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. Salmonella are a leading cause of foodborne illness and deaths in the United States and throughout the world. Outbreaks of salmonellosis are often attributed to consumption of poultry meat and eggs.

Risk Assessment Holistic Approach to Food Safety Data gap: quantitative data Contamination Cross-contamination Risk assessment is a holistic approach to food safety that has great potential for improving public health. However, important data gaps exist that result in significant uncertainty in risk assessments. Two important data gaps are: lack of information about the number of Salmonella that contaminate chicken and lack of information about the number of Salmonella that cross-contaminate other food during meal preparation and serving. There are several issues to consider when acquiring these quantitative data.

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 First, bones make it difficult to enumerate Salmonella by conventional methods. Second, Salmonella are often present at levels that are below detection by conventional methods. Third, Salmonella are associated with chicken as unattached, attached, or entrapped cells. Thus, the sampling method used must be capable of recovering all Salmonella regardless of how they are associated with chicken. To address these issues, a new approach to enumeration was developed.

Whole Part Enrichment New Approach for Salmonella Enumeration < 1 cell/ml t = 0 h > 1 cell/ml t = 6 h 6 h 42C 80 rpm In this approach, a whole chicken part is incubated in a fixed volume of buffered peptone water under standard growth conditions. During incubation, the number of Salmonella in buffered peptone water increases. If a sample is obtained during the exponential phase of growth, there will be a mathematical relationship between the concentration of Salmonella in buffered peptone water and the original number of Salmonella on the chicken part. This relationship can be used to develop a standard curve for enumeration of Salmonella on chicken parts. Throughout the incubation phase, the sample is shaken to release attached and entrapped Salmonella from the chicken part for their detection and enumeration. = Salmonella = buffered peptone water = competitors = chicken part

Enumeration of Salmonella on Chicken Parts Standard Curves Whole Part Enrichment 6 h 400 ml BPW 42°C 80 rpm These are examples of standards curves for enumeration of Salmonella on chicken that were developed using the whole part enrichment method. The standard curves were developed by inoculating chicken parts with different doses of Salmonella and then determining their concentration in buffered peptone water at 6 h of incubation by direct plating on XLT4 agar. Oscar, 2013. J Food Prot 76(1):33-39

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. Determining the number of Salmonella in enrichment media by direct plating can be problematic when the number of other bacteria is high. However, it is possible to overcome this problem using real-time PCR because there is a mathematical relationship between the cycle threshold (CT) value obtained by real-time PCR and concentration of bacteria in the sample. The CT value is the PCR cycle number at which the fluorescent signal from detection of the target DNA reaches a threshold value indicated by the horizontal blue line in the graph. The cycle threshold value is inversely related to the number of target bacteria in the sample.

Enrichment Real-Time PCR Has been used to enumerate Campylobacter in chicken rinse samples. Enrichment real-time PCR has been used to enumerate Campylobacter in chicken rinse samples. However, it has not been used to enumerate Salmonella on chicken parts. 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. Therefore, the present study was undertaken to use enrichment real-time PCR to detect and enumerate Salmonella that contaminate raw chicken parts at retail and that cross-contaminate cooked chicken during simulated meal preparation and serving for the purpose of filling important data gaps in risk assessments for Salmonella and chicken. To accomplish this objective,

Enrich in RV broth and confirm with lateral flow assay Experimental Protocol Harvest 8 raw parts from a broiler chicken carcass Inoculate parts with Salmonella Typhimurium var 5- 0 to 3.6 log10 CFU Incubate parts in buffered peptone water (400 ml) 8 h, 40ºC, 80 rpm Collect 1 ml samples for RT-PCR and Salmonella isolation Determine cycle threshold value a sterilized knife and cutting board were used to remove eight raw parts from a broiler chicken carcass obtained at retail. In standard curve experiments, parts were inoculated with different levels of Salmonella followed by incubation in fixed volume of buffered peptone water under standard growth conditions. After incubation, two one ml samples were collected. One was used for real-time PCR and one was used for Salmonella isolation. A real-time PCR kit was used to determine CT values. Salmonella-positive samples were enriched in RV broth and then confirmed by lateral flow assay, isolation on XLT4 agar, and serotyping. iQ-Check™ (Bio-Rad) Enrich in RV broth and confirm with lateral flow assay Reveal® 2.0 (Neogen) Isolate on XLT4 agar Serotype (NVSL)

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. To study cross-contamination, the unwashed knife, cutting board, and latex gloves used to harvest the raw parts from the whole chicken were used to partition a sterilized and cooked chicken breast into two portions. The portions were then used to swab the cutting board surface to provide a “worst-case” scenario for assessing cross-contamination of other food with Salmonella from raw chicken during meal preparation and serving.

