Tài liệu Practical Food Microbiology 3rd Edition - Part 9 pptx

Live bivalve molluscs and other shellfish Council Directive 91/492/EEC [1] sets out the designation of production areas of bivalve molluscs based on levels of Escherichia coli or faecal c

Live bivalve molluscs and

other shellfish

Council Directive 91/492/EEC [1] sets out the designation of production areas of

bivalve molluscs based on levels of Escherichia coli or faecal coliforms/100 g of

flesh and intravalvular liquid A three-dilution, five-tube most probable number (MPN) method is specified for testing without precise details, but other bacteri-ological methods of equivalent accuracy are permitted In the UK testing for

E coli is performed because it is considered to be a more specific indicator

of faecal pollution than faecal coliforms The microbiological criteria for the classification of shellfish harvesting areas are shown in Table 9.1

In addition to the classification criteria, diarrhoetic shellfish poison (DSP) must be absent from the shellfish flesh and levels of paralytic shellfish poison (PSP) must be below 80 mg/100 g of flesh If these levels are exceeded fishing

is prohibited in that harvesting area until compliance is achieved Since the publication of the Directive another shellfish poison known as amnesic shellfish poison (ASP) has been identified; levels of ASP should be below 20 mg/g of flesh [2]

9

Testing for DSP, PSP and ASP is normally performed by reference laboratories Routine monitoring of shellfish harvesting areas in the UK for marine biotoxins is a statutory responsibility of the Food Standards Agency who can advise on the specialist laboratories currently contracted to undertake this task In England the reference facility for outbreak related samples is the Food Safety Microbiology Laboratory, Central Public Health Laboratory Tel: 020 82004400, ext 3521/4113 The UK National Reference Laboratory for biotoxins is the Fisheries Research Services (FRS) Marine Laboratory, Aberdeen AB11 9DB Tel: 01224 876544

An end-product standard is defined for shellfish intended for immediate

human consumption For faecal coliforms and E coli this is given as category A

in Table 9.1 In addition, the shellfish should meet the standards defined above

for biotoxins and Salmonella should be absent in 25 g of shellfish flesh The

Di-rective recognized the absence of routine virus testing procedures and this is still true today However consumption of molluscs containing viruses, in particular Norwalk-like virus (NLV; also known as small round structured virus or SRSV) is the most common cause of illness from this type of food At present, methods for the direct detection of viral pathogens (NLV and hepatitis A virus or HAV)

in shellfish are all based on the polymerase chain reaction (PCR) However,

processing of shellfish extracts to recover low levels of contaminating virus and

to remove PCR inhibitors is difficult Currently these methods are complex,

poorly standardized and restricted to specialist facilities [3] The relationship

between the levels of E coli and the presence of virus particles in depurated

shell-fish is poor, but studies have shown a much better relationship between the levels of certain types of phage, in particular F-specific RNA bacteriophage [4],

and the risk of viral contamination Phage detection methods are much simpler

to perform than virus detection methods and might be incorporated more easily into routine examination of live bivalve molluscs

Table 9.1 Classification of harvesting areas.

Category Escherichia coli/100 g Faecal coliforms/100 g Interpretation

A <230 <300 May go for direct consumption

B 90% of samples not 90% of samples not Must be depurated or relayed to

to exceed 4600 to exceed 6000 meet Category A (may also be

heat treated by approved method)

C Must not exceed Must not exceed Must be relayed for long period

46 000* 60 000 (>2 months) to meet Category A or

B (may also be heat treated by approved method)

D >46 000* >60 000 Prohibited (may also be prohibited

on health grounds rather than monitoring results)

*Figures not included in EEC regulations

Testing for viral contamination (NLV and HAV) is also currently performed by reference

facilities The Enteric Virus Unit at the Central Public Health Laboratory, Tel 0208 200

4400, and other peripheral Public Health Laboratory Service (PHLS) laboratories can

advise on analysis of clinical samples associated with shellfish outbreaks The UK National

Reference Laboratory for bacteriological and viral contamination of shellfish is the

