Recovery, detection and sanitizer susceptibility of Listeria spp. from

meat processing environments

-by--by-Jovana KovaJovana Kovačevićčević

June 15June 15thth 2010 2010

Outline

Listeria Listeria spp. backgroundspp. background Project OverviewProject Overview

Comparison of sampling methodsComparison of sampling methods Evaluation of detection methodsEvaluation of detection methods Occurrence and distribution of Occurrence and distribution of Listeria Listeria spp. in meat spp. in meat

processing environmentsprocessing environments Sanitizer susceptibility Sanitizer susceptibility

Summary Summary

22

3

Project Members Food Safety Division, Alberta Agriculture, Food Food Safety Division, Alberta Agriculture, Food

and Rural Developmentand Rural Development Dr. Valerie Bohaychuk – Project ManagerDr. Valerie Bohaychuk – Project Manager Gary Gensler – Supervisor (Food Microbiology)Gary Gensler – Supervisor (Food Microbiology) Robin King – Supervisor (Molecular Biology)Robin King – Supervisor (Molecular Biology) Deana Rolheiser – Technician (Food Microbiology) Deana Rolheiser – Technician (Food Microbiology) Sonja Marshall – Technician (PFGE)Sonja Marshall – Technician (PFGE) Dr. Pablo Romero Barrios – EpidemiologistDr. Pablo Romero Barrios – Epidemiologist

University of Alberta, Department of Agricultural, University of Alberta, Department of Agricultural, Food and Nutritional Science, University of AlbertaFood and Nutritional Science, University of Alberta Dr. Lynn McMullen – Food Microbiology Professor, Graduate Dr. Lynn McMullen – Food Microbiology Professor, Graduate

Student SupervisorStudent Supervisor Jovana Kovacevic – Graduate StudentJovana Kovacevic – Graduate Student

Six species:Six species:• L. monocytogenesL. monocytogenes• L. ivanoviiL. ivanovii• L. seeligeriL. seeligeri• L. innocuaL. innocua• L. welshimeriL. welshimeri• L. grayiL. grayi

–Subspecies Subspecies L. murrayL. murray

Genus Listeria

44

Genus Listeria

Gram positive, facultative anaerobic rodsGram positive, facultative anaerobic rods Motile at 10 to 25Motile at 10 to 25°C°C Grow Grow between -0.4ºC and 50ºCbetween -0.4ºC and 50ºC Isolated from:Isolated from:

Environmental sources: soil, water, effluentsEnvironmental sources: soil, water, effluents Food processing environments Food processing environments Variety of foodsVariety of foods Feces of humans and animalsFeces of humans and animals

55Clin. Micorbiol. Rev. 14(3):584-640.Clin. Micorbiol. Rev. 14(3):584-640.

Project GoalsPhase 1: Phase 1: To compare 3 methods for recovery of To compare 3 methods for recovery of

Listeria Listeria spp. in the environment of meat processing spp. in the environment of meat processing facilitiesfacilities

Phase 2:Phase 2: To evaluate 4 methods for detection of To evaluate 4 methods for detection of Listeria Listeria spp.spp.

Phase 3:Phase 3: To assess the occurrence and distribution To assess the occurrence and distribution of of ListeriaListeria spp. in meat processing environments spp. in meat processing environments

Phase 4:Phase 4: To determine the susceptibility to sanitizers To determine the susceptibility to sanitizers of persistent and nonpersistent strains of of persistent and nonpersistent strains of L. L. monocytogenesmonocytogenes

66

7

Project Overview • Two Federally inspected meat processing facilities

• Edmonton, Alberta• Two time points:

• After cleaning and sanitation but prior to processing (ACS)

• During or after processing but before sanitation (PRO)

• Sampling in 2005 and 2006 for 19 months, 3 week intervals1) Comparison of environmental sampling

methods: – 5 months – Ten sampling locations:

* 5 where RAW meat was processed

* 5 where COOKED meat was handled

– Total of 720 samples

8

Project Overview 2) Comparison of detection methods:

– Until 100 positive and 100 negative samples collected per method

– 14 sampling locations: *7 where RAW meat was

processed*7 where COOKED meat

was handled

– Criteria for 3 methods reached after 11 samplings; 1 method required 14 samplings

