Low-Temperature Survival of Salmonella spp. in a Model Food System with Natural Microflora

Abstract

The United States Department of Agriculture requires chilled poultry carcass temperature to be below 4°C (40°F) to inhibit the growth of Salmonella and improve shelf life. Post-process temperature abuse of chicken leads to proliferation of existing bacteria, including Salmonella, which can lead to the increased risk of human infections. While models predicting Salmonella growth at abusive temperatures are developed using sterile media or chicken slurry, there are limited studies of Salmonella growth in the presence of background microflora at 4–10°C. Experiments in this study were conducted to determine the growth of Salmonella Typhimurium and Heidelberg at 4–10°C in brain heart infusion broth (BHI) and non-sterile chicken slurry (CS). Nalidixic acid–resistant Salmonella Typhimurium and S. Heidelberg (3 log CFU/mL) were inoculated separately in CS and sterile BHI in a 12-well microtiter plate and incubated at 4°C, 7°C, and 10°C, following which samples were taken every 24 h for up to 6 days. Samples from each well (n=5) were spread plated on XLT4 agar+nalidixic acid and incubated at 37°C for 24 h. Bacterial populations were reported as CFU/mL. No significant differences (p>0.05) were observed in the survival of both strains in CS and BHI over the period of 6 days at all temperatures except S. Heidelberg at 7°C. Survival populations of both strains at 4°C were significantly different (p≤0.05) than at 7°C and 10°C in both media types. S. Heidelberg showed a maximum growth of 2 logs in BHI at 10°C among all the treatments. Growth patterns and survival of Salmonella at near refrigeration temperatures during carcass chilling can be useful to develop models to predict Salmonella growth post-processing and during storage, hence assisting processors in improving process controls.

Introduction

S almonella (nontyphoidal) is the leading cause of hospitalization (35%) and deaths (28%) related to foodborne infections in the United States (Scallan et al., 2011). Foods such as raw poultry and eggs are implicated in foodborne illnesses and are reported as a significant source of Salmonella. The United States Department of Agriculture (USDA) regulations for reducing the risk of Salmonella from these products, such as the title 9 of the Code of Federal Register (CFR) 381.66, are aimed at preservation and controlling Salmonella in poultry and mandates slaughtered chicken to be cooled at or below 40°F (approximately 4°C) before it leaves the chiller. This regulation requires processors to chill chicken carcasses weighing up to 4 lbs., 4–8 lbs., and above 8 lbs. within 4, 6, and 8 h of slaughter, respectively. Implementation of this regulation requires tremendous efforts in terms of time and energy to cool the carcasses, ultimately reducing throughput and increasing costs. Poultry processors are interested in increasing their production and reducing costs for carcass chilling via reducing residence time of carcasses in the chiller. Taking these hurdles into consideration, the Salmonella Initiative Program (SIP) of the Food Safety and Inspection Service (FSIS) has suggested a waiver to this CFR for those processors providing an effective alternative to time-temperature regulation which can control Salmonella as per regulations (USDA-FSIS, 2007; Curtis, 2009).

Chilling and refrigerated storage alone at 4–8°C has shown to inhibit the growth of Salmonella Typhimurium on chicken (Oscar, 2011). In addition to chilling, antimicrobial interventions such as electrolyzed oxidizing water, chlorine, acetic acid, trisodium phosphate, peracetic acid, and sodium hypochlorite as sprays or in chiller are effectively used against Salmonella during poultry processing (Fabrizio et al., 2002; Bauermeister et al., 2008). Other novel intervention strategies such as high-pressure treatment (O'Brien and Marshall, 1996; Morales et al., 2009), gamma radiation (Thayer et al., 1992), and ultraviolet radiation (Wallner-Pedleton et al., 1994) have been effective in reducing Salmonella in poultry. While it has been well documented that Salmonella cannot grow at temperatures of 4–8°C on chicken and that antimicrobials or other interventions can reduce the pathogen, further investigations are needed to provide an optimum chilling temperature above 4°C without changing the growth rate of Salmonella, hence maintaining shelf-life, increasing number of chickens processed, and reducing processing cost. Thus, it is important to study the growth patterns of different serovars of Salmonella at or near chilling temperatures of 4°C without antimicrobials.

