation, volume, contamination and other changes changes (pH, layer thickness, color, moisture; incubate to detect contamination). .

Some comments on Culture Media

EN ISO 6887EN ISO 6887--1:1999 1:1999 Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 1: General rules for the preparation of the initial suspension and decimal dilutions (ISO 6887-1:1999)

EN ISO 6887EN ISO 6887--2:20032:2003 Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 2: Specific rules for the preparation of meat and meat productsmeat and meat products (ISO 6887-2:2003)

EN ISO 6887EN ISO 6887--3:20033:2003 Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 3: Specific rules for for the preparation of fish and fishery productsfish and fishery products(ISO 6887-3:2003)

EN ISO 6887EN ISO 6887--4:20034:2003 Microbiology of food and animal feeding stuffs - Preparation of test samples, initial suspension and decimal dilutions for microbiological examination - Part 4: Specific rules for the preparation of products otherother than milk and milk products, meat and meat products, and fish and fishery products (ISO 6887-4:2003)

Laboratory StandardISO Norms for diluting






VIBRIO CHOLERAE VIBRIO VULNIFICUSVIBRIO

PARAHAEMOLYTICUS

The major Culprits

Kingdom Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Vibrionales

Family: Vibrionaceae

Genera: Vibrio, Photobacterium and 4 morerelated: Aeromonas, Plesiomonas

Species: Vibrio parahaemolyticus

Classification

+Sheathed polar flagellum

+

57—63

(+)

AeroAero--monasmonas

+

+

+

51

(+)

PlesioPlesio--monasmonas EnteroEntero--bacteriabacteria--ceaeceae

PhotoPhoto--bacteriumbacteriumVibrioVibrioTest or PropertyTest or Property

+Enterobacterial common antigen (eca)

++D-Mannitol fermentation

dd+Lipase production

++Sensitive to the vibriostatic compound 0/129

++Na+ is required for growth or stimulates growth

+Accumulate poly-f3-hydroxy-butyrate, but do not utilize ß-hydroxybutyrate

+Peritrichous flagella when grown in liquid media

38—6040—4438—51Mol% G + C content of DNA

++Associated with diarrhea and extra-intestinal infections in humans

Properties of the four genera originally classified in the family Vibrionaceae by Veron (1965); comparison with

Enterobacteriaceae (from Bergey's Manual)

++++Blood agar plus ampicillin

Aeromonas agar:++++Rimler-Shotts medium

++++Dextrin-fuchsin-sulfite medium

++++++++Bile salts-brilliant green agar

Selective media for Aeromonas and Plesiomonas

++++ (?)++++Vibrio agar

no growthno growth++++++TCBS agar

Selective media for Vibrio:++++++++++++Photobacterium broth

++++++++++++Photobacterium agar

++++++++++++Marine agar

++++++++++++Marine broth

Nonselective "marine" media: ++++++++++++Alkaline peptone water

++++++++++MacConkey agar

++++++++++++++Sheep blood agar

PlesiomonasAeromonas Photo-bacterium Vibrio Medium

General and selective media for the genera Vibrio, Photobacterium, Aeromonas, and Plesiomonas

Bergey's Manual VII, PartB

102 (immunocompromisedpeople, antiacids)

to >106

no carriers

not exactly known, probably >106

(SCVMPH 2001, FAO/WHO 2001)

immunocompromised, antiacids lower

no carriers

106 (-1011) WHO 2006human carriers

infective dose

Oysters contain 101-103 cfu/g V.vulnificus depending on

water temperature and salinity. 0,4-6% of commercial

samples of raw oysters contain V.vlunificus

101-103 cfu/g V. para-haemolyticus in Oysters

Occurrence in natural waters ≈ 46 cfu/ltr. (Cowell & Spira

1992), almost absent in shrimp and oysters (WHO

2006).

Density

SeawaterZooplankton

Shrimp, Oysters, Fish

SeawaterZooplankton

Shrimp, Oysters, Fish

Sweet WaterEstuarine-/Seawater

ZooplanktonShrimp, Oysters, Fish

Humans

Habitat

Vibrio vulnificusVibrio vulnificusrecognized first in 1979

Vibrio parahaemolyticusVibrio parahaemolyticusrecognized as pathogenic in

1953

Vibrio choleraeVibrio choleraeCholera known before 800

Bc, recognized 1884

Properties of pathogenic Vibrio

26-28°C0.84 log(10)/h broth

0.18 log(10/h) oystersno growth in oystersOptimal Growth rate

0.5 – 5.03.0 0–4.0 0.5 NaCl (%)

anaerob-aerobaerobanaerob-aerobaerobAtmosphere

0.940 – 0.996 0.981 0.970 – 0.998 0.984 aW

4.8 – 10.0 7.5 – 8.5 5.0 – 9.6 7.6 pH

(5–)10 – 43 37 10 – 43 37 Temperature (°C)

RangeOptimum RangeOptimum

V. parahaemolyticus &V. vulnificus V. parahaemolyticus &V. vulnificus V. cholerae V. cholerae

Properties

11.011.0

9.09.0

7.07.0

5.05.0

3.03.0

1.01.0

App

les

App

les

Gra

pes

Gra

pes

Car

rots

Car

rots

Ban

ana

Ban

ana

Cor

nC

orn

Jogh

urt

Jogh

urt

Che

ese

Che

ese

But

ter

But

ter

Milk

Milk

Bee

fB

eef

Ham

H

am

Veal

Veal

Poul

try

Poul

try

Fish

Fish

Oys

ters

Oys

ters

Shrim

pSh

rimp

Cra

bmea

tC

rabm

eat

SalmonellaSalmonella7.0 7.0 –– 7.57.5

S. aureusS. aureus6.0 6.0 -- 7.07.0

V. choleraeV. cholerae7.67.6

9.69.6

5.05.0

V. V. parahparah..V. V. vulvul..7.57.5--8.58.5

10.010.0

4.84.8

11.011.0

1.01.0

pHpHwild shrimp seawater environments (pH 8.5, salinity of 30 ppt, WHO 2006)

pH Value of food and growth of pathogens

---10%10% NaCl

-+-8%8% NaCl

++-6%6% NaCl

+++3%3% NaCl

--+0%0% NaCl

Growth in (w/v):

V. vulnificusV. para-haemolyticusV. cholerae

Salt tolerance



•• Basic properties of Vibrio relevant to Basic properties of Vibrio relevant to the analytical laboratorythe analytical laboratory



Medical samplesMedical samples(Feces, blood)(Feces, blood)

EpidemiologyEpidemiology

Food samplesFood samples(Shrimp, Mussels)(Shrimp, Mussels) Food safetyFood safety

Water samplesWater samples Food safetyFood safetyAquatic farmingAquatic farming

SampleSample QuestionQuestion sample characteristicssample characteristics

Feces: solid/semisolid, homoFeces: solid/semisolid, homo--genousgenous, 10, 1099--10101111 cfu/gcfu/gEnterobacteriaceae, Lactic Enterobacteriaceae, Lactic acid bacteria, Enterococci, acid bacteria, Enterococci, Staphylococci .... Staphylococci ....

Shrimp: solid, heterogeneous, Shrimp: solid, heterogeneous, 101022--101099 (10(1055 -- 101077) cfu/g) cfu/gPseudomonas, Vibrio, MicroPseudomonas, Vibrio, Micro--

coccuscoccus, Aeromonas, , Aeromonas, StaphyStaphy--lococcuslococcus, Bacillus, , Bacillus, ColiformsColiforms.. ..

Water: liquid, homogenous, Water: liquid, homogenous, 101011--101055 cfu/gcfu/gPseudomonas, Vibrio, MicroPseudomonas, Vibrio, Micro--

coccuscoccus, Aeromonas, , Aeromonas, ColiformsColiforms.. ..

The Purpose determines the Test Method

Feces:Feces:

after suspecting diagnosishigh total bacterial cell numbersmany related species

therefore:

Direct plating of dilution series on selective agars.Subsequent isolation (restreaking on other selective agars) and characterization.Highly selective (immuno-logical) tests with original sample.no enrichment!no enrichment!

Vibrio isolation and identification from feces(Bergey's Manual)

Biochemical differentiation between Vibrio cholerae and fecal pathogens

from: Laboratory methods for the Diagnosis of Epidemic Dysentery and Cholera, CDC 1994

Quantitative: Enumerate or estimate directly or indirectly the bacterial load in the sample.Direct enumeration

Microscopic countColony Forming Unit (CFU) (CFU) count

o Non-selective media (eg. nutrient broth)o Non-selective differential media (containing an indicator, eg. for pH)o Selective media (containing a selective agent, eg an antibiotic)o Selective differential media (containing both, indicator and selective agent, eg. TCBS)

Indirect DeterminationMost Probable Number Method (MPN)Enumeration of Injured Cells by Selective MediaOverlay MethodThin Agar Layer Method

Qualitative: Determine the possible presence of certain microorganisms.Pre-enrichment step on selective or not selective media.Testing on medium containing selective and/or differential agents

Bacteriological detection methods

Factors affecting selectivity of enrichment:

OutgrowthType of accompanying microflora (species).Density of accompanying microflora.Growth rate and metabolism of accompanying species in medium (e.g. acidification, oxygen depletion, nutrient depletion).

EliminationInteraction of accompanying flora with target species (bacteriocin production, signal molecules, inhibitory or lethal substances).

Unselective and selective enrichment media

with modifications adapted from Corry, Curtis and Baird 2003, Handbook of culture media for food microbiology, Elsevier

membrane solubilizer; bactericidal for many bacteriaDetergentSodium lauryl (dodecyl)

sulphate

NaCl (0.5–1%) required by most Vibrio; higher levels inhibit many Gram-negative bacteria

Osmolarity, Na+ as counter ion (for H+)Salts

Selection for Vibrio (optimum 8.4–8.6)alkaline environment necessary for Sodium-bioenergetics

pH

bactericidal towards many Gram negative bacteria AntibioticPolymyxins

(including colistin)

inhibits some Gram positive bacteriaOxidantTellurite

inhibit Gram positive bacteria, some Gram-negative but not Enterobacteriaceae

Surface active, amphipathic detergents

Bile salts (including taurocholate)

Mode of ActionMode of ActionMechanismMechanismAgent Agent

Selective mechanisms exploited for Vibriodiagnostics

Polymyxin B7,600,50V. vulnificus, V. cholerae EI TorStarch-gelatin-polymyxin B broth

High salt, Teepol, Water blue, Alizarin yellow6,903.0V.

parahaemolyticusWater blue-alizarin yellow broth

Sodium dodecylsulphate, Methyl violet 8,603.0V,

parahaemolyticusSaline-glucose-sodium dodecyl sulphate medium

High pH, Teepol (Sodium dodecylsulphate may be substituted for Teepol)

9,402.0V. parahaemolyticusGlucose-salt-Teepol

Colistin7,402.0V, parahaemolyticusSalt-colistin broth

Polymyxin B7,402.0V. parahaemolyticusSalt-polymyxin broth

High pH, Potassium tellurite8,601.0Vibrio cholereaAlkaline taurocholate-peptone water

High pH, Potassium tellurite9,20probably 1.0Vibrio spp.Alkaline tellurite peptone water

High pH, Potassium tellurite, Sodium taurocholate9,201.0Vibrio spp.Monsur tellurite-taurocholate

peptone water

High pH8,601.0Vibrio spp.Alkaline peptone water with electrolyte supplement

High salt and High pH8,603.0halophilic Vibrio spp.Salt-alkaline peptone water

High pH8,600.5-1.0Vibrio spp.Alkaline peptone water

Selective agentspHSalt content %Intended useMedium

Unselective and selective enrichment media

In 1992, Sloan et al. compared five enrichment broths for their ability to detect and enumerate V. vulnificus in oysters. The log MPN/g of V. vulnificus detected in oysters decreased with enrichment broth in the sequence

Alkaline Peptone Water (APW), (average of MPN 4.2/g)Marine (MRN) Broth (ZoBell, 1941),Horie Arabinoseethyl Violet (HAE/HB) Broth,Monsur’ (MNS) Broth (Monsur, 1963) and

Glucose Salt Teepol (GST) broth (average of MPN 1.3/g)

Moreover, V. vulnificus strains were isolated more frequently from CPC agar (81%, 580/468) than from SPS (SDS, Polymyxin-<b-Sucrose)agar (61%, 42/207).These results were consistent with FDA’ rules.