3) Occurrence and Distribution of Listeria spp.– All the samples collected by SS method and

analyzed by culture method (n=820)– 19 months period

Phase 1: Comparison of Environmental Sampling Methods

99

soaked in 1 ml of soaked in 1 ml of neutralizing neutralizing

bufferbuffer

pre-wettedpre-wetted soaked in 10 ml of soaked in 10 ml of neutralizing bufferneutralizing buffer

1)1) Cotton Swab (CS) Cotton Swab (CS) 2)2) Sterile Sponge (SS)Sterile Sponge (SS) 3)3) Composite tissue (CT)Composite tissue (CT)

Phase 1: Sample Collection

• Sampling every 3 weeks, for 5 months in 2005/2006:

• 6 visits • 120 samples / method / facility

• 240 samples for each method

Total = 720 samples collected

1010

11

Phase 1: Sample Collection Table 1. Areas sampled for recovery of Listeria spp. in two federally inspected meat processing facilities.

a Sampling locations added for the comparison of detection methods.

Sample Processing: ISO 11290-1

Confirmation

Environmental Sample

+Demi-Fraser Broth

PALCAM OxfordFB Confirmation

35°C for 24 to 48 h

35°C for 24 to 48 h

PALCAM Oxford

1212

Phase 1: Results

1313

Figure 1. Number of samples that tested positive for presence of Listeria spp. obtained using a cotton swab (CS), a sterile sponge (SS) and a composite tissue (CT) sampling method.

*Represents the total number of times samples were positive for Listeria spp.

Evaluated Evaluated sensitivitysensitivity and and specificityspecificity of detection methods of detection methods using culture method as “gold standard”using culture method as “gold standard”

PetrifilmPetrifilm™ Environmental ™ Environmental ListeriaListeria Plates (EL Petrifilm Plates (EL Petrifilm™™)) Lateral flow immunoprecipitation (LFI) device - Lateral flow immunoprecipitation (LFI) device -

RevealReveal®® Listeria Listeria Test SystemTest System Automated polymerase chain reaction (PCR) Automated polymerase chain reaction (PCR)

BAXBAX® ®

Conventional culture ISO 11290-1Conventional culture ISO 11290-1

Samples collected until 100 positive100 positive and 100 negative100 negative samples were obtained

Phase 2: Detection Methods

1414

Phase 2: Materials and Methods1)1) PetrifilmPetrifilmTMTM Environmental Environmental Listeria Listeria Plates (3MPlates (3M™ Microbiology, London, ON)™ Microbiology, London, ON)

Tests were performed according to the Health Canada MFLP-11 method

1515

Listeria spp.

A

2) Lateral flow immunoprecipitation - Reveal® Listeria Test System (Neogen Corporation, Lansing, Mich.)

Lateral flow device combining immunoassay with chromatography (45 h)

1616

1717

3)3) Polymerase chain reaction (BAXPolymerase chain reaction (BAX®®, DuPont , DuPont Qualicon, Wilmington, DE)Qualicon, Wilmington, DE)

Tests were performed according to the Health Canada MFLP-15 method

http://www2.dupont.com/Qualicon/en_US/assets/downloads/baxproc.pdf

Phase 2: Results

Table 3. Sensitivity, specificity, and kappa values of the EL Petrifilm™, LFI and PCR methods relative to the culture method for identification of Listeria spp. in samples obtained from the environment of two federally inspected meat processing facilities.

EL Petrifilm™

LFI PCR

Sensitivity (%) 50.6 95.5 99.1

Specificity (%) 91.5 100 100

Kappa 0.457 0.958 0.993

Kappa (95% CI) 0.370 – 0.544 0.935 – 0.995 0.980 – 1.00

19

21

Phase 3: Occurrence and Distribution

Environmental samples collected for 19 Environmental samples collected for 19 months in 2005 and 2006months in 2005 and 2006

Sampling method: SS onlySampling method: SS only Detection method: ISO culture Detection method: ISO culture

method onlymethod onlyTotal: 820 samples testedTotal: 820 samples tested 249 249

(30.4%) positive for (30.4%) positive for Listeria Listeria spp. spp.

22

Phase 3: Results Table 4. The number of samples collected from two meat processing facilities either ACS or PRO from either an area where raw meat was processed or an area where cooked products were handled that tested positive for Listeria spp.

