GENERIC STANDARD OPERATING PROCEDURE

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1 European Community Reference laboratory for monitoring bacteriological and viral contamination of bivalve molluscs The Centre for Environment, Fisheries & Aquaculture Science Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset DT4 8UB UK Tel: +44 (0) , Fax +44 (0) GENERIC STANDARD OPERATING PROCEDURE DETECTION OF VIBRIO PARAHAEMOLYTICUS IN BIVALVE MOLLUSCAN Issued by Technical Manager, Microbiological Food Safety Whereas every precaution has been taken in the preparation of this document, Cefas cannot be held responsible for the accuracy of any statement or representation made nor the consequences arising from the use of or alteration to any information contained within. This procedure is intended solely as a general resource for professionals in the field operating within the European Union and specialist advice should be obtained if necessary. All references to Cefas must be removed if any alterations are made to this publication 1

2 INDEX HISTORY OF PROCEDURE INTRODUCTION SCOPE PRINCIPLE SAFETY PRECAUTIONS EQUIPMENT MEDIA AND REAGENTS MICROBIOLOGICAL REFERENCE MATERIALS PROCEDURE SAMPLE RECEIPT SAMPLE STORAGE SAMPLE SELECTION SAMPLE PREPARATION Oysters and clams Mussels and cockles HOMOGENISATION INOCULATION AND INCUBATION OF PRIMARY ENRICHMENT BROTH SUBCULTURE OF PRIMARY ENRICHMENT BROTHS ISOLATION AND CONFIRMATION OF VIBRIO SPP BIOCHEMICAL SCREENING TESTING Oxidase test VP, 0% NaCl, Thornley's, ONPG Identification by API 20E RESULTS AND REPORTING UNCERTAINTY OF TEST RESULTS REFERENCE APPENDICES.. 8 2

3 HISTORY OF PROCEDURE Controlled document title: DETECTION OF VIBRIO PARAHAEMOLYTICUS IN BIVALVE MOLLUSCAN Controlled document reference: SOP 1333 Issue Number Date Issued Sections involved All 3

4 1.0 INTRODUCTION Infectious human diseases acquired from the consumption of bivalve molluscan shellfish are internationally recognised. These health hazards are largely due to the phenomenon of filter feeding where-by bivalve molluscs concentrate and retain bacterial and viral pathogens often derived from the contamination of their surrounding waters. The risks of exposure to infectious agents are compounded by the traditional consumption of bivalve shellfish raw, or only lightly cooked. Vibrio parahaemolyticus is a halophilic, oxidase positive, Gram-negative, rod-shaped bacterium that is commonly associated with gastro-enteritis following the consumption of inadequately cooked, raw or post process contaminated seafoods. It can be isolated from both fish and shellfish originating from warm and temperate coastal waters and is not related to sewage contamination. Most strains of V. parahaemolyticus are sucrose negative and produce green colonies with a diameter of 2-3mm on thiosulphate citrate bile salt sucrose agar (TCBS). Pathogenicity is thought to be associated with the production of thermostable haemolysins. Most clinical strains (i.e. those isolated from patients with diarrhoea) express thermostable direct haemolysin (TDH) and/or thermostable related haemolysin (TRH). Thermostable haemolysins are infrequently present in environmental samples thus the public health significance of total V. parahaemolyticus in bivalve molluscan shellfish is uncertain. 2.0 SCOPE This procedure has been produced with reference to ISO 8914:1990 with modifications and describes the method for the detection and identification of total V. parahaemolyticus in bivalve molluscan shellfish. It does not enable the differentiation of TDH or TRH positive strains. The methods described herein are designed to detect and identify V. parahaemolyticus in bivalve molluscan shellfish. However, in the hands of a trained analyst, these procedures may be applicable for application on a variety of seafoods. 3.0 PRINCIPLE The method used to enumerate V. parahaemolyticus in molluscan shellfish involves an initial selectiveenrichment in alkaline salt peptone water (ASPW), followed by direct plating of the sample onto thiosulphate citrate bile sucrose agar (TCBS) and triphenyltetrazolium chloride soya tryptone agar (TSAT). Presumptive colonies of V. parahaemolyticus isolated from both agars are subcultured onto marine agar (MA) and then subjected to biochemical and serological tests to confirm their identity. 4.0 SAFETY PRECAUTIONS Standard microbiology safety precautions should be applied throughout. Risks of cuts and minor physical injury exist when performing this procedure, particularly when using sharp oyster knifes to open shellfish. Appropriate measures to reduce these risks should be taken. Vibrio spp. should be handled in accordance with ACDP category 2 guidelines. 5.0 EQUIPMENT Stomacher Stomacher bags Timer Laminar air flow cabinet (class ll) Refrigerator at 3±2 C Incubator, 35±1 C Incubator at 30±1 C Balance Shucking knives Safety/electric Bunsen system Latex gloves Safety gloves Pipette - automatic or manual for use with 1ml and 10ml open-ended pipette tips Pipette, single channel, variable volume, 2 20µl, µl Sterile plastic spreader rods Sterile scissors Sterile glassware Expender tubes sterile - 2ml Sterile bijoux and universal bottles Fine tip water proof marker (black) Sterile loops, 1µl and 10µl 4