Centre for Environment, Fisheries and Aquaculture Science (CEFAS), Weymouth DT4 8UB

Tel: 01305 206600

Method 1 Multiple tube method for Escherichia coli

This method is the standard procedure used in the UK [5] Minerals modified

gluta-mate broth is used for the first stage of the test based on the detection of acid

produc-tion followed by detecproduc-tion of b-glucuronidase activity at 44°C using a chromogenic

agar for confirmation of the presence of Escherichia coli.

continued

A pooled sample of at least six shellfish are required for testing to overcome the variability associated with individual shellfish Additional shellfish should be sub-mitted by the sampling authority to allow for rejections All samples should be stored dry at 4°C and examined preferably within 6 h of collection but no later than 24 h after collection Samples should not be frozen

Sample size

Oysters/clams 10–15

Mussels 15–30

Cockles 30–50

Equipment

Stomacher (optional)

Rotary blender (optional)

Shucking knives

Balance with resolution 0.1 g or greater

Incubators at 37 ± 1°C and 44 ± 1°C

Reagents

0.1% peptone solution (in water), pH 7.2 ± 0.2

0.1% peptone/0.85% sodium chloride solution

Minerals modified glutamate medium, double and single strength

5-bromo-4-chloro-3-indolyl b-D-glucuronide (BCIG) agar Tryptone bile agar contain-ing 144 mmol/L 5-bromo-4-chloro-3-indolyl-b-Dglucuronic acid (e.g 0.075 g/L of cyclohexylammonium salt)

Control cultures

NCTC 9001 Escherichia coli Positive

NCTC 13216 Escherichia coli b-glucuronidase weak positive

NCTC 9528 Klebsiella aerogenes b-glucuronidase negative

Sample preparation

(a) Select at least 10 oysters and clams, 15 mussels or 30 cockles Discard any gaping shellfish and those with obvious signs of damage

(b) Clean the molluscs by scraping, scrubbing and washing under cold running water

of potable quality and allow to drain on clean paper towels

(c) Open the molluscs with a flamed and cooled shucking knife, as follows:

Oysters/clams

Insert the knife between the two shells towards the hinge end of the shellfish, push further into the shellfish and prise open the upper shell Allow any liquor to drain into

a sterile weighed bag or beaker Push the blade through the shellfish and sever the muscle attachments by slicing across Remove the upper shell and scrape the contents

of the lower shell into the sterile bag or beaker Repeat for at least 10 oysters/clams to obtain the required weight and add to the same bag or beaker

continued

Insert the knife between the shells through the byssal opening of the shellfish and separate the shells by twisting the knife Collect any liquor in a weighed sterile bag or beaker Cut the muscle between the two shells and scrape the contents into the sterile bag or beaker Repeat for a minimum of 15 mussels or 30 cockles to obtain the required weight, adding the contents to the same bag or beaker

Preparation of homogenate

Using stomacher

(d) Place the bag containing the shellfish meat and liquor inside two more bags to prevent puncture from shell

(e) Place the bag in the stomacher and operate the machine for 2–3 min

(f ) Transfer 50 g of the homogenate to another stomacher bag and add approxi-mately 100 mL from a measured 450 mL volume of 0.1% peptone solution (g) Place the bag in the stomacher and operate the machine for 2–3 min Add the re-mainder of the 0.1% peptone solution and mix well This gives the 10-1dilution

or:

Using blender

(d) Weigh the shellfish flesh and liquor and add two parts by mass of 0.1% peptone solution

(e) Homogenize mixture in a rotary blender for sufficient time to achieve 15 000–

20 000 revolutions The duration should not exceed 2.5 min

(f) Stand for 30 s

(g) Swirl briefly, then transfer 30 mL of the homogenate to a measured 70 mL of 0.1% peptone solution and mix well This gives the 10-1dilution

Preparation of dilutions

(h) Prepare a 10-2dilution by transferring 1 mL of 10-1dilution to 9 mL of 0.1% peptone/0.85% sodium chloride solution Further dilutions may also be required when raw molluscs are being examined for classification of shellfish harvesting areas, i.e 10-3and 10-4