Growth patterns of Salmonella serovars at various time-temperature combinations, pH, salt concentrations, water activity, and antimicrobials have been studied (Basti and Razavilar, 2004; Oscar, 1999, 2005) in sterile media. Although the results from these studies are well documented, studies on growth patterns of Salmonella spp. on chicken matrix with background microflora are warranted. Additionally, spoilage microorganisms on refrigerated chicken such as Pseudomonas spp., lactobacilli, Enterobacteriaceae, and yeast (Hinton et al., 2007; Jimenez et al., 1997) can interfere with the growth of Salmonella and can give different pathogen growth predictions in situ. Therefore, the current study was undertaken to study the in vitro survival and growth patterns of Salmonella in sterile media as compared to non-sterile chicken slurry with background microflora and validating this on chicken carcasses without any antimicrobial treatment. Our objective was to study the survival and growth patterns of Salmonella Typhimurium and Heidelberg at near chilling temperatures (4°C) to support alternatives to waive 9 CFR 381.66.

Methods

Bacterial cultures

Nalidixic acid–resistant (NAL; 60 ppm) serotypes, Salmonella Typhimurium and Heidelberg (obtained from Nelson Cox, USDA-ARS, Athens, GA) were cultured separately in Brain Heart Infusion (BHI; Acumedia, MI) broth at 37°C for 18 h to obtain a population of ∼8 log CFU/mL. These cultures were then serially diluted in 0.1% peptone water (Acumedia, MI) to get a final population of ∼5 log CFU/mL that was used to inoculate either BHI or chicken slurry.

In vitro study

Chicken slurry

Refrigerated (4°C) ground chicken was stomached with sterile peptone water (4°C) in the ratio of 1:3 for 30 s. The homogenate was then centrifuged (Sorvall Legend RT+Centrifuge, Thermo Scientific) at 860×g (2000 rpm) for 10 min, supernatant pooled in sterile flasks, and stored at 4°C, 7°C, or 10°C before inoculation.

Inoculation and sample distribution

Chicken slurry and BHI flasks were separately inoculated with 1 mL of either Salmonella Typhimurium or Heidelberg suspensions to get a final concentration of ∼3.5 log CFU/mL. The inoculated medium was introduced in a 12-well microtiter plate (Falcon®, Becton Dickinson Labware, NJ) with five wells each filled with 5 mL of chicken slurry or BHI. Non-inoculated chicken slurry and BHI were introduced in the remaining two wells as controls. Microtiter plates were then incubated at 4°C, 7°C, or 10°C for up to 6 days.

Microbiological analysis

Samples were collected every day for up to 6 days from each growth medium and temperature. One microtiter plate from each temperature for S. Typhimurium and S. Heidelberg was removed at each sampling time; contents of each well were mixed thoroughly using a pipette before collecting the samples (1.0 mL), serially diluted, and spread-plated onto Xylose Lysine Tergitol4 (XLT4; Acumedia, MI)+nalidixic acid (NAL 60 ppm; Sigma Aldrich, St. Louis, MO) agar for the recovery of NAL resistant marker Salmonella serotypes and plate count agar (PCA; Acumedia, MI) to determine the changes in the total aerobic plate counts (APC). The agar plates were incubated at 37°C for 24 h, and viable colonies counted were reported as log CFU/mL.

In vivo study

Chicken carcasses

Freshly processed chilled chicken carcasses without any antimicrobial treatment were procured from the Auburn University Poultry Research Unit. The carcasses were maintained under refrigeration (4°C) until inoculation.

