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Poult Sci 2007. 86:1431-1435
© 2007 Poultry Science Association
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PROCESSING, PRODUCTS, AND FOOD SAFETY

In Vitro Penetration of Egg Yolks by Salmonella Enteritidis and Salmonella Heidelberg Strains During Thirty-Six-Hour Ambient Temperature Storage

R. K. Gast1, R. Guraya, J. Guard-Bouldin and P. S. Holt

Egg Safety and Quality Research Unit, Russell Research Center, Agricultural Research Service, USDA, Athens, Georgia 30605

1 Corresponding author: Richard.Gast{at}ars.usda.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Although Salmonella deposition inside yolks is uncommon in naturally contaminated eggs, migration through the vitelline membrane into the nutrient-rich yolk contents could enable rapid bacterial multiplication. Egg refrigeration restricts both penetration and growth, but a recently proposed national Salmonella Enteritidis control program would allow unrefrigerated ambient temperature storage of eggs on farms for up to 36 h. The present study used an in vitro egg contamination model to assess the ability of small numbers of 4 Salmonella Enteritidis strains and 4 Salmonella Heidelberg strains to penetrate the vitelline membrane and multiply inside yolks during 36 h of storage at either 20 or 30°C. After inoculation onto the exterior surface of the vitelline membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean log10 bacterial concentration of 2.2 cfu/mL) during incubation at 30°C. Significant differences in penetration frequency and yolk multiplication were observed between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20°C. These results demonstrate that controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness.

Key Words: Salmonella Enteritidis • Salmonella Heidelberg • yolk • vitelline membrane • penetration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Attributed principally to contaminated eggs, human illness caused by Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) has been an important international public health concern for more than 20 yr (Gillespie et al., 2005; Braden, 2006). Although the prevalence of Salmonella Enteritidis in commercially produced table eggs in the United States has been estimated to be only 1 in 20,000 (Ebel and Schlosser, 2000), USDA calculations have suggested that 182,060 human illnesses were transmitted by contaminated eggs in 2000 (Schroeder et al., 2005). State governments and the egg industry have already made significant commitments of resources to testing and quality assurance programs for controlling Salmonella Enteritidis (Mumma et al., 2004), and a national regulatory plan for commercial egg-laying flocks has been proposed (US Food and Drug Administration, 2004). The epidemiology of foodborne salmonellosis has been further complicated by the recent implication of Salmonella enterica serovar Heidelberg (Salmonella Heidelberg) as another significant egg-associated pathogen (Chittick et al., 2006). Both Salmonella Enteritidis and Salmonella Heidelberg are deposited inside eggs after invading to the reproductive tissues of infected hens (Gast et al., 2004).

Freshly laid eggs are typically reported to contain no more than a few hundred Salmonella cells (Gast and Holt, 2000a; Chen et al., 2002), so prompt refrigeration to internal temperatures of 7.2°C or lower protects consumers by preventing bacterial multiplication to more dangerous levels inside eggs during storage. Infected hens can deposit Salmonella in either the yolk or albumen of developing eggs because of the colonization of different regions of the reproductive tract (Bichler et al., 1996; Gast and Holt, 2000a; Gast et al., 2007). Although Salmonella can survive or slowly multiply in egg albumen (Chen et al., 2005; Guan et al., 2006; Kang et al., 2006), rapid multiplication occurs in egg yolk (Humphrey and Whitehead, 1993; Braun and Fehlhaber, 1995; Gast and Holt, 2000b). Accordingly, egg refrigeration must achieve growth-inhibiting internal temperatures more rapidly when bacteria are present in the yolk than when they are found in the albumen. Because high levels of contaminants have seldom been reported in freshly laid eggs, initial Salmonella deposition inside nutrient-rich yolks appears to be relatively uncommon. In a study by Gast and Holt (2001a) of eggs from experimentally infected hens, Salmonella Enteritidis was isolated far more often in association with the yolk (vitelline) membrane than with the interior contents of the yolk. However, even if Salmonella deposition occurs on the exterior surface of the yolk membrane or in the adjacent albumen, migration through that membrane to reach the yolk contents could then allow rapid and extensive bacterial multiplication (Braun and Fehlhaber, 1995; Gast and Holt, 2000b; Gast et al., 2005; Murase et al., 2005). Declining vitelline membrane integrity during nonrefrigerated storage could promote this process (Chen et al., 2005).