Procedure

(i) Prepare 15 tubes of minerals modified glutamate medium, five containing

10 mL of double strength medium and 10 containing 10 mL of single strength medium

(j) Add 10 mL of 10-1dilution to each of the five tubes containing double strength medium

(k) Add 1 mL of 10-1dilution to each of five tubes of single strength medium (l) Add 1 mL of 10-2dilution to each of five tubes of single strength medium (m) Repeat step (l) with further dilutions if necessary

(n) Incubate all tubes at 37°C for 24 ± 2 h

(o) Examine all tubes for acid production, signified by a colour change to yellow The presence of any acid, regardless of quantity, is regarded as a positive result

Absence of acid production after 24 ± 2 h constitutes a negative result for E coli.

continued p 237

Table 9.2 Most probable number (MPN) of organisms [5] Tables for multiple tube method

using 5 ¥ 1 g, 5 ¥ 0.1 g, 5 ¥ 0.01 g

Category A (<230 Escherichia coli)

Category B (>230 E coli, <4600 E coli)

Table 9.3 Most probable number (MPN) of organisms [5] Tables for multiple tube method

using 5 ¥ 0.1 g, 5 ¥ 0.01 g, 5 ¥ 0.001 g

Category A (<230 Escherichia coli)

Category B (>230 E coli, <4600 E coli)

Table 9.2 continued.

Category C (>4600 E coli, <46 000 E coli )

*Needs further dilutions to clarify classification

Table 9.3 continued.

Category C (>4600 E coli, <46 000 E coli)

Prohibited (>46 000 E coli)

Table 9.4 Most probably number (MPN) of organisms [5] Tables for multiple tube method

using 5 ¥ 0.01 g, 5 ¥ 0.001 g, 5 ¥ 0.0001 g

Category B (>230 Escherichia coli, <4600 E coli)

Category C (>4600 E coli, <46 000 E coli)

Prohibited (>46 000 E coli)

Table 9.4 continued.

(p) Subculture each tube showing acid production to a section of BCIG agar, and streak to obtain isolated colonies

(q) Incubate the BCIG agar plates at 44°C for 20–24 h

(r) Examine the plates for the presence of blue colonies, typical of b-glucuronidase

positive E coli.

(s) Consider tubes that yield growth of blue colonies on BCIG agar as positive for the

presence of E coli.

Calculation

(t) For each dilution, count the number of positive tubes

(u) If dilutions of 10-3or higher were used, select the highest dilution having five positive tubes and the next two higher dilutions If no dilution contains five positive tubes, select the three highest dilutions amongst which at least one positive result was obtained

(v) Use the number of positive tubes at each dilution selected to determine the MPN by reference to the MPN table for the appropriate dilution range (Tables 9.2–9.4) 10 mL of the 10-1dilution is equivalent to 1 g of flesh, 1 mL of the

10-1dilution is equivalent to 0.1 g of flesh, etc

Method 2 Salmonella spp.

The sample should be prepared for examination as described in steps (a)–(c) in method 1 Homogenize the sample as described in steps (d)–(g) using either a stomacher or a blender, but use buffered peptone water instead of 0.1% peptone solution Then proceed as described in Section 6.12, method 2

Method 3 Phage detection

F-specific RNA bacteriophages are bacterial viruses that have analogous morphology and genetic structure to human pathogenic viruses (NLV, enteroviruses and HAV) found in sewage This, allied to their abundance in sewage and ease of enumeration, make them a good indicator of viral contamination in the marine environment Their presence in shellfish is indicative of sewage pollution and potential contami-nation by human pathogenic viruses They are particularly useful indicators of potential viral contamination in shellfish after treatment where traditional bacterial indicators are removed more readily than human viruses F-specific RNA bacterio-phages are capable of infecting a specified F-pili producing bacterial host strain Infection produces visible plaques on a confluent lawn grown under appropriate culture conditions with the infectious process being inhibited in the presence of ribonuclease (RNase) in the plating media

