Efficacy of Recovery and Detection of Sublethally Stressed

Listeria Using Different Enrichment Media: from in vitro to in situ

Sergiy Olishevskyy1, Carolina Mejia-Wagner1, Ariane Favreau1, Alex Eyraud1 and Michael Giuffre2

1FoodChek Laboratories Inc., St-Hyacinthe, Quebec, Canada 2FoodChek Systems Inc., Calgary, Alberta, Canada

The ability of Listeria monocytogenes to proliferate in various foods at refrigerationtemperatures and survive even after deep freezing makes the occurrence of this foodborne

pathogen in ready-to-eat (RTE) foods of particular concern. It is especially threatening to thedeli meat and dairy industries if fast and reliable detection methods are not applied. SinceL. monocytogenes in RTE food can be present at low concentration with sub-lethal injury

during food processing, an enrichment step is crucial to resuscitate injured cells and allowsufficient growth for detection.

The goal of the study was to determine the effectiveness of selective enrichment broth for

the recovery and detection of sublethally stressed L. monocytogenes in ready-to-eat foods.

RT-PCR Detection Culture Detection

T im e (H o u rs )

OD

60

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0 .6

T im e (H o u rs )

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Fig. 1. Growth of uninjured (A) and sublethally heat-injured (B) L. monocytogenes

A. B.

Table 1. Growth Kinetics Parameters of L. monocytogenes

Bacterium Kinetics Parameter Actero Listeria BLEB UVM

Uninjured ListeriaLPD 9.3 ± 0.41 12.2 ± 0.56** 16.4 ± 0.75***

GR 0.352 ± 0.056 0.274 ± 0.020* ND

Heat-injured ListeriaLPD 11.1 ± 0.94 16.0 ± 1.26** 18.1 ± 0.88**

GR 0.368 ± 0.061 ND ND

Table 2. Food Matrices and Inoculating Microorganisms

Food MatrixSample

Size

L. monocytogenesBackground Flora,

log10 CFU/gSerotype Origin Stress

Pasteurized Milk 25 g 4c Beef manure Heat < 2.0

Soft Cheese 25 g 1/2a Sheep’s head None 7.5–8.0

Frankfurter 125 g 1/2a Milk Heat < 2.0

Smoked Turkey 125 g 3b Raw turkey Heat 4.7–5.0

Cured Ham 125 g 1/2a Animal tissues Heat < 2.0

Table 3. Performance Parameters for Detection of L. monocytogenes Using BAX SystemRT-PCR Assay in Dairy and Deli Meat Products Enriched with Actero Listeria

Food MatrixTotal

TestedP N FP FN

Relative Sensitivity,

%

Relative Specificity,

%

FP Rate, %

FN Rate, %

Test Efficacy, %

Pasteurized milk 60 43 16 0 1 97.7 100 0.0 2.3 98.3

Soft cheese 60 36 24 0 0 100 100 0.0 0.0 100

Frankfurter 30 19 11 0 0 100 100 0.0 0.0 100

Smoked turkey 30 12 18 0 0 100 100 0.0 0.0 100

Cure ham 30 16 14 0 0 100 100 0.0 0.0 100

TOTAL: 210 126 83 0 1 99.2 100 0.0 0.8 99.5

Notes: P – positive, N – negative, FP – false positive, FN – false negative.

Fig. 3. Recovery of L. monocytogenes Using Actero Listeria

Notes:

T im e (H o u rs )

OD

60

0n

m

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

0 .0

0 .2

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T ML is te r ia

2 4 L is te r ia E n r ic h m e n t B ro th (O x o id )

U VM

L E B O xo id

B L E B (O x o id )

A N S R (N e o g e n )

BLEB

Actero Listeria

T im e (H o u rs )

OD

60

0n

m

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4

0 .0

0 .2

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T ML is te r ia

B L E B (O x o id )

T im e (H o u rs )

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60

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0 .0

0 .2

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T ML is te r ia

2 4 L is te r ia E n r ic h m e n t B ro th (O x o id )

U VM

L E B O xo id

B L E B (O x o id )

A N S R (N e o g e n )

UVM

Culture Conditions

• Initial Inoculum – 20 CFU/well• Medium Volume – 200 µL• Temperature – 35°C• Time – 22 hours

Fig 2. Method Comparison Matrix Studies Flowchart

Food Samples

Dairy ProductsDeli Meat Products

Actero Listeria Method US FDA BAM10 Method

125 g sample 25 g sample25 g sample

500-1125 mL

Actero Listeria

24-26hrs, 35°C

225 mL

BLEB

48hrs, 30°C

150mL

Actero Listeria

22hrs, 35°C

Morphological, biochemical and

serological identification

up to 72 hrs

BAX System Assay,

Listeria Genus

2 hrs

BAX System Assay,

L. monocytogenes

2 hrs

Total Time

24-28 hrs

Total Time

up to 120 hrs

Direct Plating

Rapid’L.mono / MOX

24 hrs

Total Time

46-50 hrs

Total Time

up to 120 hrs

USDA FSIS MLG 8.10 Method

125 g sample

1125 mL, UVM

23-26hrs, 30°C

10 mL, MOPS-BLEB

18-26hrs, 35°C

Morphological, biochemical and

serological identification

up to 72 hrs

Notes: LPD – Lag Phase Duration; GR – Growth Rate.ND – Not Determined: Kinetics parameters could not be defined because all growth stages were not observed during the incubation period.*P<0.05, **P<0.01, ***P<0.001 as compared to the corresponding values obtained for Actero Listeria.

The performance parameters of relative sensitivity, relative specificity, false positiverate (FP), false negative rate (FN), and test efficacy were calculated for the ActeroListeria method and are presented in Table 3. Only one false negative outcome out of210 analyzed samples was observed.

Food Samples

9855

***

4285

Actero Negative Outcomes

Actero Positive Outcomes

RM Positive Outcomes

RM Negative Outcomes

A c te r o P o s it iv e O u t c o m e s

A c te r o N e g a t iv e O u tc o m e s

R M P o s it iv e O u tc o m e s

R M N e g a t iv e O u tc o m e s

Actero Listeria shows shorter lag phase and higher growth rate for L. monocytogenes as compared to the reference method media.

Enrichment with Actero Listeria results in a rapid and efficient repair of growing capacities of sublethally-injured L. monocytogenes.

Deli meat and dairy samples were artificially contaminated with healthy or sub-lethally heat-stressed L. monocytogenes belonging to different serotypes (Table 2) and stabilized for 48–72 hours at 2–8°C. Samples were enriched in Actero™ Listeria Enrichment Media, then processed using Hygiena’s BAX® System Real-Time PCR Assays for L. monocytogenes and ListeriaGenus as well as by direct plating. A total of 420 artificially contaminated food samples were examined to evaluate performance of the candidate method in comparison with theUSDA-FSIS or US FDA reference methods (Fig. 2).

Overall, 140 fractionally contaminated food samples containing low numbers ofsublethally injured L. monocytogenes and tested by the alternative method using ActeroListeria yielded 40% more positive results as compared to the samples analysed by theUSDA-FSIS or US FDA methods.

Single step enrichment with Actero Listeria allows for significantly reducingincubation time due to efficacious recovery of injured L. monocytogenes.

BAX System Assays show high accuracy and reliability for detection ofL. monocytogenes in deli meat and dairy samples enriched with Actero Listeria.

Performance of the Actero Listeria method for deli meat and dairy products issuperior to the reference methods.

In situ Matrix Studies

Method PrincipleIntroduction

In vitro Culture Studies

Conclusions

***P<0.001 as compared to the corresponding valuesobtained for Actero Listeria.