The proposed national Salmonella Enteritidis control plan for commercial egg producers in the United States would require refrigeration of eggs that are held on farms for more than 36 h but would allow unrefrigerated storage of eggs for shorter periods of time (US Food and Drug Administration, 2004). Previous research has not clearly indicated whether Salmonella strains commonly found in eggs are likely to penetrate through yolk membranes and begin multiplying inside egg yolks during 36 h of unrefrigerated storage. The objective of the present study was to determine whether small numbers of 4 strains of Salmonella Enteritidis and 4 strains of Salmonella Heidelberg were able to migrate through the yolk membrane to reach the yolk contents during 36 h of incubation at either 20 or 30 °C in an in vitro egg contamination model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Preparation of Salmonella Enteritidis and Salmonella Heidelberg Cultures
Eight Salmonella cultures were resuscitated by transfer into tryptone soy broth (Oxoid Ltd., Basingstoke, Hampshire, UK) for 2 successive cycles of 24-h incubation at 37°C. These cultures included 4 Salmonella Enteritidis isolates, designated as strains 6 (phage type 13a), 8 (phage type 8), 23 (phage type 14b), and 437 (phage type 4), and 4 Salmonella Heidelberg isolates, designated as strains 1, 4, 6, and 11. All Salmonella strains were originally isolated from contaminated eggs or from infected humans in egg-associated disease outbreaks. After cell numbers in the incubated cultures were estimated by determining their optical densities at 600 nm, further dilution in saline produced the desired final cell concentration (confirmed by subsequent plate counts).

Preparation, Inoculation, and Incubation of Egg Content Samples
Freshly collected eggs from the specific pathogen-free flock of Single Comb White Leghorn chickens at the Southeast Poultry Research Laboratory (Athens, GA) were aseptically broken, their contents (yolk and albumen) were separated, and each yolk was transferred into the bottom of a sterile 50-mL plastic centrifuge tube. Each yolk was then inoculated with Salmonella by using a pipette to dispense 0.1 mL of the appropriate broth culture (containing approximately 100 cfu) onto the exterior surface of the vitelline membrane. After the inoculated yolk samples were held for 5 min at room temperature (approximately 24°C), the albumen from a single egg was poured gently into each tube. Forty-eight egg samples were inoculated with each of the 8 Salmonella strains, and 8 uninoculated samples were retained as negative controls. After preparation, 24 egg samples inoculated with each culture were incubated for 36 h at 20°C, and the other 24 samples were incubated for 36 h at 30°C. Negative control samples were similarly divided between the 2 incubation temperatures.

Enumeration of Salmonella Enteritidis and Salmonella Heidelberg Inside Egg Yolks After Incubation
Each incubated egg sample was poured out into a sterile plastic petri dish. A small area of the yolk membrane was seared with a flame-heated steel spatula to destroy any surface bacteria present in that region. A sterile syringe was then inserted through the seared area of the membrane to remove 5 mL of interior yolk contents (free of membrane material), typically representing 35 to 40% of total yolk volume. The concentration of Salmonella Enteritidis or Salmonella Heidelberg in the yolk contents was determined by making 10-fold dilutions of each sample in 0.85% saline and spreading aliquots of each dilution onto plates of brilliant green agar (Becton, Dickinson, and Co., Sparks, MD). The agar plates were incubated for 24 h at 37°C, and typical Salmonella colonies were identified (Waltman et al., 1998) and counted. The detection threshold of this procedure was 10 cfu/mL.

Statistical Analysis
Significant differences (P < 0.05) between treatment groups in the frequency of isolation of Salmonella strains in the interior contents of yolk samples after incubation were determined by applying Fisher’s exact test, and significant differences (P < 0.05) between treatment groups in the mean concentration of Salmonella cells inside egg yolks after incubation were determined by applying the Kruskal-Wallis test. Data were analyzed using Instat biostatistics software (GraphPad Software, San Diego, CA).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Seven of the 8 Salmonella isolates used in this study were able to penetrate through the vitelline membrane to reach the yolk contents during 36 h of incubation of inoculated egg samples at 20°C and all isolates penetrated during incubation at 30°C (Table 1Go). Migration into yolks occurred infrequently at 20°C (in only 4.69% of all samples), and no significant differences (P < 0.05) between strains or serotypes were observed at this temperature. However, penetration to the yolk contents occurred significantly (P < 0.0001) more often at 30°C for both serotypes. Significant differences (P < 0.05) were observed between the frequencies of yolk membrane penetration at 30°C by individual Salmonella strains, with values ranging from 20.83 to 70.83%. The overall frequency of migration into yolks at 30°C by the 4 Salmonella Enteritidis isolates (56.25%) was significantly greater (P = 0.002) than by the 4 Salmonella Heidelberg isolates (29.17%). Similar trends were observed in the concentrations of Salmonella cells inside yolks after incubation. The overall mean base-10 logarithm Salmonella concentration inside yolks incubated at 20°C was only 0.120 cfu/mL, and no significant differences (P > 0.05) were evident between strains or serotypes. However, significantly higher (P < 0.0001) numbers of Salmonella cells of both serotypes were found within yolks after incubation at 30°C. Significant differences (P < 0.01) between individual strains were observed at this temperature, with mean base-10 logarithm bacterial concentrations in yolks after incubation ranging from 0.575 to 4.183 cfu/mL. The overall mean base-10 logarithm concentration of the 4 Salmonella Enteritidis strains inside yolks after 30°C incubation (3.165 cfu/mL) was significantly greater (P < 0.0001) than the corresponding value for the 4 Salmonella Heidelberg strains (1.292 cfu/mL). None of the uninoculated negative control samples were Salmonella positive after incubation.