5 6.0 MEDIA AND REAGENTS Ethanol Alkaline salt peptone water (ASPW); formula per litre - de-ionised water 1±0.01 litre, peptone water (Oxoid CM0009) 30±0.5g, NaCl 20±0.5g, ph 8.5±0.1. Thiosulphate citrate bile sucrose agar (TCBS); - formula per litre - de-ionised water 1±0.01 litre, thiosulphate citrate bile sucrose agar (Oxoid CM333) 88±0.5g, ph 8.6±0.2. Triphenyltetrazolium chloride soya tryptone agar (TSAT); - formula per litre - de-ionised water 1±0.01 litre, tryptone (Oxoid L42) 15±0.5g, soya peptone (Oxoid L44) 5±0.5g, sodium chloride 30±5.5g, sucrose 20±0.5g, bile salt (Oxoid L55) 0.5±0.05g, technical agar no.3 (Oxoid L13) 15±0.5g, 1% triphenoletrazolium chloride solution 3±0.1ml. ph 7.15±0.35. Marine agar (MA); formula per litre - de-ionised water 1 ± 0.01 litre, marine agar (Difco 2216) 55.1±0.1g, ph 7.6±0.2. Methyl Red Vogues-Proskauer with 1% NaCl (VP); formula per litre - de-ionised water 1±0.01 litre, Methyl Red Vogues-Proskauer (Oxoid CM43) 17±0.5g, ph 6.75±0.25. Thornley s arginine dihydrolase (Thornley s); formula per litre - de-ionised water 1±0.01 litre, bacteriological peptone (Oxoid LP37) 1±0.1g, sodium chloride 5±0.5g, Potassium phosphate (K 2 HPO 4 ) 0.3±0.05g, 1% phenol red solution 0.6±0.1ml, L (+) arginine hydrochloride 10±0.5g, bacteriological agar No.1 (Oxoid LP11) 3±0.3g, ph 7.2±0.5 1% triphenoletrazolium chloride solution; formula per 10ml - de-ionised water 10±1ml, triphenoletrazolium chloride 0.1±0.02g. 0% NaCl 1% peptone water (0% NaCl 1% PW); formula per litre - de-ionised water 1±0.01 litre, bacteriological peptone (Oxoid LP37) 10±0.5g, ph 6.9±0.5. Phosphate buffered saline (PBS); formula per 100ml - de-ionised water 100±5ml, 1 phosphate buffered saline tablet (Oxoid BR14) 40% Potassium hydroxide; formula per 100ml - de-ionised water 100±1ml, potassium hydroxide 40±0.5g. 5% α-napthol; formula per 100ml - Iso-propanol 100±1ml, α-napthol 5±0.2g. API 20E strips (Biomerieux) Identification sticks oxidase (Oxoid BR64A) ONPG discs - (Oxoid DD13) 7.0 MICROBIOLOGICAL REFERENCE MATERIALS Vibrio fluvialis NCTC Vibrio cholerae NCTC 8042 Vibrio parahaemolyticus NCTC Pseudomonas aeruginosa NCTC Proteus mirabilis NCTC Escherichia coli NCTC Enterobacter aerogenes NCTC PROCEDURE 8.1 SAMPLE RECEIPT Samples must be received in an intact food grade plastic bag and properly packed in a cool box with ice packs - packed in this manner they should reach a temperature of less than 8 C within 4 hours and then maintain this for at least 24 hours. Such samples should not be received frozen. Samples from harvesting areas should have been rinsed, but not immersed, and drained at time of sampling and should be regarded as unsatisfactory if they are received in the laboratory if the sample container is leaking, the shellfish are covered in mud or immersed in water or mud/sand. 8.2 SAMPLE STORAGE Upon receipt in the laboratory the temperature of the samples should be recorded. Samples should be examined immediately or stored at 3±2 C for no more than 24 hours until examination. 8.3 SAMPLE SELECTION Choose shellfish that are alive according to the following points: - If any flesh is exposed and reacts to touch using a sterile shucking knife with movement of any kind. - If the shellfish are open and then close of their own accord. - If a tap on the shell causes closing or movement. - Tightly closed shellfish. 5