Principle of method

A culture of host strain is mixed with a small volume of molten nutrient medium Shellfish homogenate is added and the mixture flooded on a solid nutrient agar base and allowed to set This is then incubated at 37°C during which time the host multi-plies to produce a confluent lawn Visible plaques form where bacteriophage is pres-ent It is assumed that each plaque is derived from one bacteriophage Where necessary, simultaneous examination of parallel plates with added RNase for confir-mation by differential counts is carried out The results are expressed as the number of plaque forming units (pfu)/100 g of shellfish [6]

Sample size

As for method 1

Equipment

As for method 1, and in addition:

Centrifuge

Water bath at 45 ± 2°C

Spectrophotometer

Colony counter

Sterile glassware

Reagents

1.0% calcium-glucose solution

12.5% w/v nalidixic acid solution

Tryptone yeast extract glucose broth (TYGB)

Tryptone yeast extract glucose 2% agar (TYGA2) as plates

Tryptone yeast extract glucose 1% agar (TYGA1) in 100 mL volumes

100% w/v RNase (store at –20°C)

0.1% peptone solution (negative control)

MacConkey agar

Glycerol

Chloroform

continued

Microbiological reference materials

Salmonella typhimurium strain WG49, phage type 3 Nalr(F¢ 42 lac:Tn5), NCTC 12484

Bacteriophage MS2 NCO12487 (for positive control; obtainable from NCTC) (see Appendix C)

Sample preparation

(a) Prepare the sample as described in method 1 Prepare the homogenate using

a blender, as described in steps (d) and (e) of method 1, to produce a 1/3 homogenate

(b) Centrifuge 30–50 mL of homogenate, prepared as above, at 2000 ± 200 g for 5 min

at room temperature

(c) Make any decimal dilutions, if required, by adding 1 ± 0.1 mL of supernatant to

9 ± 0.2 mL of 0.1% peptone solution

Agar overlay preparation

(d) Melt TYGA1 then cool to 45°C in the water bath

(e) Add 1 ± 0.1 mL of 1.0% calcium-glucose solution per 100 ± 2 mL of TYGA1 If high levels of background bacteria are expected add 400 ± 2 mL of 12.5% w/v nalidixic acid solution

(f) Aliquot 2.5 ± 0.1 mL of TYGA1 for each replicate sample into bijoux held at 45°C

in the water bath (If DNA plaques are expected, for example in samples from faecally polluted areas, perform confirmatory tests in the presence of 100 ± 1 mL 100% w/v RNase)

Preparation of host

(g) Add 1 ± 0.1 mL of calcium-glucose solution to 100 ± 1 mL of TYGB at 37°C

(h) Inoculate this medium with 1 ± 0.1 mL of S typhimurium WG49 working culture.

(i) Incubate at 37°C for 4 ± 2 h to achieve a cell density of 7–40 ¥ 107cfu/mL at

600 nm, using sterile TYGB as the blank

Assay

(j) Immediately after incubation add 1 ± 0.1 mL of WG49 host culture to all test bijoux followed by 1 ± 0.1 mL of the sample under test Mix contents by inverting the bijoux

continued

To prepare a working culture of S typhimurium WG49, inoculate TYGB and incubate

for 18 ± 2 h at 37°C Subculture to MacConkey agar and incubate at 37°C for 18 ± 2 h;

then select five to seven lactose positive colonies and inoculate into 100 mL of

pre-warmed TYGB Incubate for 5 ± 1 h at 37°C, then add 20 mL glycerol After mixing

thoroughly aliquot into plastic vials and freeze at –70°C

Positive MS2 controls are produced by inoculating an exponentially growing culture

of S typhimurium WG49 with MS2 NCO12487 Incubate at 37°C After 4–5 h add

5 ± 1 mL of chloroform to lyse bacterial cells, then incubate at 5 ± 3°C for 18 ± 2 h

Centrifuge the mixture to remove cell debris The MS2 culture is then titrated with

S typhimurium WG49 The dilution required to give 30–500 plaques is calculated

and aliquots stored at –70°C