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  		Table 1. Isolation and enumeration of Salmonella Enteritidis and Salmonella Heidelberg strains inside egg yolk samples1
 
Prompt refrigeration of eggs has been identified as a critical risk reduction practice for preventing the eggborne transmission of Salmonella to consumers (Hope et al., 2002). Mandatory egg refrigeration to an ambient temperature of 7.2°C is a common feature of most quality assurance or regulatory programs for shell egg production in the United States (Mumma et al., 2004; US Food and Drug Administration, 2004). The refrigeration of freshly laid eggs reduces opportunities for small populations of microbial pathogens to grow to more dangerous levels (Chen et al., 2002). The effectiveness of egg refrigeration for restricting bacterial multiplication is influenced by the initial level and location of bacterial contamination, the movement of bacteria or nutrients within eggs during storage, and the rate at which growth-limiting temperatures are achieved. Experimental infection studies have suggested that systemically infected laying hens are far more likely to deposit Salmonella in the albumen or on the outside of the vitelline membrane of their eggs than inside the nutrient-rich yolk contents (Gast and Holt, 2001a; Gast et al., 2003). Although slow bacterial multiplication may occur in the albumen or on the outside of the yolk membrane (Fleischman et al., 2003; Chen et al., 2005), migration into the yolk contents could allow rapid multiplication of Salmonella until refrigeration achieves growth-inhibiting internal temperatures.

Several in vitro egg contamination models have been used to demonstrate the migration of Salmonella through yolk membranes at warm temperatures (Humphrey and Whitehead, 1993; Braun and Fehlhaber, 1995; Gast and Holt, 2000b; Gast et al., 2005; Murase et al., 2005). Although bacterial penetration into the yolk contents has been observed at a very low frequency after as little as 2 h of incubation (Gast et al., 2006), the likelihood of penetration increases significantly when inoculated eggs are held for several days at warm temperatures (Guan et al., 2006; Murase et al., 2006). The relatively modest numbers of Salmonella Enteritidis cells detected inside the nutritionally abundant yolk after inoculated eggs were incubated for 24 h at 30°C in a previous study by Gast et al. (2005) indicated that migration into the yolk had proceeded rather slowly during the first day of storage. Likewise, the characteristically small numbers of cells reported inside freshly laid, naturally contaminated eggs suggest that rapid penetration into the yolk contents is uncommon (Humphrey et al., 1991). However, access to the yolk may become easier over time as albumen viscosity and vitelline membrane integrity decline, especially at elevated temperatures (Humphrey and Whitehead, 1993; Hara-Kudo et al., 2001; Messens et al., 2004). Refrigeration significantly reduces the movement of Salmonella Enteritidis across the vitelline membrane to reach the interior contents of egg yolks (Gast et al., 2006). Egg refrigeration has also been shown to promote better retention of yolk membrane integrity against physical rupture than what occurs at warmer temperatures (Chen et al., 2005).

The present study determined that Salmonella strains can sometimes differ significantly in their ability to migrate inside egg yolks and multiply. The higher propensity of the Salmonella Enteritidis isolates to penetrate yolks and grow within them is consistent with the epidemiological preeminence of this serotype as an egg-associated pathogen, but the small number of strains evaluated in this study precludes broad generalizations about differences between serotypes. Previous studies have also reported significant differences between individual Salmonella strains in their growth properties in eggs (Gast and Holt, 2001b; Cogan et al., 2004; Gast et al., 2005). Strains of Salmonella Enteritidis obtained from eggs have been reported to differ from isolates from other poultry sources in the composition of their surface lipopolysaccharide (Guard-Bouldin et al., 2004). Nevertheless, all 8 Salmonella Enteritidis and Salmonella Heidelberg strains tested in the present study penetrated through the vitelline membrane, and most strains grew to significantly higher numbers in the yolk contents during 36 h of incubation at 30°C. The recently proposed national Salmonella Enteritidis control plan for shell egg producers in the United States would permit eggs to be held on farms without refrigeration for up to 36 h (US Food and Drug Administration, 2004). However, this interval appears sufficient to enable Salmonella deposited outside yolks to gain access to yolk nutrients and multiply to much more dangerous levels if eggs are stored at very warm temperatures. The current study highlights the pivotal importance of prerefrigeration storage temperatures, because penetration and extensive multiplication were rare during incubation at 20°C. Although unrefrigerated storage of eggs for 36 h at moderate temperatures appears to pose only a relatively modest risk of supporting extensive bacterial growth, storage at warmer temperatures increases this risk very dramatically. Therefore, controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness.


   ACKNOWLEDGMENTS
 
We thank Otis R. Freeman for excellent technical assistance.

Received for publication December 14, 2006. Accepted for publication March 7, 2007.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
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