Inoculation of carcasses

Based on results from the previous study suggesting no significant differences (p>0.05) between the survival patterns of S. Typhimurium and S. Heidelberg in BHI or chicken slurry, carcass inoculations were performed with S. Typhimurium. Inoculation was performed within 1 h of procurement by spraying individual carcasses with S. Typhimurium (∼5 log CFU/mL) in a laminar flow biosafety cabinet (NuAire^™ Biological Safety Cabinets, NuAire Laboratory Equipment Supply, MN). The breast, backside, left, and the right wings of the carcasses were sprayed using a garden spray bottle with a total volume of 10 mL. These inoculated carcasses were then vacuum-packed in sterile carcass rinse bags (390×520 cm; Nasco, WI) and incubated at either 4°C or 10°C for up to 6 days.

Microbiological analysis

Carcasses (n=3) were removed from each incubation temperature every day for up to 6 days and rinsed as per the USDA-FSIS method (USDA-FSIS, 2011). In short, the carcasses were taken out of the incubator; 400 mL of 0.1% peptone water was added as a diluent to each bag and rinsed thoroughly for 1 min. The rinsate (approximately 350 mL) was recovered in sterile flasks from which 1 mL of sample was taken for serial dilutions and 0.1 mL was spread plated on XLT4+NAL (60 ppm) agar followed by incubation at 37°C for 24 h to estimate the concentration of S. Typhimurium marker strains on the carcass. Samples were also plated on plate count agar to determine the total aerobic plate counts. After incubation, viable colonies were counted and reported as log₁₀ CFU/mL of rinsate.

Statistical analysis

Survival and growth patterns in laboratory media (BHI) and meat model (chicken slurry) are reported as an average of two replications with five sub-samples within each replication. All experiments with carcasses (in vivo) were conducted in triplicate with counts being reported as an average of three sub-samples within each replication. Analysis of variance (with temperature of growth and growth media as independent variables) was conducted followed by Tukey's multiple comparison test using SAS statistical software (SAS Institute Inc., Gary, NC) and significant differences were reported at p≤0.05.

Results and Discussion

Salmonella growth patterns affected by background microflora

When comparing survival of S. Heidelberg and Typhimurium at 4°C, 7°C, and 10°C over a 6-day storage period, results suggested that both these serovars survived better in sterile medium (BHI) as compared to the non-sterile medium (chicken slurry) irrespective of storage temperatures (data not shown). This could be due to the presence of background microflora in the non-sterile growth medium (chicken slurry) that can potentially suppress the growth of Salmonella. Aerobic plate counts of the chicken slurry inoculated with S. Typhimurium and Heidelberg were not different (p>0.05; data not shown); therefore, data only from the APCs of non-inoculated chicken slurry and chicken slurry inoculated with S. Typhimurium are shown in Figure 1A,B. The results indicated that background microflora significantly (p≤0.05) affected the survival of Salmonella serovars at 4°C, 7°C, and 10°C. Studies have suggested that background microflora consists of organisms that can support or suppress the growth of Salmonella to various extents. Microorganisms exhibit antagonistic behavior by lowering pH, excretion of antimicrobial peptides or metabolites like ammonia and nutrient competition. Some microorganisms like yeast and lactic acid bacteria have antagonistic effect against Salmonella in pork stored under modified atmosphere (Liu et al., 2006). Specific spoilage organisms like Pseudomonas fluorescence can produce siderophores and have a competitive edge to sequester iron leading to suppression of Salmonella growth (Cheng et al., 1995; Gram et al., 2002; Liu et al., 2006). Similarly, Birk et al. (2011) reported that the background microflora in non-sterile pork extended the generation time of Salmonella by twofold than the sterile samples. Comparatively, Szczawinska et al. (1991) found minor effect of microflora on Salmonella growth in mechanically deboned chicken (MDC) at 10°C and further suggested in their study that removal of competing microorganisms can support Salmonella growth.