Experiment 1 Relationship 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. In the first experiment, the relationship between CT values and Salmonella counts was determined by subjecting serial dilutions of a Salmonella culture to the real-time PCR procedure. When CT values were graphed as a function of Salmonella concentration, a slightly non-linear and concave downward curve was obtained that fitted well to the power law model with a shape parameter of 1.23. The Y-intercept, which was the CT value for a single cell of Salmonella per ml, was projected to be around 41. Thus, as expected, there was an inverse relationship between CT value determined by real-time PCR and number of Salmonella in the buffered peptone water sample.

Experiment 2 Standard Curve for Enumeration of Salmonella on Chicken Parts Y = 34.3 – (X/0.0047)0.436 Experiment 2 was designed to develop a standard curve for enumeration of Salmonella on chicken parts by enrichment real-time PCR. Here, chicken parts were inoculated with 0 to 3.6 logs of Salmonella. As expected, CT values were inversely related to initial number of Salmonella inoculated. The standard curve was non-linear and concave upward and fitted well to the power law model. At the lowest dose inoculated, which was 1 cell per part as determined by drop plating on XLT4 agar, variation of CT values was high and was likely due to expression of variability of lag times among individual cells of Salmonella.

Experiment 3 Salmonella Prevalence, Number, and Serotype 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 1,1,1,1 Typhimurium (2); 4,5,12:Nonmotile (1); not identified (1) Breast 25 (5/20) 32,28,31,42,33 1,2,1,1,1 Typhimurium (1); 4,5,12:Nonmotile (1); Typhimurium var 5- (1); 4,12:i:- (1); not identified (1) Thigh 15 (3/20) 40,31,31 1,1,1 Typhimurium var 5- (1); Kentucky (1); 4,12:Nonmotile (1) Drumstick 35,32,30 Typhimurium var 5- (2); Kentucky (1) Cooked 10 (2/20) 30,34 1,1 Kentucky (1); 4,12:i:- (1) In Experiment 3, the standard curve was used to determine number of Salmonella on naturally-contaminated chicken parts. A total of 100 chicken parts, 80 raw parts and 20 cooked parts, were examined. Raw parts were from 10 different whole chickens of the same brand and from the same retail store. Seven of ten whole chickens had one or more parts that were contaminated with Salmonella for a prevalence of 70% whereas 15 of 80 raw parts were contaminated with Salmonella for a prevalence of 19%. CT values ranged from 28 to 42. The standard curve predicted a Salmonella level of 1 or 2 cells per part. The predominant serotypes were Typhimurium and its variants, which are highlighted in blue, and Kentucky. Incidence of cross-contamination of cooked chicken was 10% and in both cases corresponded to transfer of a single cell of Salmonella from raw chicken to cooked chicken.

Experiment 3 Distribution 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. Chicken #7 4,5,12: Nonmotile 4,12:i:- Kentucky 4,5,12:Nonmotile 1 2 3 4 5 Distribution of Salmonella among parts of individual chickens was examined by mapping. There were six different patterns of contamination among seven chickens that tested positive for Salmonella. In some cases, more than one serotype was found. For example, chicken #7 was contaminated with three different serotypes of Salmonella.

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% In conclusion, results indicated that whole part enrichment combined with real-time PCR can be used to develop a standard curve for enumeration of Salmonella on naturally-contaminated chicken parts. Although Salmonella prevalence of whole raw chickens and parts were high, the number of Salmonella that contaminated raw parts at retail and that cross-contaminated cooked chicken during simulated meal preparation and serving was low (1 or 2 cells per part). Thus, as long as consumers properly stored and handled the chicken studied, it should have presented a low risk of salmonellosis.