No. of positive samples/total number collected

ACS* PRO†

Raw Product

Area

Cooked Product

Area

Raw Product

Area

Cooked Product

Area

Facility A (n=652)

95/163 22/163 117/163 14/163

Facility B (n=168)

0/42 0/42 1/42 0/42

Total 95/205 22/205 118/205 14/205*ACS, After Cleaning and Sanitation and prior to processing†PRO, During or after Processing

23

Phase 3: Results

Figure 2. The number of samples that tested positive for different species of Listeria collected either after cleaning and sanitation (ACS) or during or after processing (PRO), from the areas where raw and cooked products were processed in two meat processing facilities.*Represents the total number of times samples were positive for Listeria spp.

n= 410 n= 410

24

Figure 3. The distribution and number of the samples that tested positive for species of Listeria and the total number of times samples were positive for Listeria spp. in meat processing Facility A.

Raw food areas Cooked food areas

25

Phase 3: Results

Facility AFacility A Overall contamination with Overall contamination with Listeria Listeria spp. was spp. was

38.0% (248/652)38.0% (248/652) L. monocytogenesL. monocytogenes recovered from an area recovered from an area

where raw meat was processed where raw meat was processed (157/326)(157/326) and and an area where cooked products were handled an area where cooked products were handled (25/326)(25/326)

Facility BFacility B Only one sample (n=168) was positive for Only one sample (n=168) was positive for

L. innocuaL. innocua

Phase 4: Susceptibility to Sanitizers

Persistent and nonpersistent strains identified using Persistent and nonpersistent strains identified using Pulsed Field Gel Electrophoresis (PFGE) as part of a Pulsed Field Gel Electrophoresis (PFGE) as part of a concurrent studyconcurrent study

Sanitizer susceptibility of Sanitizer susceptibility of L. monocytogenesL. monocytogenes Planktonic cells in tryptic soy broth (24 h)Planktonic cells in tryptic soy broth (24 h) Growth on stainless steel (48 h)Growth on stainless steel (48 h) Growth in the MBECGrowth in the MBEC™ HTP assay (4 days)™ HTP assay (4 days)

SanitizersSanitizers E-SanE-San®® 10% 10% (5% N-alkyl dimethyl benzyl (5% N-alkyl dimethyl benzyl

ammonium chloride and 5% N-alkyl dimethyl ethyl ammonium chloride and 5% N-alkyl dimethyl ethyl benzyl ammonium chloride)benzyl ammonium chloride)

Perox-EPerox-E®® (hydrogen peroxide and acetic acid) (hydrogen peroxide and acetic acid)

2626

27

Phase 4: Materials and Methods

L. monocytogenesL. monocytogenes strains strains

Two persistentTwo persistent Two nonpersistentTwo nonpersistent L. monocytogenesL. monocytogenes ATCC 19115 (American Type ATCC 19115 (American Type

Culture Collection, Manassas, VA)Culture Collection, Manassas, VA) Stainless steel couponsStainless steel coupons

Type 304, No. 4 finish, 12 mm diameterType 304, No. 4 finish, 12 mm diameter MBEC™ assayMBEC™ assay

High-throughput device (Innovotech High-throughput device (Innovotech Incorporated, Edmonton, AB)Incorporated, Edmonton, AB)

28

1) Susceptibility to Sanitizers: Planktonic Cells

Centrifuged and decanted

1.9 ml of sanitizer or 0.85% saline for controls in each well

1 x 109 CFU/ml

30 s exposure time

Serially dilutedTSB

100 µlDEB

100 µl

35°C for 24 – 48 h

Resuspended in10 ml of saline 1 x 107 CFU/ml

Sanitizer concentrations: • E-San®: 50, 100, 200, 300, 400, 800 ppm• Perox-E®: 70, 200, 500, 800, 1100 ppm

29

2) Susceptibility to Sanitizers: Cells Grown on Stainless Steel Coupons

1 x 109 CFU/ml

Sanitizer concentrations: • E-San®: 100, 200, 300, 400, 500, 600 ppm• Perox-E®: 900, 1100, 1300 ppm

5 min exposure time

DEB

35°C for 48 h

TSB

2 ml into each well

Serially diluted

1 x 105 CFU/ml

TSB

2 ml of sanitizer or 0.85% saline for controls in each well

35°C for 24 – 48 h

30

3) Susceptibility to Sanitizers: Biofilms Grown on MBEC™ Devices

Sanitizer concentrations:

• E-San®: 5,000 39 ppm• Perox-E®: 19,200 150 ppm

Two-fold dilution series

Set up the sanitizer challenge

plate

Expose biofilms to sanitizers (10 min)

Use challenge plates to obtain MIC (planktonic cultures) streak contents of each well onto TSA

Incubate recovery plate (DEB) at 35°C for 24 – 48 h

31

Phase 4: Results

Planktonic cells in TSB

Cells adhered to SS coupons (48 h)

Planktonic cells -MBEC™

Biofilms cells -MBEC™

Perox-E® (ppm)

50 100 200200 300 400 500 600 700 800 900 1,000 5,000

70

100 300 500 700 1,300900 1,1001,100 19,200

E-San® (ppm)

1,500

800

>

4,800

150

39

>

MRC*

MRC*

*MRC, manufacturer recommended concentration for sanitation

Planktonic cells in TSB

Cells adhered to SS coupons (48 h)

Planktonic cells -MBEC™

Biofilms cells -MBEC™

Perox-E® (ppm)

50 100 200200 300 400 500 600 700 800 900 1,000 5,000

70

100 300 500 700 1,300900 1,1001,100 19,200

E-San® (ppm)

1,500

800

>

4,800

150

39

>

MRC*

MRC*

50 100 200200 300 400 500 600 700 800 900 1,000 5,000

70

100 300 500 700 1,300900 1,1001,100 19,200

E-San® (ppm)

1,500

800

>

4,800

150

39

>

MRC*

MRC*

*MRC, manufacturer recommended concentration for sanitation

32

Summary Sterile sponge and composite tissue superior Sterile sponge and composite tissue superior

environmental sampling methods than cotton swabenvironmental sampling methods than cotton swab LFI and PCR-based methods excellent alternatives to LFI and PCR-based methods excellent alternatives to

conventional culture method for detection of conventional culture method for detection of ListeriaListeria spp., spp., while EL Petrifilmwhile EL Petrifilm™ is easy to use but less efficient ™ is easy to use but less efficient

Perox-EPerox-E®® is more efficient in inactivation of cells of is more efficient in inactivation of cells of L. L. monocytogenesmonocytogenes adhered to stainless steel than E-San adhered to stainless steel than E-San®®

Concentrations far greater than the MRC for both E-SanConcentrations far greater than the MRC for both E-San®® and Perox-Eand Perox-E®® required for inactivation of biofilms grown for required for inactivation of biofilms grown for four days on MBEC™ devicesfour days on MBEC™ devices

NO DIFFERENCENO DIFFERENCE in susceptibility to sanitizers among in susceptibility to sanitizers among persistent, nonpersistent and control strains of persistent, nonpersistent and control strains of L. L. monocytogenesmonocytogenes

33

Acknowledgements

• Dr. Lynn McMullenDr. Lynn McMullen

• Dr. Valerie Bohaychuk and Dr. Valerie Bohaychuk and Listeria Listeria in Meat Plants (LMP) teamin Meat Plants (LMP) team

• The financial support fromThe financial support from• The Alberta Agricultural Research Institute, The Alberta Agricultural Research Institute, • The Alberta Chicken Producers, and The Alberta Chicken Producers, and • The Food Safety Division, Alberta Agriculture and FoodThe Food Safety Division, Alberta Agriculture and Food

• The participation of two meat processing facilitiesThe participation of two meat processing facilities

• Phases 1 and 2 of the project were published in the Journal of Food Phases 1 and 2 of the project were published in the Journal of Food Protection: Protection:

Kovačević, J., V. M. Bohaychuk, P. Romero Barrios, G. E. Gensler, D. L. Rolheiser and L. Kovačević, J., V. M. Bohaychuk, P. Romero Barrios, G. E. Gensler, D. L. Rolheiser and L. M. McMullen. 2009. Evaluation of environmental sampling methods and rapid detection M. McMullen. 2009. Evaluation of environmental sampling methods and rapid detection assays for recovery and identification of assays for recovery and identification of ListeriaListeria spp. from meat processing facilities. spp. from meat processing facilities. J. J. Food Prot.Food Prot. 72(4): 696-701. 72(4): 696-701.

34http://www.umanitoba.ca/faculties/afs/soil_science/MSSS/Ecology/Cartoons/Cartoons%20Images/Microbes%20multiplying.jpg