Sloan, E.M., Hagen, C.J., Lancette, G.A., Peeler, J.T., Sofos, J.N. (1992).Comparison of five selective enrichment broths and two selective agars for recovery of Vibrio vulnificus from oysters. J. Food Protect. 55,356-359.

Enrichment Media: Caveat

http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/complete.pdf

Laboratory methods for the Diagnosis of Vibrio choleraeLaboratory methods for the Diagnosis of Epidemic Dysentery and Cholera

Centers for Disease Control and Pre-vention(CDC)

ISO/TS 21872-1

ISO/TS 21872-2

Laboratory Procedures for the Microbiological Analysis of Foods

Bacteriological Analytical Manual (BAM)

Manual

only commercially available

Detection of Vibrio parahaemo-lyticus and Vibrio choleraeDetection of other species

International Organization for Standardization

http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/index-eng.php

Laboratory procedures MFLP-37 (1) Halophilic VibriosMFLP-37 (2) V. choleraeMFLP-72 (V. cholerae) andMFLP-73 (V.vulnificus)

Health Canada

http://www.cfsan.fda.gov/~ebam/bam-9.htmlChapter 9: VibriosUS Food and Drug

Administration

SourceNameInstitution

Official Methods for Food & Environmental samples

USA: Safe Quality FoodUSA: Safe Quality Food UK: British Retail StandardUK: British Retail StandardG, F, many European countries:G, F, many European countries:

International food standardInternational food standard

Accredited LaboratoryAccredited Laboratory

Retail Standards

http://www.cfsan.fda.gov/~ebam/bam9.html

FDA

ISO/TS 21872-1:2007(E)Microbiology of food and animal feeding stuffs — Horizontal method for the detection of potentially enteropathogenic Vibrio spp.

Part 1: Detection of Vibrio parahaemolyticus and Vibrio choleraePart 2: Detection of species other than Vibrio parahaemolyticus and Vibrio cholerae


Standards: ISO (1)

The Vibrio are very fragile organism, and are difficult to detect in mixed culture. This especially applies for sublethally injured cells, e.g. in frozen samples. While rescuing and the detection of VNC Vibrios requires longer incubation times, they are easily overgrown by other competing microflora during longer enrichment periods.

Therefore two steps become necessary.

A pre-selective enrichment in ASPW for just 6-8 hs at both, 37°C and 41.5 °C for fresh and frozen samples, respectively.

A second selective enrichment, for further 18 h at 37 °C

Only 20% of V. vulnificus20% of V. vulnificus propagate at 42 42 °°CC but 90% at 40 °C and 99%99% at 37 37 °°CC;

99%99% of V. parahaemolyticusV. parahaemolyticus grow at 37, 40 and 42 °C.

A good selective enrichment broth has not been developed for V. cholerae. However, due to its rapid generation timerapid generation time (20 min.20 min.), short short incubation periods are effective for isolation.

APW provides suitable enrichment for incubation periods of 6 to 8 h, but Overnight periods (16 to 18 h), although not desirable, have been used to facilitate sample analysis during work hours. If the product was subjected to a processing step, i.e. heating, freezing, drying, or low densities are expected, incubation overnight is recommended to thoroughly resuscitate injured cells.

Analytical aspects (1)

Vibrio parahaemolyticus and Vibrio cholerae can be present in small numbers and are often accompanied by a much larger number of other microorganisms belonging to the Vibrio-naceae family or to other families. Consequently, two successive selective enrichmentstwo successive selective enrichments are necessary in order to detect the target organisms.

First enrichment: Alkaline saline peptone water, (ASPW)Alkaline saline peptone water, (ASPW) is inoculated with the test portion at ambient temperature. It is incubated at 37,0 37,0 °°C for 6 h C for 6 h ±± 11 h for deep frozen productsfor deep frozen products, or at 41,5 41,5 °°C for 6 h C for 6 h ±± 1 h1 h for fresh productsfor fresh products.

Transfer 1 ml of the culture above taken from the surface into a tube containing 10 ml of ASPW

Second enrichmentAlkaline saline peptone water, (ASPW) Alkaline saline peptone water, (ASPW) is inoculated with an aliquot of the first enrichment and is incubated at 41,5 41,5 °°C for 18 h C for 18 h ±± 1 h1 h

ISO (2): Enrichment

The incubation of raw oyster samples in APW at 42°C for 6 to 8 h has proven effective for isolation of V. cholerae and is recommended . However, DePaola and Hwang found that enrichment incubation for 18 to 21 h, instead of 6 to 8 h, gave a higher recovery of O1 O1 V. V. choleraecholerae when low inoculalow inocula were used. Because of these considerations, it is recommended to streak APW enrichments both after 6 to8 h and after overnight incubation.

It is also recommended for raw oystersraw oysters to use a 1:1001:100 ratio of oyster to APW (dilution!).

http://www.cfsan.fda.gov/~ebam/bam-9.html

FDA BAM

Second enrichment

inoculate with a sampling loopinoculate with a sampling loop

First enrichment

inoculate with a sampling loopinoculate with a sampling loop

Thiosulfate Citrate Bile Sucrose Agar

Selective Agars

ISO (3): Isolation and identification

Oliver 2005, The Journal of Microbiology, 43(1),93-100

In naturenature, bacteria enter the VBNC state in response to some form of environmental stress, such as starvation, incubation outside the temperature range of growth, elevated osmotic concentrations (e.g. seawater), oxygen, or exposure to white light.

When in this state, these bacteria cannot be detectedcannot be detectedby conventional culture methodsby conventional culture methods as they are incapable of the sustained cellular division required for colony formation.

Processes which are normally assumed to be bactericidal bactericidal for bacteria may instead result in cells which reside in the VBNC state. These include such treatments as pasteurization of milk (e.g. Gunasekeraet al., 2002) and chlorination of wastewater (Oliver et al., 2005).

Entry of V. vulnificus into the VBNC state on incubation at 5oC. Shown are total cell counts (□), culturable counts (○), and viable counts (●).

Viable but nonculturable (VNC) Bacteria(Oliver 2005)

Binsztein et al. 2004, Applied and Environmental Microbiology 70 (12),. 7481-7486Vora et al. 2005, Proc. Natl. Acad. Sci. USA 102 (52), 19109-19114

Like other bacteria, the Vibrionaceae enter a VBNC stateVBNC state as a survival strategysurvival strategyin response to environmental stresses such as low temperatureslow temperatures.

CulturableCulturable V. cholerae non-O1 was identified predominantly during warmer warmer months and in the lowermonths and in the lower--salinity waterssalinity waters of the estuary. This finding is in agreement with reports for other areas.

A similar relationship was notnot evident for the presence of nonculturablenonculturable V. cholerae O1, since this organism was found during all seasons at both the marine and estuarine stations. Thus, it can be assumed that the VNC state makes it possible for this bacterium to persist at a wide range of salinities and persist at a wide range of salinities and temperatures. temperatures.

The transition to the non-culturable state might also occur through human activities, as in the lab after storage for prolonged times without nutrients, after after cooling or freezing (seafood processing!)cooling or freezing (seafood processing!) or insufficient inactivation (heat, pressure).

Vibrio in the VBNC state remain metabolically active Vora et al. 2005), retain their virulence properties, and under the appropriate conditions (such as ingestion) recover to become fully vegetative, culturable (Oliver & Bockian 1995) and pathogenic (Colwellet al. 1996).

Fluorescent antibody stained V.cholerae on

Microplankton

Water

Zooplankton

Viable but nonculturable (VNC) Vibrio(Binsztein et al 2004; Vora et al. 2005)

Alexandre Leclercq 2006

Viable but not cultivable

Whitesides & Oliver. 1997 Appl. Environ. Microbiol. 64, 3025-3028.

The VNC state might be reversed and bacteria be induced to revert to a cultivable form by prolonged incubation under appropriate conditions (media, temperatures)

Therefore enrichment in liquid media is very Therefore enrichment in liquid media is very recommendable for the detection of recommendable for the detection of VibrionaceaeVibrionaceae from water samples and frozen from water samples and frozen or chilled food.or chilled food.

Liquid cultivation is superior to solid cultivation.Microtiter plates are more convenient than tubes for assessing microbial counts by MPN methods.

Resuscitation(Whitesides and Oliver, 1997)

Thank You

Vibrio (Tuesday)

Properties of Properties of VibrioVibrioV. choleraeV. choleraeV. parahaemolyticusV. parahaemolyticusV. vulnificusV. vulnificus

IdentificationIdentificationSelective solid mediaSelective solid mediabiochemical toolsbiochemical tools

One more note on enrichment protocols:Confusion

• For oysters only, incubate two containers containing the same APW-homoge-nate,one at 35at 35°°CC (flask A) and the other (flask B) at 4242°°CC, both for 6-8 h and for 24 h.

• For all other foods, incubate one APW-homogenate at 3535°°C for 6C for 6--8 h8 h and for24 h24 h.

• For frozen foods (oysters only), incubate two containers, one at 3535°°CC and other at 4242°°CC for 6-8h.

V. choleraeV. cholerae

• (APW or ASPW). Incubate tubes 1616--18 h at 3518 h at 35°°CC. MPNV. vulnificus &V. vulnificus &V. parahaemolyticusV. parahaemolyticus

Health Canada

• Incubate the ASPW at 41,5 41,5 °°CC for 18 h ± 1 h.

• Incubate the initial suspension (9.1) at 37 37 °°C for 6 h C for 6 h ±± 1 h1 h for deep-frozen products,

• or at 41,5 41,5 °°C C ±± for6 h for6 h ±± 1 h for fresh1 h for fresh, dried or salted products.V. cholerae &V. cholerae &V. parahaemolyticusV. parahaemolyticus

• Incubate APPW at 35 35 ±±22°°C for 6 to 8 h.C for 6 to 8 h.• Reincubate the jar overnight if the sample had been processed in some way.• For analysis of raw oysters, include a second tared flask with 25 g of product

plus 2,475 ml APW. This flask should be incubated 18 to 21 h at 42 42 ±±0.2 0.2 °C in a water bath .


• no enrichment, dilution series Incubate APW 3. overnight at 35 35 ±±22°°CC..V. parahaemolyticusV. parahaemolyticus

enrichmentenrichment--MPN: growth: numbers multiply MPN: growth: numbers multiply and ratios between species shift and ratios between species shift

Dilution series in Dilution series in saltsalt--water (saline).water (saline).

no growthno growth

direct plating:direct plating:number on plate corresponds number on plate corresponds directly to numbers in dilution directly to numbers in dilution

tubetube

growth: growth: here was here was >1 colony>1 colony

no growth: no growth: assumed less assumed less

than one colonythan one colony

Microtiter methods

Vibrio (Tuesday)

Properties of Properties of VibrioVibrioV. choleraeV. choleraeV. parahaemolyticusV. parahaemolyticusV. vulnificusV. vulnificus

IdentificationIdentificationSelective solid mediaSelective solid mediabiochemical toolsbiochemical tools

500-400 BC: Sushruta Samshita from India, written in Sanskrit describes Cholera

500 BC: Hippocrates200 AD: Galen900 AD: Rhazes, Islamic physician describes "El-Houwa"

(from the air)1364 AD: Persian records describe the Cholera1563 AD: Garcia del Huerto, a Portuguese physician at

Goa describes Cholera

Ancient Texts Describe Cholera

John Snow`s Map Memorable Scientists

1854 1854 Filippo PaciniPacini (1812-1883)

is the first to discover Vibrio cholerae Vibrio cholerae in a sample of intestinal mucosa from a Cholera victim. His finding is ignored by the scientific community.

18841884 Robert KochKoch (1843-1910)

rediscovers V. cholerae and is the first to cultivate thebacterium (on beef broth agar). He describes it as "Komma-Bacillus". For his work on tuberculosis he isto recieve the Nobel-Price in 1905.

