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Microsoft word - issc 2005 summary of actions fda concurrence.doc

Proposal Number: 05-114

Proposal Subject
Method to determine the presence of Male Specific Coliphage in shellfish meats and the Microbiology Specific NSSP
NSSP Guidance Documents Chapter II. Growing Areas .10 Approved Laboratory Tests Guide Reference

Key Words
Microbiology Method Isolation of Male-specific Coliphage, MSC Public Health
FDA is submitting a proposal to ISSC to allow MSC to be used as a re-opening criterion in Significance
cases where unexpected, unusual sewage contamination occurs that may have impacted shellfish harvest areas (not for conditional re-openings). The MSC method must be reviewed and adopted prior to use in determining the acceptability of shellfish growing waters for reopening. Cost Information
(if available)

Action by 2005
Recommended referral of Proposal 05-114 to the appropriate committee as determined by the Laboratory Methods
Review Committee

Action by 2005 Task
Recommended adoption of the Laboratory Methods Review Committee recommendation on Action by 2005
Adopted recommendation of 2005 Task Force I. General Assembly
Action by USFDA
Proposal Number: 05-114
Enumeration of Male- specific bacteriophage in water and shellfish tissue What are male- specific (f-specific) bacteriophage? •Lytic viruses of bacteria- (killing of host bacteria) (production of E. coli pili) •Requires a piliated host cell for adsorption, they do not attach to cell surface (somatic). •Requires host cell in log- phase of growth- cells do not produce pili at < 30°C •Optimal growth temperature: 35- 37°C. •Plaque size is generally self- limiting Two Predominant Host Strains •E. coli HS(pFamp)RR - Resistant to Streptomycin and Ampicillin Result of mating: E. coli WG27 (piliated) -Resistant to Naladixic acid and Kanamycin --Pili production in each strain is plasmid mediated Media Composition E. coli Famp Bottom Agar •Tryptone •Autoclave 121°C 15 min - temper to 50°C. •Add 0.05 g Streptomycin sulfate 0.05 g Ampicillin (aseptically) DS Soft Agar •Tryptone •Dextrose 1.0 g •NaCl 5.0 g •1M CaCl2 Proposal Number: 05-114
••Water 500 ml ••Boil- Dispense in 2.5 ml aliquots (16 x 100 ml tubes) and freeze (-20°C) ••Autoclave prior to use; Growth broth- same formulation as Bottom Agar w/o agar or antibiotics Media Composition S. typhimurium WG49 Bottom Agar •Trypticase Peptone •Autoclave 121°C 15 min - temper to 50°C. •Add 0.10 g Naladixic Acid 0.02 g Kanamycin sulfate ••Boil- Dispense in 2.5 ml aliquots (16 x 100 ml tubes) and freeze (-20°C) ••Autoclave prior to use; Growth broth- same formulation as Bottom Agar w/o agar or antibiotics Differentiation of RNA and DNA Bacteriophage •RNAse Type I-A Sigma # R4875 ••Final conc= 100ug/ ml of media •Stock concentration= 10 mg/ml (100X) ••Dissolve at a conc. of 10 mg/ml in 0.01 M Sodium Acetate (pH 5.2); Boil for 15 min and allow to cool to RT; PH by adding 0.1 vol of 1M Tris HCl (pH 7.4) ••Store @ -20C Propagation of E. coli Famp Bottom Agar Streak plate- •Broth Growth medium tempered to 35- 37°C- vortex to aerate. •Using 10ul loop collect material from of several colonies and transfer to broth medium. •Shake briefly to mix, then incubate at 35- 37°C for 4-6 hours (turbidity Proposal Number: 05-114
% RECOVERY OF BACTERIOPHAGE F-2W/ VARIOUS AGES OF FAMP CULTURE Age of a 10 ml host cell culture(h) Adapted from DeBartolomeis, 1999 For MSB density determinations in shellfish tissue 1. Homogenize by blending 12 shellfish for 1 min at high Aliquot 30- 50 g from each sample into centrifuge bottle. Centrifuged for 15 min. @ 9,000 x g; 4°C. Collect and weigh supernatant in a sterile container. Allow supernatant to warm to RT (20- 30 min) Combine 2.5 ml aliquot of supernatant, 2.5 ml DS Soft agar (tempered to 52°C) and 0.2 ml of E. coli HS(pFamp)RR Overlay onto a tryptone agar plate containing streptomycin/ ampicillin (50 µg/ml final). Plates are inverted and incubated for 18- 24 h @ 35- 37°C Ave PFU/ plate ÷ number of ml added/ plate= Average PFU/ml Average PFU/ml x grams of supernatant x 100 g . = PFU/ 100 g Example: Plate counts- 75, 73,80; 2.5 ml/ plate 76 ÷ 2.5 x 33 g supernatant x 100 grams = 2006 PFU/ 100 grams To determine level of sensitivity 3 plates containing 0, 0, 0; 2.5 ml/ plate Assume 1 plaque on 1 plate then calculate 1÷ 3 plates ÷ 2.5 ml x 33 x (100 ÷ 50) = Reported as < 9 pfu/ 100 grams Proposal Number: 05-114
For MSB density determinations in low contaminated water- Concentration technique Weigh 100 ml of water in a sterile container Add 1g tryptone and 1 g beef extract to water Add 10 ml of E. coli Famp culture- Do not shake Incubate at 35- 37°C for 50 min - rotate at 100 rpm. Centrifuged for 15 min. @ 9,000 x g; 4°C. For MSB density determinations in highly contaminated water (> 100 pfu/ 100 ml) Combine 2.5 ml aliquot of supernatant, 2.5 ml DS Soft agar (tempered to 52o C), and 0.2 ml of E. Overlay onto a tryptone agar plate containing streptomycin/ ampicillin (50µg/ml final). Plates are inverted and incubated for 18- 24 h @ 35- 37°C Problems that may arise Multiple layers are formed after centrifugation Reason- glycogen- lipids associated w/ shellfish Reason- waited too long to remove supernatant Reason- wet plates; too much condensation No plaques/ individual bacterial colonies on agar plates Reason- no phage present or inadequate amount host cell Ways of Enhancing Plaque Visibility Addition of 2,3,5- triphenyl tetrazolium chloride (TTC), 1% solution in ethanol Grams Safrin 1:100 in water- differentiates lawn from plaque Storage of E. coli Famp Selective pressure- Streptomycin and Ampicillin Bottom Agar Streak Plate •Tryptic Soy Agar Deep w/ Mineral oil overlay Storage: Room temperature in Dark (2-5 years +) •Addition of glycerol (10% final) into broth culture. Storage: Freeze at - 80°C (Indefinite?) Source of Bacterial Host Strains •E. coli HS(pFamp)R; ATCC #700891 •Salmonella choleraesuis subsp. choleraesuis (Smith) Weldin serotype Typhimurium aka WG49; ATCC #700730 Proposal Number: 05-114
Types and Sources of Positive MSB Controls Bacteriophage MS2; ATCC# 15597-B1 Bacteriophage Fd; ATCC# 15669 -B2 Municipal Wastewater Bacteriophage Stability in Shellfish Homogenate Temperature
Addition
Log Fampa
aFamp added at a density of 270 cells/ g bSignificant decrease at 95% Confidence limit Bacteriophage Stability in Shellfish Supernatant Temperature
Addition
aFamp added at a density of 270 cells/ g bSignificant decrease at 95% Confidence limit cSignificant increase at 95% Conifidence limit

Source: http://www.issc.org/client_resources/2005%20proposals%20summary%20of%20actions/05-114.pdf

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