6 Discard all dead shellfish and those with obvious signs of damage. Select the appropriate number of shellfish depending on the species (Appendix 1). More shellfish can be used, if necessary, to produce the required volumes for each analysis. 8.4 SAMPLE PREPARATION Mud and sediment adhering to the shellfish should be removed prior to opening the shellfish by rinsing/scrubbing under cold, running tap water of potable quality. Shellfish should not be re-immersed in water as this may cause them to open. Open all selected shellfish as described below with a flame sterilised shucking knife and empty meat and liquor into a beaker. To flame sterilise the shucking knife place the knife in the beaker of ethanol and sterilise using an electric Bunsen system. Allow the knife to cool before using. When opening shellfish ensure that the hand holding the shellfish is protected with a heavy-duty safety glove to prevent cuts Oysters and Clams Insert the knife between the two shells towards the hinge end of the animal. Push the knife further into the animal and prise open the upper shell, allowing any liquor to drain into the beaker. Push the blade through the animal and sever the muscle attachments by sliding across the animal. Remove the upper shell and scrape the contents of the lower shell into a beaker Mussels and Cockles Insert the knife in between the shells of the animal and separate the shells with a twisting motion of the knife. Collect the liquor from the animal in the beaker then cut the muscle between the shells and scrape the contents into a beaker. 8.5 HOMOGENISATION Weigh 25±1g of shellfish flesh and intravalvular fluid into at least three stomacher bags, to avoid small pieces of shell from puncturing the bags. Remove excess air from the bag and operate the stomacher for 3 minutes at normal speed. Add 75±1ml from a bottle of 225±5ml sterile ASPW. Stomach at normal speed for a further 3 minutes. 8.6 INOCULATION AND INCUBATION OF PRIMARY ENRICHMENT BROTH Transfer the stomached sample prepared in section 8.5 into a sterile beaker and add the remaining ASPW (150ml). Label as the 10-1 (primary suspension) and prepare decimal dilutions (10-2 and 10-3 etc) by adding 1±0.1ml of primary suspension to 9±0.1ml of ASPW in a universal. If heavily contaminated samples are expected then prepare further dilutions as necessary. For the positive control inoculate 9±0.1ml of ASPW with V. parahaemolyticus, using a 1μl loop. For the negative control leave a second 9±0.1ml of ASPW uninoculated. Incubate the primary suspension, the subsequent dilution series and the controls at 41±1 C for 7 8 hours. The sample may be stored overnight at 3±2 C if the dilution and incubation steps cannot be carried out on the same day. 8.7 SUBCULTURE OF PRIMARY ENRICHMENT BROTH After incubation subculture all dilutions of ASPW (including the controls), taking a loopful from just below the surface without mixing, onto two plates each of TCBS and TSAT spreading for single colonies. Incubate the TCBS plates at 37±2 C for 18±2 hours and the TSAT plates at 37±2 C for 22±2 hours. After incubation examine the plates for characteristic colonies of V. parahaemolyticus: TCBS: Smooth, green (sucrose negative) and 2-3mm in diameter. TSAT: Smooth, flat dark red (reduction of triphenyltetrazolium chloride) and 2-3mm in diameter. 8.8 CONFIRMATION Confirmation is carried out on 5 characteristic colonies taken from the lowest dilutions showing single colonies on both TCBS and TSAT plates. Use all colonies if there are less than 5. Subculture 5 presumptive V. parahaemolyticus colonies from the TCBS and TSAT plates onto MA plates, streaking for single colonies. Incubate the MA plates at 30±2 C for 22±2 hours. After incubation check plates for colony purity before carrying out the following biochemical screening tests. 8.9 BIOCHEMICAL SCREENING TESTING OXIDASE TEST The oxidase test is carried out using Oxoid Identification Sticks. The tip of each oxidase identification stick is impregnated with a solution on N, N-dimethyl-p-henylenediamine oxalte, ascorbic acid and α- 6