Massive public health reformsMuch smaller outbreaks later on in Europe

Results Cholera: far from over

Melinda Nugent, presentation April 6, 2004

Cholera in the world, 1960-2005

0

100000

200000

300000

400000

500000

600000

700000

1960

1962

1964

1966

1968

1970

1972

1974

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

Year

Num

ber

of C

ases

Latin AmericaAsiasub-Saharan Africa

Source: WHO Weekly Epi Record

V. cholerae Afflicted Areas (2000)

In industrialized with reported cases cholera--imported

Cholera Gravis

• Loss of 1 liter of fluid/hour• >10% of body weight• Hypotension within 1 hour

(usually 4-12 hours)• Death if untreated within 2 hours

(usually >18 hours)

Cholera differs from the other diseases caused by Vibrio`s in being a intestinal disease and lethal in most untreated infection.

Vibrio cholerae: transmission

• Infectious Dose:Infectious Dose: 102102--1011 colony1011 colony--forming units (cfu)forming units (cfu)• Water (infectious dose = 109)• Food (infectious dose = 103) • Person-to-person• Vibrio cholerae has to adapt to human host.

– Temperature– pH (acidic in stomach, alkaline in small intestine)– bile acids– penetrate mucous layer and compete with normal intestinal flora– Invasion of appropriate cells by pathogenicity systems and spread within organs.

People most at risk:those with low gastric acid levelsthose with specific or impaired immune system:

Children: 10x more susceptible than adultsElderly

Blood types O>> B > A > AB

Classification Scheme

A & B A & B A & CA & C A, B, CA, B, C

Designed using information presented in review by NS Crowcroft. 1994. Cholera: Current Epidemiology. The Communicable Disease Report. 4(13), R158-R163.

O1O1

inabainaba hikojimahikojima

O139O139

ClassicalClassical El TorEl Tor

Division into ribotypes

ToxigenicV. cholerae

Not toxigenicV. cholerae

2 epidemic serotypes

Division into 2 biotypes

ogawaogawa

Each O1 biotype can have 3 serotypes

Immunology

Microbiology

Immunology

16S RNA

O-Antigen

3 Types of Antigenic recognition:3 Types of Antigenic recognition:

O – Antigen – somatic polysaccharide of the endotoxinK – Capsular antigen -- virulence relatedF – Flagellar (H) antigen – found in motile species

For For V. choleraeV. cholerae differentiate:differentiate:Vibrio cholerae encompasses more than 200 serotypes, two of which (O1 and O139) are causative agents of epidemic Asiatic cholera. O1-Antigen

typical for most pathogenic cholera strains. O-139 Antigen

Emerging among previously immune persons in India in 1992 and in 1993 in Bangladesh. O139 organisms produce a polysaccharide capsule (K antigen) but do not produce O1 LPS or O1 antigen. Toxigenic O139 cholera originated by the horizontal transfer of the lipopolysaccharide gene cluster from an O22 serotype strain to an O1 recipient.

NA (not agglutinating) strainsNon-O1, Non-O139 Serogroup. Most - but not all -are CT (cholera toxin) negative and are not associated with epidemic disease. (but implicated in some cases of mild gastroenteritis). Such strains are autochthonous (indigenous) members of aquatic bacterial communities throughout the world .

Toxin identification:- Immunochemistry -

modified fromwww.bmsc.washington.edu cholera toxin

The Reversed Passive Latex Agglutination (RPLA) Toxin Detection Kit relies on Latex particles sensitized with antibodies to Cholera toxin. In the presence of the toxin, the latex particles agglutinate to form a clearly visible lattice structure.

Besides Vibrio cholerae enterotoxin, the test will detect E. coli heat-labile enterotoxin (and possibly other related AB5 toxins, eg. Campylobacter Toxin).

A subunit: A subunit: ADPribosylase

5xB5xB--subunitsubunit

Differentiation of Classical and EI Tor biovars

of Vibrio cholerae 01

+-EI Tor phage 5

-+Classic phage IV

Phage susceptibility:

-+Susceptibility to polymyxin 50 µg

+-Voges-Proskauer (acetoin)

+-Haemagglutination of chick cells

+ or --Haemolysis

EI Tor BiovarClassical BiovarTest

Survival of V. cholerae in water

Major reasons for survival:Major reasons for survival:VNC statusChitinolytic activity - adherence to zooplankton

T90 = the time in hours required for a 1-log reduction in V. cholerae. NOTE:

56 (8–235) Seawater (non-sterile) 53 (1–230) Fresh water (non-sterile) El Tor 95 (0.36–161) Seawater (non-sterile) 18 (0.16–36) Fresh water (non-sterile) Classical

Mean (range) Mean (range) T90 (h) T90 (h) Water Water Biotype Biotype

from WHO 2006; Feachem, Miller and Drasar, 1981.

V. parahaemolyticus

V. parahaemolyticus

Halophilic, Gram-negative bacterium, Komma-shaped.>13 O- and >71 K-serotypes, all with the same H (flagellar-) antigen.O3:K6 serotype isconsidered an emerging pathogen.Predominant in marine and estuarine environments.Occurrence depends on salinity and temperature.

about 4 cfu/100 ml seawater @ T<16°C; about 1,000 cfu/100 ml seawater @ T≈ 25°C.(DePaola et al. 1990, Appl.Envir.Microbiol. 56, 2299).

Enriched in live Oysters up to >103 cfu/g (De Paola et al. 2000, Appl.Envir.Microbiol. 66, 4649).Grows very fast (generation time ≈ 10 min.); multiplies very fast in unrefrigerated oysters.Can exceed 1,000 MPN/g in market oysters (Cook et al. 2002, J. Food Protect. 65, 79).Dies at temperatures < 5°C, but some (VNC) survive freezing at –18oCIn Asia, Vibrio parahaemolyticus is a common cause of foodborne disease, up to 70% of reported foodborne infections in the 1960’s.Several pathogenicity factors, transmitted by horizontal gene transfer.

ESR: Quantitative data for Vibrio parahaemolyticus in seafood

FAO/WHO, 2002 101-103/100g, 69% +veJapan, over 1 year

88/g Grouper 82/g Rabbitfish96/g Crab 32/g Prawn

Chan et al., 1989 Hong Kong, June to October <3 MPN/100g Minimum

150 MPN/100g Maximum 24 MPN/100g Mean Brazil (Palmas), mussels <3 MPN/100g Minimum

210 MPN/100g Maximum 31 MPN/100g Mean Brazil (Flamengo), mussels <3 MPN/100g Minimum

24,000MPN/100g Maximum Matté et al., 1994 6,100 MPN/100g Mean Brazil (Ubatuba), mussels

Reference Counts Samples tested

Lake et al. 2003, Vibrio parahaemolyticus in seafood; New Zealand Food Safety Authority (“NZFSA”)

Vibrio vulnificus Vibrio vulnificus

Koenig KL, Mueller J, Rose T 1991 : Vibrio vulnificus-Hazard on the half shell. Western Journal of Medicine 155:400-403

Vibrio vulnificus is a a lactose-fermenting (biogroups 1 and 2; difference to V. parahaemo-lyticus), halophilic, Gram-negative bacterium.Three biotypes (biogroups), designated 1, 2, and 3, have been established based upon characteristics, such as indole production, host specificity, serotype (Tison et al., 1982), and genetic subtyping.V vulnificus is most frequently isolated from seawater with a temperature greater than 20°C and a salinity of 0.7% to 1.6%, it is rarely found in seawater cooler than 17°C.It is associated with various marine species such as plankton, shellfish (oysters, clams, and crabs), and finfish. Surveys have shown that more than 50% of oysters and 10% of crabs might be culture-positive for this organism.V. vulnificus might exist in a VNC state. It will proliferate in seafood kept at room temperature but will be killed by boiling or freezing.V. vulnificus, described first in 1960 and characterized not earlier than 1976, is rapidly acquiring new (pathogenic) traits and is counted among the emerging pathogens of humans.

V. vulnificus growth and survival in oysters

DePaola & Bowers 2004, Risk Assessment for Vibrio vulnificus, ISSC

-0.002 5.7 Cook et al. 2002

no growth13.0 Kaspar and Tamplin, 1993

0.025 18.0 Cook, 1994

0.175 28.0 Cook, 1997

Growth rate (log10 per hr)

Holding temperature (Celsius)

Study

Biogroups of V. vulnificus

–+ (+) ONPG –+ (+) Lactose –+ + Cellobiose –+ + Salicine–+ (+) Citrate (Simmons) –+ –Sorbit––d Mannit+ –+ Indole + –d

Ornithindecarboxylase

BiogroupBiogroup3 3

BiogroupBiogroup2 2

BiogroupBiogroup1 1 Test Test

Vibrio vulnificus virulence factors

Electron micrograph of Vibrio vulnificus. The arrows mark fimbriae (pili) of the bacterium. Dr. Mark Strom at the NOAA Northwest Fisheries Science Center.

© 2005 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology.Gulig et al. 2005, Molecular Pathogenesis of Vibrio vulnificus, The Journal of Microbiology 43, 118-131

Capsule Capsule The most important virulence factor for V. vulnificus is its capsular polysaccharide (CPS), the noncapsulated form is nonvirulent. Under laboratory conditions, acapsular variants arise at a fairly high frequency (~1/100), with certain environmental stresses dramatically increasing this switch rate.

LPS LPS AV. vulnificus lipopolysaccharide (endotoxin) is expected to play a role in fever and septic shock brought on by infection. On the basis of lipopolysaccharide (LPS) antigens, the species can be organized into three biotypes. Biotype 1 is the predominant human pathogen; biotype 2 is associated with eels; and biotype 3 was recently isolated from fish handlers in Israel. LPS type (1/5) is significantly more prevalent among clinical strains.

Extracellular enzymesExtracellular enzymesinclude proteases, collagenase, mucinase, esterase, chondroitinase, hyaluronidase, DNAase and sulfatase.

RTX type toxinRTX type toxinRTX toxins cause pore formation in red blood cells, necrotic death of Hep2 cells, and depolymerization of actin in HeLa cells.

Properties of the halophilic Vibrio

Thermostable hemolysins TDH, TRH ;TTSS (2)ProteasePhospholipases

Extracellular enzymes, eg. Metalloprotease/ElastaseSiderophoresRTX-ToxinPolysaccharide capsule

Virulence factors

Maximum: 10 (FDA) Minimum salt 0.5

Maximum: 6.0 Minimum: 0.1 Salt tolerance (%)

Maximum: 11Minimum: 4.5-5

Maximum:10 Minimum: 5 (FDA) pH range

Gram-negative curved or straight rods with flagellum.

0.5-0.8 x 1.4-2.6 um

Gram-negative straight or curved rods.

0.5-0.8 x 1.4-2.6 um Morphology

4-96 h Short, 7 h to several days (median 16-38 h). Incubation period

Particularly important in raw seafood. Assumed to be responsible for 50-70% of

enteritis cases in Japan.