FIG. 1.

Aerobic plate counts (log CFU/mL) of non-inoculated chicken slurry (A) and chicken slurry inoculated with Salmonella Typhimurium (B) incubated at 4°C (●), 7°C (▪), and 10°C (▴) for 6 days.

Salmonella growth was significantly (p≤0.05) suppressed as temperature increased to 10°C corresponding to significant (p≤0.05) increase in aerobic plate counts (APC) of chicken slurry at 10°C during 6 days of incubation as compared to 4°C and 7°C (Figs. 1 and 2). Aerobic plate counts increased from approximately 5 to 9 log CFU/mL in chicken slurry incubated at 10°C within 6 days competing with Salmonella in chicken slurry while serovars in sterile BHI grew unabated. Spoilage microflora of chicken is diverse consisting of Pseudomonads, Coliforms, Yeast, and Molds and can change with processing parameters and storage (Freeman et al., 1976). The composition as well as the total counts of the spoilage microflora affects the growth of Salmonella in chicken (Becker et al., 1987).

FIG. 2.

Survival patterns (log CFU/mL) of Salmonella Heidelberg (A) and Salmonella Typhimurium (B) in blood heart infusion (BHI) (solid symbols) and chicken slurry (open symbols) incubated at 4°C (O), 7°C (□), and 10°C (Δ) for 0–6 days.

Several Salmonella growth models have been developed in sterile broth. Koutsoumanis et al. (1998) modeled antimicrobial efficacy of different combinations of oleuropein, temperature, and pH on the growth of S. Enteritidis in BHI. Similarly, Oscar (1999) developed response surface models in BHI to understand the growth characteristics of S. Typhimurium at various temperature-pH combinations. Although broths give the most optimum conditions for bacterial propagation, they are not a true indicator of the environments that an actual food system can present for bacterial behavior. Salmonella is a major pathogen in poultry mainly composed of water (approximately 70%), fat (approximately 10%), and protein (approximately 18%) (Saucier et al., 2000). This food matrix can affect the survival and growth of bacteria (Ahmed et al., 1995). Leuschner and Samparini (2002) found that the antimicrobial activity of garlic against S. Enteritidis was faster in broth as compared to the food system (mayonnaise) due to the steric hindrance posed by food components. Oscar (2007) suggested that growth models should be developed in medium with natural microflora since they can give reliable predictions as compared to the ones developed using sterile medium. Similar inferences can be drawn from the present observations which reiterate the importance of studying growth patterns of Salmonella in its respective food matrix with competitive microflora.

Survival and growth patterns of Salmonella spp. in BHI and chicken slurry

In vitro growth patterns of Salmonella Heidelberg and Typhimurium inoculated in BHI and chicken slurry incubated at 4°C, 7°C, and 10°C for 6 days are shown in Figure 2A,B. Survival populations of both S. Heidelberg and Typhimurium in BHI and chicken slurry were significantly lower (p≤0.05) at 4°C and 7°C as compared to 10°C over the 6-day storage period. Furthermore, populations of both these serovars were significantly lower (p≤0.05) on days 5 and 6 of the storage period at 4°C as compared to 7°C in BHI, whereas no differences (p>0.05) were observed in the chicken slurry. Populations of S. Typhimurium and Heidelberg increased (p≤0.05) by 1.98 and 1.83 log CFU/mL, respectively, from day 0 to 6 at 10°C in BHI, whereas their populations significantly (p≤0.05) decreased by 1.43 and 1.59 log CFU/mL at 4°C, and 0.43 and 0.37 log CFU/mL at 7°C, respectively. In contrast to BHI, growth in chicken slurry was lower (p≤0.05): 0.95 and 0.70 log CFU/mL for S. Typhimurium and Heidelberg, respectively, at 10°C after 6 days of storage indicating the importance of background microflora while determining the growth patterns of Salmonella. Both serovars exhibited minimal growth at 10°C from 0-2 days in both media. Slightly lower growth was observed in this study as compared to approximately 1.1 and 2 log on the 6^th and 11^th day of incubation at 10°C reported by Oscar (2011) and Burnette and Yoon (2004), respectively, in chicken meat matrix. These differences can be attributed to the experimental conditions, growth medium, and probably strain differences. Observations similar to our study were made by Mansfield and Farakas (1980), who reported that various Salmonella serovars did not grow on meat at 7–8°C as compared to 10°C. Moreover, Matches and Liston (1972) showed that Salmonella cannot grow below 5.2°C, whereas Oscar (2009, 2011) did not observe any growth at 4–8°C. Supporting evidence of no growth at 8°C was reported by Zaher and Fujikawa (2011), who found an 8-h lag time at 12°C and a <1-h lag time at 16–32°C. Low temperatures are known to impact bacterial membrane fluidity, lowered diffusion rates, enzymatic reactions, and metabolism, leading to a reduced growth rate (Weber and Marahiel, 2003).