7 napthol. Remove the oxidase sticks from the refrigerator and allow to stand for 5 minutes at room temperature. Carry out controls on the sticks using colonies from the P. aeruginosa for the positive control and E. coli for the negative control. Choose a well separated representative colony from the MA purity plate and touch the colony with the impregnated end of the oxidase stick. Rotate the stick gently on its edge, picking off a small mass of cells and then place the stick upright between the lid and the base of the inverted plate. Examine the impregnated end of the stick after 30 seconds and if no colour change has occurred examine again after 3 minutes. A positive reaction will lead to a blue-purple colour and a negative reaction will show no colour change. Reject any plates that show oxidase negative colonies and continue identification of all plates that show oxidase positive colonies VOGES-PROSKAUER (VP), 0% NaCl 1% PEPTONE WATER, ARGININE DIHYDROLASE TESTS AND ORTHO-NITROPHENYL-β-D-GALCTOPYRANOSIDASE (ONPG). Subculture each positive oxidase plate using a 1μl sterile loop without recharging the loop. First inoculate 10±0.1ml of VP broth, then 10±0.1ml of 0% NaCl 1% PW, 200±10μl of PBS tube, 3±0.1ml of Thornley s and finally streak onto a MA plate, spreading for single colonies. Overlaying the surface of the Thornely s medium with sterile mineral oil will overcome the problem of differentiation between weak positives from negatives. Add a single ONPG disc to each inoculated tube of PBS. For the positive and negative controls inoculate using the microbiological reference culture in table 1. Table 1. CONTROLS FOR BIOCHEMICAL SCREENING TESTING Controls Voges-Proskauer 0% NaCl 1% Thornley Arginine Ortho-Nitrophenyl-βpeptone water Dihydrolase D-Galctopyranosidase Positive E.aerogenes E.coli V.fluvialis V.cholerae Negative V.parahaemolyticus V.parahaemolyticus V.parahaemolyticus V.parahaemolyticus Incubate VP, 0% NaCl 1% peptone water, Thornley s and MA at 30±2 C for 24±2 hours and ONPG tubes at 37±2 C for 24±2 hours. After incubation check the MA purity plate for single colonies, which should be of uniform size and appearance. After incubation take 1±0.2ml of the incubated VP solution and place into a universal to provide a larger surface air-liquid interface. To this add 200±20μl of 40% potassium hydroxide (KOH) and 600±20μl of α-napthol solution. Leave for 15 minutes. Repeat for all VP tests. A positive VP reaction is indicated by a red colouration whereas a negative reaction is indicated by a yellow - brown colouration. Bacterial growth (turbidity) in the 0% NaCl should be recorded as a positive reaction while absence of turbidity is a negative reaction. A positive reaction for the presence of arginine dihydrolase is indicated by a red colouration throughout the medium. A negative reaction is no colour change to pale pink. If a negative reaction occurs it should be re-incubated at 30±2 C and examined daily up to (5 days) to allow for slow growing bacteria. A positive ONPG reaction for the presence of β-galactosidase activity is indicated by a yellow colouration. No colour change indicates a negative result. Table 2. EXPECTED RESULTS OF SCREENING TESTS Test V. cholerae V. parahaemolyticus V. vulnificus Other Vibrios E. coli Oxidase (-) - Thornley Arginine V dihydrolase Growth in 0% NaCl 1% (+) + peptone water Voges- Proskauer V - - V V ONPG IDENTIFICATION BY API 20E If screening tests indicate that V. parahaemolyticus is present then identification must be carried out by the inoculation of API 20E. The API test acts as a confirmatory test but does not necessarily rule out possible pathogenic Vibrios if strips are negative or yield uncertain results. Carry out an API for each of the presumptive V. parahaemolyticus colonies. 7

8 9.0 RESULTS AND REPORTING If at least one colony from the sample tested conforms to the expected criteria of V. parahaemolyticus (table 2) then report the sample as V. parahaemolyticus detected in 25g. If no colonies from the sample tested conforms to the expected criteria of V. parahaemolyticus (table 2) report the sample as " V. parahaemolyticus not detected in 25g" UNCERTAINTY OF TEST RESULTS Uncertainty inherent in any test method, i.e. instruments, media, analysts performance etc can be assessed by the repeatability and reproducibility of test results. These should be monitored through control tests analysed alongside sample tests, through in-house comparability testing between analysts and through external inter-comparison exercises to highlight any uncertainties within the test methods REFERENCES Anon ISO 8914:1990 Microbiology General guidance for the detection of Vibrio parahaemolyticus. 12 APPENDICES 12.1 APPENDIX 1: SUB-SAMPLE SIZES OF REQUIRED FOR VIBRIO SPP. ANALYSIS The following sub-sample sizes are recommended for inclusion in the homogenisation step: King scallops (Pecten maximus) Horse mussels (Modiolus modiolus) Sand Gapers (Mya arenaria) Razor clams (Ensis spp.) Oysters (Crassostrea gigas and Ostrea edulis) Hard clams (Mercenaria mercenaria) Queen scallops (Aequipecten opercularis) Mussels (Mytilus spp.) Manila clams (Tapes philippinarum) Palourdes (Tapes decussatus) Cockles (Cardium edule) Thick trough shells (Spisula solida)