Invasive and rapidly lethal pathogen. 56% fatal. Significance

Seafood, coastal marine environments, intestines of marine animals

Salt water, molluscs, seafood. Habitat

V. parahaemolyticusV. parahaemolyticusV. vulnificusV. vulnificus

Epidemiology

Report for 2006Report for 2006

Stool 9Blood 64Wound 31other 4

Stool 359Blood 6Wound 27other 12

site of isolation

31

1

000

Death

79

66

400

hospitalized

99

403

O-1: 8O-141 2O-75 1

Patients

2 other food44 raw oysters16 other oysters

Fruit, Water, CrabRaw OystersClams, Oysters

Sources (Food)

V. vulnificusV. vulnificus

62V. parahaemolyticusV. parahaemolyticus


Out-breaksSpecies

Simple tests to differentiate Vibrio

S (R have been reported) SS150 ugS (R have been reported) SR10 µg

Susceptibility toO129---H2S---Gas

AcidAcidAcidButt

AcidAlkaline or rarely acidAlkalineSlant

Appearance on TSI-Agar--+ or variableArabinose fermentation+- or rarely +-Sucrose fermentation++-ONPG (ß-Galactosidase) test

Classical - EI Tor +--Voges-Proskauer (VP) test--+8-++6+++3+--0

% Salt tolerance

V. choleraeV. vulnificusV. parahaemolyticusCharacteristics

Reference laboratories

National Reference Laboratory for Vibrio cholerae O1 and O139Epidemic Investigations and Surveillance LaboratoryFoodborne and Diarrheal Diseases Laboratory SectionCenters for Disease Control and Prevention1600 Clifton Rd., N.E., MS C03Atlanta, GA 30333USA

Standards: ISO (1)

ISO/TS 21872-1:2007(E)Microbiology of food and animal feeding stuffs — Horizontal method for the detection of potentially enteropathogenic Vibrio spp.Part 1: Detection of Vibrio parahaemolyticus and Vibrio choleraePart 2: Detection of species other than Vibrio parahaemolyticus and Vibrio cholerae


ISO: Isolation and identification(1)

First enrichment

ASPW at ambient temperatureXXg Product + 9XX ml ASPW (1:10 dilution)

Incubation for 6±1 hours

@ 41.5 ± 1°C for fresh products

@ 37.0 ± 1°C for frozen products

1 ml culture + 10 ml ASPW@ 41.5 ± 1°C

for 18 ± 1 hours

Second enrichment

First isolation

Streak with a sampling loop aliquot on TCBS platesIncubate @ 37°C for 24 ± 1 hours

Streak with a sampling loop aliquot on second selective agar plateIncubate @ 37°C for 24 ± 1 hours

Second isolation

Pick at least 5 colonies and subculture on TCBS platesIncubate @ 37°C for 24 ± 1 hours

Pick 5 colonies from the second selective agar plate and subcultureIncubate @ 37°C for 24 ± 1 hours

ISO: Isolation and identification (1)

First isolation

Streak with a sampling loop aliquot on TCBS platesIncubate @ 37°C for 24 ± 1 hours

Streak with a sampling loop aliquot on second selective agar plateIncubate @ 37°C for 24 ± 1 hours

Second isolation

Pick at least 5 colonies and subculture on TCBS platesIncubate @ 37°C for 24 ± 1 hours

Pick 5 colonies from the second selective agar plate and subcultureIncubate @ 37°C for 24 ± 1 hours

Confirmation

Pick at least 5 colonies each and subculture on Saline nutrient AgarIncubate @ 37°C for 24 ± 3 hours

Biochemical confirmationOxidase test, API 20E and 20NE, O-129, Wagatsuma Agar, String test

and many more. PCR and immunological methods possible.

Evaluate Results

TCBS AgarStandard solid medium for foodborne Vibrio. High pH for selection, ox bile and NaCl to suppress nontarget bacteria (Enterobacteria-ceae, Pseudomonas, Aeromonas).Sucrose fermentation as indicator (acid turns indicator yellow, thus sucrose metabolizing species form yellow colonies). Yellow colonies from TCBS agar give poor oxidase reactions and might contain substances inhibitory for further tests (PCR).Results varyResults vary between commercial batches from different suppliers.

3-4yellowV. metschnikovii

2-3 blue-greenV. vulnificus

3-5blue-greenV. parahaemolyticus

2-3 yellow, flatV. cholerae

colony dia-meter (mm)

Appearance on TCBSSpecies

Plate from Hara-Kudo et al. 2001Appl. environ. Microbiol. 67, 5819-5823

Selective mechanisms exploited for Vibrio diagnostics

with modifications adapted from Corry, Curtis and Baird 2003, Handbook of culture media for food microbiology, Elsevier

membrane solubilizer; bactericidal for many bacteriaDetergentSodium lauryl (dodecyl)

sulphate

NaCl (0.5–1%) required by most vibrios; higher levels inhibit many Gram-negative bacteria

Osmolarity, Na+ as counter ion (for H+)Salts

Selection for Vibrios (optimum 8.4–8.6)alkaline environment necessary for Sodium-bioenergetics

pH

bactericidal towards many Gram negative bacteria AntibioticPolymyxins

(including colistin)

inhibits some Gram positive bacteriaOxidantTellurite

inhibit Gram positive bacteria, some Gram-negative but not Enterobac-teriaceae

Surface active, amphipathic detergents

Bile salts (including taurocholate)

Mode of ActionMode of ActionMechanismMechanismAgent Agent

Examples for special selective agars in Vibrio diagnostics

Appropriate carbo-hydrates and phenol red may be added to identify particular species

Potassium iodide, Sodium thiosulphate0,5Pathogenic Vibrio spp.,

Not V. vulnificusThiosulphate-chloride-iodide (TCI) agar

Sucrose, sulphataseNa dodecyl sulphate2.0V, vulnificusSodium dodecyl-sulphate-polymyxin-sucrose (SPS) agar

Cellobiose, Lactose; x-galpH 8.5, Ox bile, Bile salts, Potassium tellurite2.0V. vulnificusV. vulnificus enumeration

(VVE) agar

SalicinpH 8.6, Ox bile; Potassium tellurite, Crystal violet1.0V. vulnificusVibrio vulnificus (VV) agar

CellobiosePolymyxin B, Colistin, Raised incubation temperature (40°C)2.0V. vulnificus, V. choleraeCellobiose-polymyxin B-

Colistin (CPC agar)

Mannose, Polymyxin B, Potassium tellurite, Na dodecyl sulphate

pH 8.41.0Vibrio spp., Differentiation of V. cholerae 01 and non-01

Polymyxin-Mannose-tellurite (PMT) agar

Sucrose, Triphenyltetrazolium-chloride (TTC)

Bile salt3.0Vibrio parahaemolyticus, Vibrio spp.

Tryptone-Soya-tri-pheny-ltetrazolium agar (TSAT)

Potassium telluritepH 8.5, Na taurocholate, Potassium tellurite1.0Vibrio spp.Gelatin-taurocholate-tellurite

agar (GTT - Monsur)

SucrosepH 8.6, Na thiosulphate, Na taurocholate, Ferric citrate1.0Vibrio spp.

(not V. hollisae)Thiosulphate-citrate-bile salt agar (TCBS)

Diagnostic agentsSelective agentsSalt (%)Intended useMedium

Special agars

ChromAgarChromAgar VibrioVibrio

Thiosulphate-Chloride-Iodide (TCI) Agar

Polymyxin-Mannose-Tellurite (PMT) AgarTellurite-Lauryl Sulphate

Agar (TLS)

TSAT agar was formulated by Kourany to assist the detectionofVibrio parahaemolyticus when present together with large numbers of V. alginolyticus in seawater. V. parahaemolyticus colonies are generally 3-4 mm diameter and dark-red in colour. V. alginolyticus colonies have a diameter of 2-3 mm and are white, sometimes with a pin-point pink centre. Enteric bacteria resistant to the bile in the medium, with the exception of Proteus spp., are readily distinguished from V. parahaemolyticus by colony appearance. Proteus spp. may be distinguished from V. parahaemolyticus by their biochemical reactions on triple sugar-iron agar.

ISO:5.2.2 Second mediumThe selection of the second medium is left to the choice of the test laboratory. Preparation of the medium should be strictly according to the manufacturers' instructions.

EXAMPLES Soya peptone triphenyl tetrazolium chloride agar (TSAT) and sodium dodecyl sulfate polymyxin sucrose

(SDSPS) agar (mCPC, CPC, CC agars are not recommended for isolation of V. parahaemolyticus).

Oxidase Test

APIs 20E, 20NE Swarming

Swimming in liquid is promoted by a single polar flagellum. The polar flagellum is produced continuously.

Swarming over surfaces or in viscous environments is enabled by the produc-tion of numerous peritrichous, or lateral, flagella. The polar flagellum is produced continuously, while the lateral flagella are pro-duced under conditions that dis-able polar flagellar function. Thus at times, two types of flagellar organelles are assembled simultaneously.

Sheathed polar flagellum of V. parahaemolyticus.

Polar and lateral flagella of surface-grown bacteria

L. McCarter 2001; Microbiology And Molecular Biology Reviews 65 (3), 445–462

O-129

OO--1291292,42,4--diaminodiamino--6,76,7--diisopropylpteridine (Phosphate)diisopropylpteridine (Phosphate)

O-129 inhibits Vibrio. It is applied at two concentrations and the different susceptibility of different species might serve as a diagnostic tool.

Inhibition can also be used, to distinguish Aeromonads (resistant) from Vibrio.

Nutrient agar containing NaCl should be used for testing. in contrast, special media for antibiotic resistance testing like Isosensitest agar are not suited for O-129 testing due to their low salt content.

There are more bacterial species resistant to O-129. Incubation at 41°C might enhance selectivity.

Vibrio variants resistant to O-129 have been reported.

String Test

The string test is a simple test for V. cholerae. It relies on its mucoid capsule.

A colony is emulsified in a drop of 5% sodium deoxycholate in 0.85% NaCl solution. V. cholerae forms a mucoid mass, which exhibits a "cheesefondue" effect when lifted wit a loop.

Salt O/F Medium; Tryptone Broth; Blood Agar

TCBS AgarAlkaline Peptone waterPathogenic Vibrio spp. Detection & Enu-meratiion in Food.

Nordic Committee on Food Analysis (NMKL) No. 156

Salt nutrient Agar; Salt TSI Agar; Salt-Lysine-decarb-oxylaseMedium; Salt-Tryptone Medium

TCBS AgarTSAT Agar

Alkaline Peptone waterSaline-glucose-Sodium dodecylsul-phate Broth

V. parahaemolyticusBritish Standard/ISO standard BS 5763 Part 14: 1991

Various biochemical test media, Wagatsuma Aga

TCBS Aga CPC AgarAlkaline Peptone waterPathogenic VibrioUSA Food and Drug

Administration. FDA.

Various biochemical test media.Wagatsuma Agar.

TCBS Agar

Bismuth sulphite BrothSalt-polymyxin BrothGlucose-salt-TeepolBroth

V. parahaemolyticus detection, enumeration

Canada. Health Protection Branch MFHPB - 15 1997 MFLP - 57 1995

Tryptone water Broth; Buffered glucose Agar; Nutrient Agar; TSI Agar;Bromocresol purple-cello-biose Broth; All with3% sodium chloride

TCBS AgarAlkaline Peptone waterV parahaemolyticus

Australian/New Zealand Standard AS/NZS 1766.2.9.1997

IdentificationPlatingEnrichmentSpeciesOfficial Body

Standard Methods (1) Standard Methods (2)

TI NI Agar; Kligler Agar;Tryptone Broth; O/F Medium;Purple carbohydrate Broth;Moeller decarboxylase Medium

TCBS AgarAlkaline Peptone waterV. choleraeUSA. AOAC Official Method 988 - 20 1990

Kligler Agar; Tryptone soya Agar; Glucose Agar

TCBS AgarSalt gelatin phosphateAgar

Alkaline Peptone waterSaline-glucose-Sodium dodecyl sulphate Broth

V. cholerae

Italian Istituto Superiore diSanita Rapporti istison96/35

Salt-nutrient Agar; Salt-TSI Agar;Salt- Lysine-decarboxylase Medium; Salt-Tryptone Medium

TCBS AgarTSAT Agar

Alkaline Peptone waterSalt-polymyxin B BrothSaline-glucose Sodium dodecyl sulphate Broth

V. parahaemolyticusItalian Istituto Superiore diSanita Rapporti istison96/35

Salt-Tryptone Medium;Salt-nutrient Agar; Salt-meat yeast Agar; Salt-Lysine-decarboxylase Medium; Salt-Tryptone Medium

TCB Sugar TSAT Agar

Alkaline Peptone waterSalt-polymyxin BrothSaline-glucose-Sodium dodecy lsulphate Broth

V. parahaemolyticusNorme Franpaise (AFNOR)NF ISO 8914 1991

IdentificationPlatingEnrichmentSpeciesOfficial Body

ISO ISO/TS 21872-1:Criteria

a) + indicates 76 % to 89 % positive

−−10 % NaCl + −8 % NaCl + −6 % NaCl + + 2 % NaCl

−+ liquid mediumGrowth in peptone water with 0 % NaCl

+ + Kovacs' reagent.Production of indole−+ API or ß-galactosidaseONPG hydrolysis −−API or AgarADH + + API or AgarLDC + + API or AgarODC −+ saline TSI agar red/yellowSucrose −−saline TSI agar red/yellowLactose −−saline TSI agar buttProduction of gas (glucose) + + Test stickOxidase

V. parahaemo-lyticus a V. cholerae aMethodTests (media containing 1 % of NaCl)

FDA: Minimal Number of Characters Needed to Identify V. cholerae and V.

parahaemolyticus Strains

From Hugh and Sakazaki (48)

c V. parahaemolyticus

b V. cholerae (and V. mimicus)

a No sodium chloride added.

