USE OF ENRICHMENT REAL TIME-POLYMERASE CHAIN REACTION TO ENUMERATE SALMONELLA ON CHICKEN PARTS

Thomas P. Oscar, Ph.D.

USDA, ARS

Room 2111, Center for Food Science and Technology

University of Maryland Eastern Shore

Princess Anne, MD 21853

410-651-6062

thomas.oscar@ars.usda.gov

Salmonella

• Leading cause of foodborne illness.

• Chicken is an important source of Salmonella for humans.

Risk AssessmentHolistic Approach to Food Safety

• Data gap: quantitative data• Contamination• Cross-contamination

Chicken Parts Salmonella Enumeration Issues

• Bones• Viable count and MPN

• Low Number• One cell per part

• Association• Unattached

• Surface water layer

• Attached• Colonies

• Entrapped• Skin crevices• Feather follicles• Deep tissues

Whole Part EnrichmentNew Approach for Salmonella Enumeration

= Salmonella= competitors

= buffered peptone water= chicken part

< 1 cell/mlt = 0 h

> 1 cell/mlt = 6 h

6 h42C

80 rpm

Enumeration of Salmonella on Chicken PartsStandard Curves

Oscar, 2013. J Food Prot 76(1):33-39

Whole Part Enrichment6 h

400 ml BPW42°C

80 rpm

Real-Time Polymerase Chain Reaction

• CT is inversely related to the number of target bacteria.

• Cycle threshold (CT) is the number of PCR cycles for the fluorescent signal to reach a threshold value.

Enrichment Real-Time PCR

• Has been used to enumerate Campylobacter in chicken rinse samples.

Josefsen et al., 2004, Appl. Envrion. Microbiol. 70:3588-3592.

Objective

• To use enrichment real-time PCR to:

• enumerate Salmonella on chicken parts at retail; and

• that cross-contaminate cooked chicken during simulated meal

preparation and serving.

Harvest 8 raw parts from a broiler chicken carcass

Inoculate parts with Salmonella Typhimurium var 5-

Incubate parts in buffered peptone water (400 ml)

Collect 1 ml samples for RT-PCR and Salmonella isolation

Determine cycle threshold value

Enrich in RV broth and confirm with lateral flow assay

Isolate on XLT4 agar

Serotype(NVSL)

Experimental Protocol

0 to 3.6 log10 CFU

8 h, 40ºC, 80 rpm

iQ-Check™ (Bio-Rad)

Reveal® 2.0 (Neogen)

Cross-contamination“Worst-case Scenario”

• Sterilized, cooked chicken breast was cut in half and then the portions were used to swab the drip on the cutting board.

Experiment 1Relationship between cycle threshold (CT) value and Salmonella counts

• Determined CT for serial dilutions of Salmonella culture.

• Graphed CT versus Salmonella counts in BPW.

• Fitted data to power law model.

• CT at 0 log/ml was projected to be 41.

Experiment 2Standard Curve for Enumeration of Salmonella on Chicken Parts

Y = 34.3 – (X/0.0047)0.436

Experiment 3Salmonella Prevalence, Number, and Serotype

Salmonella prevalence 70% (7/10) for whole chickens

19% (15/80) for raw chicken parts

10% (2/20) for cooked chicken

  Prevalence Number  

Part % CT CFU/part Serotypes

Wing 20 (4/20) 36,35,30,39 1,1,1,1Typhimurium (2); 4,5,12:Nonmotile (1); not identified (1)

Breast 25 (5/20) 32,28,31,42,33 1,2,1,1,1Typhimurium (1); 4,5,12:Nonmotile (1); Typhimurium var 5- (1); 4,12:i:- (1); not identified (1)

Thigh 15 (3/20) 40,31,31 1,1,1Typhimurium var 5- (1); Kentucky (1); 4,12:Nonmotile (1)

Drumstick 15 (3/20) 35,32,30 1,1,1 Typhimurium var 5- (2); Kentucky (1)

Cooked 10 (2/20) 30,34 1,1 Kentucky (1); 4,12:i:- (1)

Chicken #7

4,5,12:Nonmotile

4,12:i:-4,12:i:-Kentucky

Kentucky

Kentucky

4,5,12:Nonmotile

1 1

22

3 3

44

5 5

Experiment 3Distribution of Salmonella among parts

• Six different patterns of contamination among seven contaminated chickens.

• 1 = wing

• 2 = breast

• 3 = cooked

• 4 = thigh

• 5 = drumstick

• More than one serotype was present at times.

Conclusions

• Enrichment real time-PCR can be used to enumerate low to high levels of Salmonella on chicken parts.

• Salmonella prevalence was high but Salmonella number was low (1 or 2 cells per part).

70% 19% 10%