Although no significant (p>0.05) differences were observed in the survival patterns of Salmonella at 4°C and 7°C, our results suggest that these low temperatures are important factors in determining survival and growth of both serovars. However, it is not clear from the results whether nutrient content of sterile laboratory medium or the chicken slurry were important factors in determining survival and/or growth of both serovars. Moreover, similarities in growth patterns of S. Typhimurium and Heidelberg under all experimental conditions indicate that any one serovar can be used to validate the in vitro growth patterns in an in vivo system, which is in agreement with results reported by Oscar (2000).

Survival and growth patterns of Salmonella on chicken carcass

Based on the results from in vitro studies, where no significant differences (p>0.05) were observed in the Salmonella growth patterns at 4°C and 7°C, an experiment was conducted to determine the growth patterns of S. Typhimurium at 4°C and 10°C on raw chicken carcasses that were not treated with any antimicrobials (Fig. 3). Growth patterns of S. Typhimurium inoculated on carcasses followed similar patterns as the in vitro experiments with a significant (p≤0.05) increase (approximately 2.26 log) in pathogen numbers at 10°C, whereas there was no (p≥0.05) growth at 4°C. This reiterates the fact that Salmonella cannot grow at 4°C either in laboratory medium, in chicken slurry, or in whole carcasses. Although the increase in Salmonella populations on chicken carcasses was higher than in chicken slurry at 10°C for 6 days, statistical analysis indicated BHI as the best medium, followed by chicken slurry and then carcasses for Salmonella growth. As mentioned previously, BHI is a well-defined medium, whereas chicken slurry was a mixture of proteins, fat, carbohydrates, and other biochemical components in the form of fine particles supporting the growth of bacteria. On the other hand, chicken carcasses with skin-on is a nutrient-rich medium, but is not as readily available as BHI or the chicken slurry. These differences in medium probably are responsible for the observed variation in growth patterns, suggesting that growth models should be validated on actual food matrices for accurate predictions.

FIG. 3.

Survival patterns of Salmonella Typhimurium (solid symbols) and spoilage microflora (open symbols) on raw chicken carcasses stored at 4°C (O) and 10°C (Δ) for 0–6 days.

Although title 9 CFR 381.66 states that poultry should be cooled at or below 4°C post-chill to prevent Salmonella growth, our observations indicate that S. Typhimurium and Heidelberg do not grow at 4–7°C. These findings support alternative time-temperature combinations as a waiver to the guidelines provided in title 9 CFR 381.66. Hence, processors should be able to chill chicken carcasses at 4–7°C along with the use of Salmonella intervention strategies after careful in-plant validations and still comply with the USDA-FSIS regulations. Such efforts can decrease energy spent on chilling, reduce holding time of the carcasses, and increase throughput and profitability of the slaughter operation.

Footnotes

Disclosure Statement

No competing financial interests exist.

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