99.199.1bb/0/0cc+ + growth in 1% tryptone brotha

98.998.9+ + L-ornithine decarboxylase

00--L-arginine dihydrolase

100100++L-lysine decarboxylase

100100±±String

100100++Oxidase

100100++Gram-negative, asporogenous rod

PercentagePositive Reaction

The Food and Drug Administration (FDA) Bacteriological Analytical Manual Online(Kaysner and DePaola, 2004; http://www.cfsan.fda.gov/~ebam/ bam-9.html)

FDA: Minimal Number of Characters Needed to Identify V. cholerae and V.

parahaemolyticus Strains


Three analytical schemes for enumerating V. parahaemolyticusV. parahaemolyticus are presented. The first is the MPN procedureMPN procedure commonly used by many laboratories. In addition, this procedure is nearly identical for enumeration of V. vulnificus.The second is a membrane filtrationmembrane filtration procedure using hydrophobic grid membrane filter (HGMF).The third is a direct platingdirect plating method using DNA probesDNA probes for identification of the total V. parahaemolyticuspopulation and pathogenic (TDH containing) strains. In addition, a TRH gene probe procedure and aPCR confirmationPCR confirmation analysis are also included.

Enumeration of putative V. vulnificusV. vulnificus in shellfish. Two basic approaches are outlined: a most probable number (MPNMPN) enrichment series coupled with biochemical or molecular confirmation of V. vulnificus;the second specifies direct isolation on minimally selective mediadirect isolation on minimally selective media followed by identification of V. vulnificus via colony lift and DNA probe. Previous BAM versions (Elliot et al., 1995) had included the species-specific monoclonal antibody (FRBT37) for confirmation of V. vulnificus after enrichment and isolation on mCPC agar; however, that is omitted in the 2004 edition.

Health Canada Method

3 steps:3 steps:MPN enrichmentSubcultivation on selective agars

Thiosulfate citrate bile salts sucrose (TCBS) agarModified cellobiose-polymyxin B-colistin (mCPC) agarBlood agarMannitol-maltose agar

Rapid biochemical tests (selection)API 20E strips or equivalent rapid identification kitsOxidase testMotility testTriple sugar iron agar slantO/129 Vibriostat sensitivityONPG testArginine dihydrolase, lysine decarboxylase and ornithine decarboxylase

Salt toleranceVoges-Proskauer (V-P) test

http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/mflp-37-eng.php

Vibrio isolation and identification from feces

(Bergey's Manual)Feces:Feces:

high total bacterial cell numbersmany related species

therefore:

Direct plating of dilution series on selective agars.Subsequent isolation (restreaking on other selective agars) and characterization.Highly selective (immuno-logical) tests with original sample.no enrichment!no enrichment!

Testing Methods

Culture-dependent techniques

Total heterotrophic plate count Growth on selec-

tive media

Sample

Culture-independent techniques

Direct Observation

Immunological Immunological MethodsMethods

Nucleic AcidNucleic Acid--BasedBased

Chemical

Most ProbableNumber

Reversed Passive Latex Agglutination Test Antibody techniques

FluorophoresFluorophoresfluorescein, rhodamine,

phycoerythrin

Beads/Solid support Beads/Solid support Latex beads, Latex beads, agarose,

magnets, ProteinA

EnzymesEnzymesAP, HRP

BiotinBiotin--StreptavidinStreptavidin

Reversed Passive Latex Agglutination Test

Principle:Principle:Microtiter assay.polyvalent antibodies bound to latex matrix.Agglutination in presence of toxin results in precipitate.

SensitivitySensitivity2 ng/ml

OxoidRPLA TD0920A (VET-RPLA) Vibrio cholera Vibrio cholera

New Horizon Agglutination choleraScreen

New Horizon AbpptcholeraSMART

DiffchambELISA Transia Plate SE

UnipathRPLA SETRPLA enterotoxin

TECRA ELISA S. aureus VIA

Becton Dickinson LA StaphyloslideStaphylococcus aureus

Wampole Labs LA BactigenShigella

TECRA CaptureEIAUNIQUEe

LUMAC AbpptPATHSTIK

GEM Biomedical ELISA Salmonella Salmonella

3M AbblotPetrifilmHEC

Difcosera O157&H7

REMEL LA RIM EHEC O157:H7

UnipathRPLA PET C. perfringens enterotoxin

ElcatechELISA ELCA Clostridium botulinum toxin Clostridium botulinum toxin Becton Dickinson LA CampyslideCampylobacter

TECRA UnipathELISA RPLA TECRA BCET Bacillus cereus toxin

Manufacturer Assay FormataTrade Name Organism/toxin

FDA/CFSAN BAM Appendix 1:Partial list of commercially-available, antibody-

based assays for the detection of foodborne pathogens and toxins

Molecular Methods

Vibrio molecular biology (Wednesday)

Molecular biology of Molecular biology of VibrioVibrioGenomesGenomesGenetic elementsGenetic elementsVirulence genesVirulence genes

IdentificationIdentificationFilter hybridizationFilter hybridizationPCR protocolsPCR protocols

Evolution of bacteria – „Gene pools“

Essential physiologicalprocesses required forsurvival in general

Physiological processesrequired for survival in specificenvironments (niches)

modified fromProf. Dr. Hubert Hilbi, ETH ZH lecture WS 04/05

Classical MicrobiologyClassical Microbiology Molecular MicrobiologyMolecular Microbiology

Vehicles of change

Phages ctx phage V.cholerae toxin

Plasmids Biotype 2 V.vulnificusfish pathogenicity

Pathogenicity islands 80-kb PI on Ch2 V. parahemolyticuswith 11 T3SS2 genes

Vibrio: 2 chromosomes

Vibrio differs from many other bacteria by the fact that it contains two chromosomes differing by many traits and being perhaps of different origin.

In order to differentiate between not pathogenic marine bacteria of Vibrio and pathogenic Variants it is necessary to know

•• locationlocation•• sequencesequence•• stabilitystability•• of virulence genes.of virulence genes.

V. parahaemolyticus O3:K6

Nair et al. 2007,Global Dissemination of Vibrio parahaemolyticusSerotype O3:K6 and Its SerovariantsClinical Microbiology Reviews20 (1), 39-48

Examples for V. vulnificus PCR

Arias nested PCRFish, Sediment, Water

23rRNA genes (Dvu)

Cambell and Wright, 2003RT-PCROysterscytotoxin gene (vha)

Brasher et al.,1998

Panicker et al., 2004multiplex PCROysters

cytotoxin-hemolysin (vvh) gene from lactose-positive V. vulnificusSiderophore (viuB, !wrong primer)

Hill et al. 1991 (FDA)PCROysterscytotoxin-hemolysin (vvh) gene from lactose-positive V. vulnificus

LiteratureMethodMatrixPrimer target

Eurachem Standard (Proficiency testing)

http://www.eurachem.org/guides/EurachemEA_Micro.pdf

Reference stocks shall be preserved by a technique such as

• freeze-drying, • liquid nitrogen • storage, beads,

etc., which maintains desired characteristics of the strains. Laboratories shall have a policy and procedures for purchase, handling, storage, preservation, maintenance and use of reference cultures and stocks.

Eurachem Standard (Proficiency testing)http://www.eurachem.org/guides/EurachemEA_Micro.pdf

Reference Strain (Master): A microorganism

• defined to at least the genus and species level,

• catalogued • and described according to its

characteristics and stating its origin.

Reference Stocks: A set of separate identical (to master) cultures obtained in the laboratory by a single subby a single sub--culture culture from the reference strain.The STOCK cultures are usually glycerol broths or beads that are stored frozen. Sufficient vials should be prepared to last 3-12 months.

Working Culture: A primary sub-culture from a reference stock.

Common Confusions

• Definitions of reference/stock/working cultures• Can you subculture and if so how many times• Are the newer delivery systems acceptable

• Reference cultures (i.e. from a collection) may be subcultured onceonce to provide reference stocks• Reference stocks shall be maintained ‘...by techniques which maintain the desired characteristics of the strains’• Examples-freeze drying, LN,deep freeze methods

• Reference stocks are used to prepare working stocksworking stocks for routine use• Working stocks can be subcultured if:-

• required by a standard method• documentary evidence exists to show changes do not occur over a defined number of subcultures, and

this has been validated

• Once thawed or otherwise reconstituted reference stocks must not be refrozen or renot be refrozen or re--usedused• Working stocks must not be sub-cultured to replace reference stocks

• Document traceability of strains including date purchased,dates sub-cultured dates beads removed etc..• Continuous monitoring of freezer temp. or at least max/min.• Place shelf life on all stocks, reference and working

Things often forgotten

‘New’ Delivery Systems

• E.g Culti loops, Quanticult etc• All previous requirements still apply• Proof of quality, traceability etc falls to the

laboratory• Treat like any other goods and service

supply but with extra ‘calibration’requirements

Advantages and Disadvantages of PCR(uncertainty of results)

1. PCR methods detect as little as 1,000 genomes, RT-PCR eventually as little as 1 cell. However, for most food applications an enrichment step is necessary, at least to remove inhibitors substances.

2. PCR can easily distinguish between pathogenic and non-pathogenic varieties.

3. The method requires strict observance of good laboratory practice to avoid contamination. Even traces of reference DNA spoils results. Clean surfaces and equipment regularly with a DNA-removing detergent (which should not contain polymerase inhibitors).

4. Appropriate positive and negative controls are indispensible for every PCR run. In particular include a positive control monitoring a species specific gene.

5. Determine sensitivity and selectivity with reference strains and defined matrices.(eg: Campbell and Wright 2003, Real-Time PCR Analysis of Vibrio vulnificus from Oysters, Applied and Environmental Microbiology, 69 (12), 7137-7144Gordon et al. 2008, Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water, Applied and Environmental Microbiology 74 (6), 1704-1709.

6. The method detects viable, VNC and dead cells as well as DNA fragments, thus it does not yield information on cells capable of multiplying.

7. The method might miss strains carrying mutations within the target sequence of one primer.

8. Only few standard methods published - auxiliary test for confirmation only.

Standard: V. parahaemolyticus PCR


V. parahaemolyticus multiplex PCR


5'-GGTATTCTGCACACAAATCAG-3P2

5'-TGAAATAAAGCAGTCAGGTG-3'P1Cholera (ctx) toxin

Three primer sets



5' tgg aat aga acc ttc atc ttc acc 3'VPTDH-R

5' gta aag gtc tct gac ttt tgg ac 3'VPTDH-Ltdh gene (270 bp)thermostable related hemolysin

5' cat aac aaa cat atg ccc att tcc g 3'VPTRH-R

5' ttg gct tcg ata ttt tca gta tct 3'VPTRH-Ltrh gene (500 bp) thermostable direct hemolysin (Kanagawa hemolysin)

5' gct act ttc tag cat ttt ctc tgc 3'R-TL

5' aaa gcg gat tat gca gaa gca ctg 3'L-TLtlh gene species specific (450 bp)thermolabile hemolysin

Three primer sets

Filter hybridization methods for Vibrio

The Food and Drug Administration (FDA) Bacteriological Analytical Manual Online 2001Chapter 24 Identification of Foodborne Bacterial Pathogens by Gene Probes

Yamamoto et al., 1990Cytotoxin-hemolysinV. vulnificus

Nishibuchi & Kaper, 1985

Thermostable direct hemolysin (tdh)

V. para-haemolyticus

Lockman &. Kaper, 1983 Cholera enterotoxinVibrio cholerae

LiteratureProbeOrganism

real-time PCR DNA molecules from Vibrio

Length: 48,508 ntGC Content: 44%% Coding: 83%

Plasmid 2

Length: 66,946 ntGC Content: 43%% Coding: 79%

small Plasmid (not cha-racterized;Wang, Leung, et al., 2006

Plasmid 1

Length: 1,857,073 ntGC Content: 47%% Coding: 89%



chromosome 2




chromosome 1

V. vulnificusV. parahaemolyticusV. cholerae

Vibrio cholerae virulence genes

Virulence related DNA sequences acquired from other speciesare shown in red:VibrioVibrio pathogenicity island (VPI)pathogenicity island (VPI) encodes the essential intestinal colonization factor toxin-coregulated pilus and a virulence regulator, ToxT.The temperate bacteriophagebacteriophage CTXCTX encodes cholera toxin.The integronintegron islandisland is a gene-capture system yet to be associated with virulence. The pathogenicity determinants that have notnot

been recently acquired (black) include:toxRtoxR, which encodes a transcriptional regulator of

several virulence loci; hlyUhlyU, which encodes a transcriptional regulator that controls the expression of the haemolysin-encoding gene hlyA;hlyA; and EPSEPS, a protein-secretion system that is required for the export of CTX , cholera toxin and several proteases, including HapHap.

Waldor and RayChaudhuri 2000, Bacterial genomics: Treasure trove for cholera research, Nature 406, 469-470

Genomic Structure: Pathogenicity Islands (PAI)

• Upon transduction, the bacteriophage (ctxΦ) brings the toxin and a specific pilus called toxin-co-regulated pilus (TCP).

• The important genes involved in intestinal colonization (tcp) and virulence gene regulation (toxT) are encoded in a 40Kb pathogenicity island.

• This PAI is present in pathogenic cholera strains.

tcp gene

ctx gene

Genetic Diversity & Virulence Potential

Faruque, Shah et al. 2004. Genetic diversity & virulence potential of environmental Vibrio choleraepopulation in a cholera endemic area. PNAS. 101(7); 2123-2129.

V. parahaemolyticus virulence genes

toxR genes from V. parahaemolyticus and V. cholerae share 52% identity. Used to differentiate between O3:K6 and other V.parahaemolyticus.

Regulator of Virulence genes in VibriotoxR

V. cholerae O1 and V. vulnificus do not carry T3SS genes while V. cholerae non-O1/non-O139 appear to contain one set. V. para-haemolyticus O3:K6 contains twotwo sets of T3SS genes, on chromosomes 1 (T3SS1)and 2 (T3SS2) respectively. T3SS1 appears to be pr-sent in all V. parahaemolyticus while T3SS2, located within an 80-kb PI that harbors two copies of the tdh gene, occurs only in pathogenic Kanagawa(+) strains.

t3ss1t3ss2

Present in all V. parahaemolyticus HemolysinThermolabile hemolysin-like gene tlh/r72h

V. cholerae O1 and V. vulnificus do not carry Iron acquisitionVibrioferrinpvuA

Ure is probable linked to trh, but no unique marker for pathogenicity.Urea hydrolysisUreaseure

About 70% nucleotide homology with tdh.Heat labile hemolysin.Tdh related hemolysintrh

Typically of clinical isolates. Only 5% of environmental isolates harbor tdh

Kanagawa-Factor: lyses cells

Thermostable direct hemolysintdh

CommentsfunctionnameGene

Sensitivity of RT-PCR methods directed against V. parahaemolyticus virulence genes

LiteratureSensitivity (cfu/g)Gene

Rizvi et al. (2006) 103

Ward et al. (2006) 104

ORF8

Takahashi et al. (2005) 36, >100 (shellfish) toxR

Rizvi et al. (2006) 103

Ward et al. (2006) 104

Kaufman et al. (2004) 102,11–104,77

Davis et al. (2004) 5,340

tlh

Ward et al. (2006) 103

Davis et al. (2004) 5,340

Fukushima et al. (2003) 104,7 (100 after enrichment)

trh

Ward et al. (2006) 103

Davis et al. (2004) 5,340

Fukushima et al. (2003) 104,7 (100 after enrichment)

tdh

Lehmacher and Hansen 2007, Real-time PCR von virulenten Vibrio parahaemolyticus in Fisch- und Krebstierprodukten J. Verbr. Lebensm. 2, 213–217

V. parahaemolyticus O3:K6

Matsumoto et al. 2000,Pandemic Spread of an O3:K6 Clone of Vibrio parahaemolyticus and Emergence of Related Strains Evidenced by Arbitrarily Primed PCR and toxRS Sequence AnalysesJournal of Clinical Microbiology 38 (2), 578-585

O3:K6 strains possessing the tdh gene but not the trh gene were detected first in India and Bangladesh in 1996. They might have emerged recently and become prevalent not only in India, but also in other parts of the world.

The toxR and toxS genes in the toxRS operon encode transmembrane proteins involved in the regulation of virulence-associated genes and are well conserved in the genus Vibrio .Intraspecies variation of the toxRS sequence can be used for confirmation of the clonality of the new O3:K6 strains.


Reference stocks shall be preserved by a technique such as

• freeze-drying, • liquid nitrogen • storage, beads,

etc., which maintains desired characteristics of the strains. Laboratories shall have a policy and procedures for purchase, handling, storage, preservation, maintenance and use of reference cultures and stocks.


Reference Strain (Master): A microorganism

• defined to at least the genus and species level,

• catalogued • and described according to its

characteristics and stating its origin.

Reference Stocks: A set of separate identical (to master) cultures obtained in the laboratory by a by a single subsingle sub--culture culture from the reference strain.The STOCK cultures are usually glycerol broths or beads that are stored frozen. Sufficient vials should be prepared to last 3-12 months.

Working Culture: A primary sub-culture from a reference stock.

ISO 17025

• Definitions of reference/stock/working cultures• Can you subculture and if so how many times• Are the newer delivery systems acceptable

• Reference cultures (i.e. from a collection) may be subcultured onceonce to provide reference stocks• Reference stocks shall be maintained ‘...by techniques which maintain the desired characteristics of the strains’• Examples-freeze drying, LN,deep freeze methods

• Reference stocks are used to prepare working stocksworking stocks for routine use• Working stocks can be subcultured if:-

• required by a standard method• documentary evidence exists to show changes do not occur over a defined number of subcultures, and

this has been validated

• Once thawed or otherwise reconstituted reference stocks must not be refrozen or renot be refrozen or re--usedused• Working stocks must not be sub-cultured to replace reference stocks

• Document traceability of strains including date purchased,dates sub-cultured dates beads removed etc..• Continuous monitoring of freezer temp. or at least max/min.• Place shelf life on all stocks, reference and working

Precausions

• E.g Culti loops, Quanticult etc• All previous requirements still apply• Proof of quality, traceability etc falls to the laboratory• Treat like any other goods and service supply but with extra ‘calibration’ requirements

• ISO 9000.... Company?• But what for -manufacture,supply? Who actually makes the products?• Traceability to national collection?• How many passages?• What preservation techniques are used?

• Validated methods and QC data• Certificate of traceability to National Collection number• Statement of uncertainty i.e. if quantitative, number of cfu +/- but also details of how this is

calculated, cumulative errors etc.

• For Vibrio: For transport, inoculate onto saline nutrient agar slopes (slant)

Fish Pathogens

(a) The sign + means 76 % to 89 % positive.−−−−−10 % NaCl −−+ −−8 % NaCl + + + −−6 % NaCl + + + + + 2 % NaCl −−−+ + 0 % NaCl

Growth in peptone water with b + + + + Production of indole+ + −+ + ONPG hydrolysis + −−−−ADH –++ + + LDC −+ + + + ODC + −−−+ Sucrose −+ −−−Lactose −−−−−Production of gas (glucose) + + + + + Oxidase

V. fluvialisV. vulni-ficus

V. parahae-molyticusV. mimicusV. cholerae aTest

Advantages and Disadvantages of PCR(uncertainty of results)

1. PCR methods detect as little as 1,000 genomes, RT-PCR eventually as little as 1 cell. However, for most food applications an enrichment step is necessary, at least to remove inhibitors substances.

2. PCR can easily distinguish between pathogenic and non-pathogenic varieties.

3. The method requires strict observance of good laboratory practice to avoid contamination. Even traces of reference DNA spoils results. Clean surfaces and equipment regularly with a DNA-removing detergent (which should not contain polymerase inhibitors).

4. Appropriate positive and negative controls are indispensible for every PCR run. In particular include a positive control monitoring a species specific gene.

5. Determine sensitivity and selectivity with reference strains and defined matrices.(eg: Campbell and Wright 2003, Real-Time PCR Analysis of Vibrio vulnificus from Oysters, Applied and Environmental Microbiology, 69 (12), 7137-7144Gordon et al. 2008, Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water, Applied and Environmental Microbiology 74 (6), 1704-1709.

6. The method detects viable, VNC and dead cells as well as DNA fragments, thus it does not yield information on cells capable of multiplying.

7. The method might miss strains carrying mutations within the target sequence of one primer.

8. Only few standard methods published - auxiliary test for confirmation only.

Standard: V. parahaemolyticus PCR






Three primer sets









Three primer sets

Filter hybridization methods for Vibrio

The Food and Drug Administration (FDA) Bacteriological Analytical Manual Online 2001Chapter 24 Identification of Foodborne Bacterial Pathogens by Gene Probes

Yamamoto et al., 1990Cytotoxin-hemolysinV. vulnificus

Nishibuchi & Kaper, 1985

Thermostable direct hemolysin (tdh)

V. para-haemolyticus

Lockman &. Kaper, 1983 Cholera enterotoxinVibrio cholerae

LiteratureProbeOrganism

Proceeding

1. 1. Introduction (Monday)Introduction (Monday)

BackgroundBackgroundPrerequisites: LabPrerequisites: LabBasic properties of Basic properties of VibriosVibrios relevant to the analytical laboratoryrelevant to the analytical laboratoryEnrichmentEnrichment

2.2. Specific properties of Specific properties of Vibrio cholerae, Vibrio parahaemolyticus Vibrio cholerae, Vibrio parahaemolyticus and and Vibrio vulnificusVibrio vulnificus (Tuesday)(Tuesday)

SSpecific properties of pecific properties of Vibrio cholerae, Vibrio parahaemolyticusVibrio cholerae, Vibrio parahaemolyticus and and Vibrio Vibrio vulnificusvulnificusIdentification by microbiological, biochemical and immunologicalIdentification by microbiological, biochemical and immunological traits.traits.

3.3. Pathogenicity (Wednesday)Pathogenicity (Wednesday)Genomes and virulence factorsGenomes and virulence factorsPCR MethodsPCR Methods

4.4. SOP'sSOP's, Quantification and uncertainty of results (Thursday), Quantification and uncertainty of results (Thursday)

MPN and plating methodsMPN and plating methodsUncertainty and evaluation of resultsUncertainty and evaluation of results

ALMOST DONE!!!

ALMOST DONE!!!

Bacteriological detection methods

Quantitative: Enumerate or estimate directly or indirectly the bacterial load in the sample.Direct enumeration

Microscopic countColony Forming Unit (CFU) (CFU) count

o Non-selective media (eg. nutrient broth)o Non-selective differential media (containing an indicator, eg. for pH)o Selective media (containing a selective agent, eg an antibiotic)o Selective differential media (containing both, indicator and selective agent, eg. TCBS)

Indirect DeterminationMost Probable Number Method (MPN)Enumeration of Injured Cells by Selective MediaOverlay MethodThin Agar Layer Method

Qualitative: Determine the possible presence of certain microorganisms.Pre-enrichment step on selective or not selective media.Testing on medium containing selective and/or differential agents

Most probable Number (1) Most probable Number (2)

Most probable Number (3)

SampleSample10 g+90ml buffer10 g+90ml buffer

(1:10)(1:10)

GrowthGrowth

GrowthGrowth

GrowthGrowth

1:101:10

GrowthGrowth

NoNoGrowthGrowth

NoNoGrowthGrowth

1:1001:100

NoNoGrowthGrowth

NoNoGrowthGrowth

NoNoGrowthGrowth

1:1,0001:1,000

33 0011

http://www.cfsan.fda.gov/~ebam/bam-a2.html



(1:10)(1:10)

might contain on averagemight contain on average10 cells/ml, but different 10 cells/ml, but different

speciesspecies

1 ml1 ml

1 ml1 ml

1 ml1 ml

9 ml9 ml

9 ml9 ml

9 ml9 ml

9 ml9 ml

9 ml9 ml

9 ml9 ml

00

00

1 ml1 ml

1 ml1 ml

1 ml1 ml

00

88

1010

1212

1 ml1 ml

1 ml1 ml

1 ml1 ml Series 1Series 1

Series 2Series 2

Series 3Series 3

22

00

11

Series 1Series 1



(1:10)(1:10)

Series 2Series 2

Series 3Series 3

NoNoGrowthGrowth

NoNoGrowthGrowth

NoNoGrowthGrowth

1:1,0001:1,000

GrowthGrowth

GrowthGrowth

GrowthGrowth

1:101:10

3/3/22

GrowthGrowth

GrowthGrowth

1:1001:100

2/2/00NoNoGrowthGrowth


Pichhardt 1993

inoculate 10 tubes conta-ining 9 ml of selective en-richment medium with 1 ml 1/10 sample.

incubate for appropriate time and check for turbidity.

Streak turbid soln. on selective solid agar plates.

Pick colonies and re-streak on other media or subject to bioche-mical or immunologi-cal testing or PCR.

MPN Scheme for V. parahaemolyticus

Pichhardt 1993

Dilution seriesDilution series

Test tubesTest tubes turbid tubeturbid tube+ next clear one+ next clear one

2 selective media

Variations

Cell numbers vary due to:Cell numbers vary due to:

Environmental fluctuationsEnvironmental fluctuationsTemperatureTemperaturesalinitysalinityecological nicheecological nichemicrofloramicroflorahosthost

Processing or other human impactProcessing or other human impactheating/chillingheating/chillingHigh pressureHigh pressuremincing, spicing, marinatingmincing, spicing, marinating

SamplingSamplingTestingTesting

methodmethodstaffstaffequipmentequipment

Variations

Cell numbers varyCell numbers vary ............

limits, critical values, limits, critical values, tresholdstresholds, legislation, legislation notnot

Fluctuations: V. vulnificus density in oysters from the US-golf coast at harvest


Monthly occurrence of V. parahaemolyticus O:K types in the Khanh Hoa province in Viet Nam

Chowdhury et al. 2004. Emergence and serovar transition of Vibrio parahaemolyticus pandemic strainsisolated during a diarrhea outbreak in Vietnam between 1997 and 1999. Microbiol. Immunol. 48, 319-327

Shellfish Associated V. vulnificus Illnesses1995 through 2001


Hazards: HarvestHarvestHarvest

2–6 log reduction 15–60 days -12° to -20°C FREEZINGFREEZINGFreezing of cooked and raw products, storage, and shipment time

>6 log reduction 0.5–1.0 minute (holding time at >90°C) >90°C

COOKINGCOOKINGCooking at processing plant

No effect 2–8 hours 4–10°C TEMPERATURETEMPERATURETemperature during processing before

freezing

2–3 log reduction 2–16 hours2–48 hours

0–7°C 0–7°C

ICING ICING Icing during transport (including aboard fishing vessel for wild-caught shrimp) to processor aquaculturewild- caught

1 log reduction 1–4 hours1–4 hours

0–7°C0–30°C

WASHINGWASHINGWashing and icing of aquaculture shrimp Washing in seawater of wild-caught shrimp

No effect 0–1 log increase

0–1 hours 0–3 hours

15–35°C10–30°C

Handling in period before icing Aquaculture shrimp Wild-caught shrimp

WHO 2006: Risk assessment of choleragenic Vibrio cholerae 01 and 0139 in warm-water shrimp in international trade

FDA & EPA Safety Levels in Regulations and Guidance

http://www.cfsan.fda.gov/~comm/haccp4x5.html

This appendix contains a listing of FDA and EPA levels relating to safety attributes of fish and fishery products published in regulations and guidance. In many cases, these levels represent the point at or these levels represent the point at or above which the agency will take legal action to remove productsabove which the agency will take legal action to remove products from the marketfrom the market. Consequently, the levels contained in this table may not always be suitable for critical limits.

ICMSF Recommended Microbial Limits

Recommended microbiological limits for V. parahaemolyticus in fish (ICMSF, 1986).

103102110Fresh and frozen bivalve mollusks

103102110Cooked, chilled, and frozen crabmeat

10310215Frozen cooked crustaceans

10310215Frozen raw crustaceans

10310225Fresh and frozen fish and cold-smoked fish

M4m3

Bacteria/gram or/cm2

c2n1Product

1 Number of representative sample units. 2 Maximum number of acceptable sample units with bacterial counts between m and M. 3 Maximum recommended bacterial counts for good qualitygood quality products. 4 Maximum recommended bacterial counts for marginally acceptable qualityacceptable quality products. Plate counts below "mm" are considered good quality.Plate counts between "m" and "M" are considered marginally acceptable quality, but can be accepted if the number of samples does not exceed "c." Plate counts at or above "M" are considered unacceptable quality (ICMSF, 1986).

EU Hazard estimate

Opinion 19-September 2001 produced for the European Commission (http://europa.eu.int/comm/food/fs/sc/scv/out45_en.pdf) Recommendations with regard to V. parahaemolyticus in shellfish were:“The practice of judging seafood exclusively based on total Vibrio counts as indicative for the presence of pathogenic Vibrios is not appropriate and should be discontinued. The practice of judging seafood based exclusively on total V. parahaemolyticus counts without consideration of the virulence factors TDH/TRH (or tdh/trh) is not appropriate and should be discontinued.Currently available data do not support setting specific standards of microbiological criteria for pathogenic V. vulnificus and V. parahaemolyticus in seafood. Codes of practice should be established to ensure that GHP has been applied.”

(iii) Live bivalve molluscs must not contain Salmonella in 25 g flesh and Vibrio parahae-molyticus 100MPN/g flesh.".It is not possible with current detection methods to routinely distinguish pathogenic from non-pathogenic Vibrio parahaemolyticus or, consequently, to set a criterion according to the principles set out in document Codex CAC/GL 21-1997"Joint FAO/WHO food standards programmeCodex committee on fish and fishery productsTwenty-ninth Session, Trondheim, Norway, 18-23 February 2008 Shigematsu et al. 2007, Biosci. Biotechnol. Biochem. 71 (12), 3093-3097

Limits of methods:Viable bacterial counts (1)

Shigematsu et al. 2007, Biosci. Biotechnol. Biochem. 71 (12), 3093-3097

Viable bacterial counts (2)(Shigematsu et al. 2007) Relevance of Measurement Uncertainty

upper limit

i iv ii iii

i) result > limit + extended measurement uncertaintyii) result > limit by less than extended measurement uncertaintyiii) result < limit by less than extended measurement uncertaintyiv) result < limit - extended measurement uncertainty

§§

opaq etransparentassimilation (Phen lacetic acid)0 8phen lacetic acidPAC

opaquetransparentassimilation (trisodium C/Trace)2.28trisodium citrateCIT

opaquetransparentassimilation (MaLaTe)1.56malic addMLT

opaquetransparentassimilation (ADlpic acid)1.12adipic addADI

opaquetransparentassimilation (CAPric add)0.78capric acidCAP

opaquetransparentassimilation (potassium GlucoNate)1.84potassium gluconateGNT

opaquetransparentassimilation (MALtose)1.4D-maltoseMAL

opaquetransparentassimilation (N-Acetyl-Glucosamino)1.28N-acetyl-glucosamineNAG

opaquetransparentassimilation (MANnitol)1.36D-mannitolMAN

opaquetransparentassimilation (ManNosE)1.4D-mannoseMNE

opaquetransparentassimilation (ARAbinose)1.4L-arabinoseARA

opaquetransparentassimilation (GLUcose)1.56D-glucoseGLU

yellowcolorlessß-galactosidase (Para-NitroPhenyl-ßD-Galactopyranosidase)0.224-nitrophenyl-ßD-

galactopyranosidePNPG

diffusion ofblack pigment

no pigmentdiffusionhydrolysis (protease) (GELatin)0.6gelatin

(bovine origin)GEL

grey/ brown/blackyellowhydrolysis (ß-glucosidase) (ESCulin)0.56

0.072esculinferric citrateESC

orange/ pink/redyellowUREase0.76

_ureaURE

orange/ pink/redyellowArginine DiHydrolase1.92L-arginineADH

yellowblue to greenfermentation (GLUcose)1.92D-glucoseGLU

pinkcolorless/pale green/ yellow

JAMES / immediateindole production (TRyptoPhane)0.2L-tryptophaneTRP

colorlesspink

Zn /5 minreduction of nitrates to nitrogen

pink-redcolorless

NIT 1 + NIT 2 / 5 minreduction of nitrates to nitrites

0.136potassium nitrateNO3

NEGATIVE POSITIVE

RESULTSREACTIONS/ENZYMES(mglcup.)ACTIVE INGREDIENTSTESTS

Results O-129 Test

38 mm25 mm45 mmGroup 4

34 mm23 mm43 mm34 mmGroup 3

40 mm27 mm44 mm34 mmGroup 2

40 mm26 mmGroup 1

150 µg10 µg150 µg10 µg

Vibrio parahaemolyticusVibrio cholera

Competence: ISO 17025

General Requirements for the Competence of Testing and Calibration LaboratoriesTo prove the competence of the testing and calibration laboratory it must be certified according to ISO 17025. For international benchmark ISO 17025 is divided into two principal parts:

Management requirementsTechnical requirements

Both contain sections relevant to testing.ISO 17025 allows laboratories to carry out procedures in their own ways, but an auditor may require the laboratory to justify using a particular method.

2 different aspects

Method:Method: must be validated (Standard, in house validation

Lab:Lab: must prove that it has expertise to run the method

Methods: six principles

1. Analytical measurements should be made to satisfy an agreed requirementsatisfy an agreed requirement.” (i.e. to a defined objective)

2. Analytical measurements should be made using methods and equipmentmethods and equipment which have been tested to ensure they are fit for purposefit for purpose.

3. StaffStaff making analytical measurements should be both qualified and competentshould be both qualified and competentto undertake the task (and demonstrate that they can perform the analysis properly).

4. There should be a regular independent assessmentregular independent assessment of the technical performance of a laboratory.

5. Analytical measurementsmeasurements made in one location should be consistent with thoseconsistent with thosemade elsewhere.elsewhere.

6. OrganizationsOrganizations making analytical measurements should have well defined quality well defined quality control and quality assurance procedurescontrol and quality assurance procedures.

EURACHEM 1998, http://www.eurachem.org/guides/valid.pdf

Test Methods &Method Validation

•• DIN EN ISO/IEC 17025DIN EN ISO/IEC 17025

• 5.5.4.2 - Selection of Methods– Preferentially use methods published in

international, regional, or national standards– Use latest valid edition unless inappropriate or

impossible– Supplement with additional details, when

necessary, to ensure consistent application– Use any of method specified by the client;

inform client if this method is inappropriate or out-of-date (document).

Measurement Uncertainty

• DIN EN ISO/IEC 17025

– 5.4 Test and calibration methods and method validation

• 5.4.6 Estimation of uncertainty of measurement

– 5.4.6.2 Testing laboratories shall have and shall apply procedures for estimating uncertainty of measurement. In certain cases the test method may preclude rigorous, metrologically and statistically valid calculation of uncertainty of measurement.

... Uncertainty is a parameter associated with the result of a measurement whichcharacterises the dispersion of the values, which can reasonably be attributed themeasurand

(International Vocabulary of Basic and General Terms in Metrology, ISO, Genf, 1993)

Test Methods &Method Validation

– The laboratory mustmust apply procedures for estimating uncertainty of measurement.

– If actual calculations of uncertainty cannot be done, attempt to identify all components of uncertainty & make a reasonable estimate based on performance, scope, experience, & validation data

–– All All uncertainty components must be taken into account using appropriate methods of analysis

– NOTE: If a well-recognized test method specifies limits to values of major sources of measurement uncertainty and if the form of presentation of calculated results is specified, then the laboratory complies with this section by following then the laboratory complies with this section by following the test method & reporting instructionsthe test method & reporting instructions

Fitness

Laboratories have to prove their competence to conduct specific Laboratories have to prove their competence to conduct specific tests by validating results.tests by validating results.

XXVerification of implementation of RRM or HCV METHOD (Single-Laboratory) Point 5.4.2. EN ISO17025 v 2005

XXXXMultiple-Laboratory Validated Methods harmonized against reference methods or not.

XXXXHarmonized Collaboratively Validated Methods (HCVHCV)

XXXXReference/Regulatory Methods (RRMRRM): methods specified by international organizations or regulatory agencies or CEN, ISO, FDA BAM

InterInterlaboratoryValidation

IntraIntralaboratoryValidation

5.2. Staff5.2. Staff

5.7. Sampling5.7. Sampling

5.6. Traceability5.6. Traceability

5.3. Facilities5.3. Facilities 5.4. Methods5.4. Methods

5.5. Equipment5.5. Equipment

5.8. Consumables 5.8. Consumables and calibrationand calibration

Training;Engagement

Milieu Method

Statistics

ResultsResults

ISO 17025 and causes of uncertainties

The “black-box“ approach to measure uncertainty for each target microorganism (ISO/PDTS 19036)

Systematic errors: eliminate by usingreference organisms

Sampling errors cannot be included in method

evaluation

.Remember?.. Uncertainty is a parameter associated with the result of a measurement whichcharacterises the dispersion of the values,

which can reasonably be attributed the measurand(International Vocabulary of Basic and General Terms in Metrology, ISO, Genf, 1993)

Random distribution

Sources for errorsSources for errors-- uncertaintyuncertaintymostly systematic, exhibiting a characteristic variancemostly systematic, exhibiting a characteristic variance

balancingbalancing

homogenizationhomogenization

dilutingdiluting • decrease of volume during autoclaving

• number of dilution steps • carryover of cells through multiple

use of same pipette/tip • adsorption on tube wall• pipette not calibrated• technique of discharging• reading errors

countingcounting • individual errors• too many colonies on plate• overlapping colonies

• composition• thickness• dryness• consistency of spreading• incubation parameters

(temperature, moisture, time)• bacterial effects

o sublethal damaged cellso synergy/antipathy of

species (metabolites, bacteriocins)

o growth and nutrient consumption of species

PlatesPlates

ISO 17025 parameters to measure uncertainties:

Repeatability means precision of analytical data obtained under identical conditions.Reproducibility means precision of analytical data obtained under different conditions (analyst, laboratory, apparatus).As the true value for microbiological samples cannot be determined with absolute certainty, the precision of a given method as performed in a laboratory will serve as criterion.The true value will be estimated considering the uncertainties involved.

Harmonized Collaboratively Validated Methods (HCV)

For many recently introduced methods (PCR), so far no standards exist. However, they might have been extensively reviewed in literature and data on selectivity and specificity might exist. Moreover, these methods might be superior to standard methods regarding time to complete, relevant information gained and costs. These methods must be validated.

Methods validated through a full collaborative study that meets standards of ISO 5725, EN ISO 16140 or AOAC Microbiology guidelines.Validated by full collaborative study.

Qualitative method - at least 10 labs with valid dataQuantitative method - at least 8 labs with valid dataPerformance parameters are specified in EN ISO 16140 or AOAC Microbiology Guidelines

Performance criteria for intralaboratoryvalidation

• Relative limit of detection

• Trueness• Intralaboratory

repeatability

• Precision• Trueness• Intralaboratory

repeatability

Comparison of Comparison of reference method to reference method to inin--house methodhouse method

• Specificity

• Internal reproducibility• Limit of Quantification• Detection limit• Linearity• Specificity

Characterization of in-house methods

Qualitative methodsQuantitative methods

Single Lab Validation for Laboratory Methods Validation criteria

Accuracy/Trueness is the closeness of agreementcloseness of agreement between test results and the accepted reference value. Tto determine method accuracy/trueness, the concentration of the target organism of interest as measured by the MPN based method under study is compared to a referencecompared to a reference concentration.

Measurement uncertainty is a single parameter, usually a standard deviation or confidence interval.expressing the possible range of values around the measured result within which the true value is expected to be with a stated degree of probability.It takes into account all recognized effects operating on the result

including: overall precisionoverall precision of the complete method, the method and laboratory biasbias and matrix effectsmatrix effects.

Validation criteria

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

Sample:> 10 animals

20 samples20 samples

Sample A1Sample A1

Sample B1Sample B1

Spike with defined number of Spike with defined number of microorganismsmicroorganisms

cfu 1cfu 1

Sample A2Sample A2 Sample A3Sample A3

cfu 2cfu 2 cfu 3cfu 3

Sample B2Sample B2 Sample B3Sample B3

Determination of cfu by dilution

plating

Determination of Determination of cfu by MPNcfu by MPN

Determination of Determination of cfu by dilution cfu by dilution

platingplating

Validation procedure

Validation procedure (2)

Results: cfu original sampleResults: cfu original sampleResults: cfu original sampleResults: cfu original sample

convert into log cfuconvert into log cfuconvert into log cfuconvert into log cfu

Sample ASample A

Sample BSample B



platingplating



platingplating

form average log cfuform average log cfu

form quotientform quotienta= 100*(MPN)/plate counta= 100*(MPN)/plate count

This gives, how many percent of the (accurate) plate count is detected by MPN. This value is the accuracy/truenessaccuracy/trueness of the method as implemented by the laboratory. Good accuracy/trueness suggests the appropriateness of the method and the laboratory’s performance of it for the intended work.

Validation procedure (2)

form quotientform quotienta= 100*(MPN)/plate counta= 100*(MPN)/plate count

The result indicates, how many percent of the (accurate) plate count is detected by MPN. This value is the accuracy/truenessaccuracy/trueness of the method as implemented by the laboratory.

Good Good accuracy/trueness suggests the appropriateness of the method and the laboratory’s performance of it for the intended work.

Poor Poor accuracy/trueness on the other hand indicates the potential unsuitability of the method and/or the laboratory’s performance of it for the intended work.

Uncertainty of measurement

(log cfu)(log cfu) (log MPN)(log MPN) (log MPN)(log MPN)

correct for matrix effectscorrect for matrix effects

log cfu log cfu (c)(c) log MPN log MPN (c)(c) ∆∆((log cfu log cfu (c)(c) -- log MPN log MPN (c)(c) ))log cfu log cfu (c)(c) log MPN log MPN (c)(c) ∆∆((log cfu log cfu (c)(c) -- log MPN log MPN (c)(c) ))log cfu log cfu (c)(c) log MPN log MPN (c)(c) ∆∆((log cfu log cfu (c)(c) -- log MPN log MPN (c)(c) ))log cfu log cfu (c)(c) log MPN log MPN (c)(c) ∆∆((log cfu log cfu (c)(c) -- log MPN log MPN (c)(c) ))

Measurement of uncertainty

Measurement uncertainty can be determined by

subtracting the MPN results for each sample from the reference values for the samples as determined from the accompanying plate counts in logs and

calculating the 95% confidence interval (2-times standard deviation) of these differences.

The confidence interval of these differences represents the range in values within which the true measurement uncertainty lies. A narrow range in values indicates that the method as implemented by the laboratory produces reliable results.

Use the log transformed data for both the plate count and the MPN results. If necessary use the sample blank to correct the MPNs of the spiked sample for matrix effects and calculate the two-sided, 95% confidence interval for the difference in log counts between the reference (plate count) and the MPN method under study. This range in counts represents the measurement uncertainty of the method as implemented by the laboratory.

Data Summary:

Calculated % accuracy/trueness ____________

Calculated measurement uncertainty ________________

More key numbers

Ruggedness In the day to day operationsday to day operations of the laboratory there will be changes in the batches/ lots of culture media and/or test reagents used to process samples. Environmental factors are also likely to change over time. None of these factors, however, should adversely impact test results if the method as implemented is sufficiently rugged to be used routinely for regulatory monitoring.

Linear Range is the range within the working range where the results are proportional to the target organisms in the sample. Plot the correlation of organisms in the spiked sample versus MPN.

Limit of Detection is the minimum concentration at which the analyte or measurandcan be identified. Assuming that the relationship between the number of target organisms present and the MPN based method used to detect and quantify them is linear, the equation of the line describing this relationship can be used to determine the limit of detection of the method as implemented.

Limit of Quantitation/Sensitivity is the minimum concentration of the analyte or measurand that can be quantified with an acceptable level of precision and accuracy under the conditions of the test.

Relevance of Measurement Uncertainty

upper limit

i iv ii iii

§§


Evaluation of MPN-Method

MPN MethodMPN Methodto be evaluatedto be evaluated

sterile, spiked samples

plate countplate countaktualaktual, true value, true value

trueness, accuracytrueness, accuracy

confidenceconfidence





rugednessrugedness


sterile, spiked samples c1

sterile, spiked samplesc2


precisionprecision

Random distribution uncertainty

1.07314104460133310

94579715012339

955168946013338

1.03601141.10023337

1.06451121.10023336

98299946013335

91814871.10023334

9365981.10023333

1.173812124003332

1.0551110521022331101000110100theoretical cfu/Test1100.010.1110gram/Test

1/1001/101/10,0001/10001/1001/10Dilution

cfu/gPlate countMPN/gMPNSample #

uncertainty

90%ACCURACY/TRUENESSACCURACY/TRUENESS3,002,70AVERAGE

3,03141042,661333102,987972,18123392,9816892,66133383,0201143,04233373,0351123,04233362,999992,66133352,9614873,04233342,975983,04233333,0781212,38033323,02111052,3222331

101000110100theoret. cfu/Test1100.010.1110gram/Test

log cfu/g1/1001/10log MPN/g1/10,0001/10001/1001/10Dilution

cfu/gPlate countMPN/gMPNSample #

uncertainty

549Uncertainty27495% Confidence443Standard deviation

6136131.0731.0731410446046013331079579594594579715015012339495495955955168946046013338--64641.0361.03601141.1001.10023337--36361.0641.06451121.1001.1002333652252298298299946046013335--18218291891814871.1001.10023334--1641649369365981.1001.100233339339331.1731.1738121240240033328458451.0551.0551110521021022331

101000110100theoret. cfu/Test1100.010.1110gram/Test

∆∆cfucfu--MPNMPN1/1001/101/10,0001/10001/1001/10Dilution

cfu/gcfu/gPlate countMPN/gMPN/gMPNSample #

Relevance of Measurement Uncertainty

upper limit

i ivii iii

§§


Abb. 5.10 Decarboxylase-Test mit der Aminosäure Lysin. Enterobacter aerogenes kann Lysin decarboxylieren (links); Proteus vulgaris kann Lysin nicht decarboxylieren (Mitte); unbeimpfteKontrolle (rechts). Bei Verwendung anderer Aminosäuren erhält man die gleichenFarbumschläge.

Trisugar-Iron media V. cholerae API 20E

API 20E Results

--------VP++-+----IND+-----+-TDA-+------60URE--------0H2S+-------2197CIT++++----100ODC++++----100LDC-+------10ADH++-+----0ONPG

Group4

Group3

Group2

Group1

Group4

Group3

Group2

Group1

V. choleraeV. parahaemolyticusVPVC

API 20E Results

0,480,410,840,671T-Wert

99,5keineAngaben

keineAngaben97,932,172,646,732,1ID %

Vibrio cholerae

Vibrio cholerae

Eikenellacorrodens

Vibrio cholerae

non-fermenter

spp.

Ochrobactrum

anthropi

Ochrobactrum

anthropi

non-fermenter

spp.Spezies

++++++++OX--+--+--810ARA--------AMY--+-----MEL++-+----SAC---+----RHA--------0SOR--------INO++-+----MAN++-+----10099GLU++-+----9899GEL

Group4Group3Group2Group1Group4Group3Group2Group1V. choleraeV. parahaemolyticusVPVC

O-129 results

38 mm25 mm45 mmGroup 4

34 mm23 mm43 mm34 mmGroup 340 mm40 mm27 mm44 mm34 mmGroup 240 mm40 mm26 mmGroup 1150 µg10 µg150 µg10 µg

Vibrio parahaemolyticusVibrio parahaemolyticusVibrio choleraVibrio cholera

V. cholerae toxin PCR




Three primer sets









Three primer sets

Results: V. parahaemolyticus

Results: V. cholerae

Documents

Proceeding Introduction (Monday) and sterilize culture media in accordance with ISO/TS 11133-1 and ISO/TS 11133-2. ... Use - if possible - standardized and certified media. Laboratory