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Poult Sci 2008. 87:2208-2214. doi:10.3382/ps.2008-00153
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

The Impact of Temperature During the Storage of Table Eggs on the Viability of Salmonella enterica Serovars Enteritidis and Virchow in the Eggs

A. Lublin*,1 and S. Sela{dagger}

* Division of Avian & Fish Diseases, Kimron Veterinary Institute, PO Box 12, Bet Dagan 50250, Israel; and {dagger} Department of Food Science, Institute of Technology and Storage of Agricultural Products, Agricultural Research Organization, PO Box 6, Bet Dagan 50250, Israel

1 Corresponding author: avishailublin{at}yahoo.com or lublina{at}int.gov.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Salmonellosis is a foodborne infection of major economic importance. Contamination of table eggs with Salmonella, especially Salmonella enterica serovar Enteritidis, is a major health concern worldwide. Recently, S. enterica serovar Virchow has emerged as a major pathogen in Israel, where it is among the 3 most prevalent serovars found in poultry and the second most prevalent serovar isolated from individuals with salmonellosis. Although there is ample knowledge regarding the role of S. enterica serovar Enteritidis in contamination of eggs, virtually nothing is known regarding the possible association of S. enterica serovar Virchow with table eggs. Therefore, our goal was to examine the capability of serovar Virchow to contaminate chicken eggs. Commercial table eggs were inoculated independently with serovar Enteritidis and with serovar Virchow cells at a concentration of 105 cfu/egg, either on the shell surface or by injection into the yolk. The numbers of live Salmonella cells on the shell and within the egg were determined at various time points. At both low (6°C) and room temperatures (25°C), S. enterica serovar Virchow was not detected on the eggshell after 2 wk, whereas S. enterica serovar Enteritidis could be detected only sporadically at 25°C. In contrast, within the eggs, S. enterica serovar Virchow survived for up to 6 wk at 6°C, and it multiplied up to 109 cfu/mL of egg content from 2 to 8 wk postinoculation at 25°C. In comparison, S. enterica serovar Enteritidis survived within the eggs up to 8 wk at 6°C and at 25°C. Our results suggest that in cold storage, serovar Virchow is able to persist for long periods (6 wk), and at room temperature, these bacteria can multiply within eggs and reach high concentrations. Therefore, eggs might be considered potential vectors for transmitting S. enterica serovar Virchow into the food chain.

Key Words: table egg • storage temperature • Salmonella enterica serovar Enteritidis • Salmonella enterica serovar Virchow • salmonellosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Salmonella infections are the most common food-borne illnesses transmitted from animals to humans, and epidemics of salmonellosis occur as a result of eating improperly cooked, contaminated foods, primarily eggs and egg products (Patrick et al., 2004; USDA-FSIS, 2005). In the United States, approximately 1 egg in every 20,000 is contaminated with the major egg-associated pathogen Salmonella enterica serovar Enteritidis (referred to henceforth as Salmonella Enteritidis; Guard-Petter, 2001; Rabsch et al., 2001; Ebel and Schlosser, 2002) and which has displaced the serovar Typhimurium as the primary cause of salmonellosis worldwide (Baumler et al., 2000). Table eggs are an important component of the human diet, and approximately 67 to 79 billion eggs are produced in the United States each year. Both serovars Enteritidis and Typhimurium are responsible for a high rate of hospitalization and death in the United States (Fierer and Swancutt, 2000; Schroeder et al., 2005) and in the European Union (Messens et al., 2005). In fact, in more than 88% of samples from patients with salmonellosis, the infective agent was identified as Salmonella Enteritidis (Durecko et al., 2004). In the United Kingdom, Salmonella was isolated from 3.4% of almost 17,000 sampled eggs between October 2002 and November 2004 (Little et al., 2007). In Israel, as the result of several strict steps taken by the veterinary services since 1994, such as the policy of periodic sampling of all breeding flocks and hatcheries, and destroying or treating those infected with Salmonella Enteritidis or Salmonella Typhimurium, the number of Salmonella Enteritidis isolations decreased in chickens from the peak of 954 in 1994 to only 15 in 2001. In laying breeding hen farms (consumption eggs), no Salmonella Enteritidis were detected in the last 4 yr of that survey (1997 to 2001). In a risk estimation conducted by the World Health Organization in Israel, it was found that the percentage of infected eggs with Salmonella Enteritidis in infected flocks was 0.03% in average; however, in more than 60% of flocks, the average was only 0.025% (E. Berman, Israeli Veterinary Services, Bet Dagan; personal communication).

There are 2 possible routes for Salmonella contamination of intact eggs: the trans-ovarian route (Messens et al., 2005) and the trans-shell route (Miyamoto et al., 1998). Whereas several other serovars have been isolated from eggshells, serovar Enteritidis has been isolated primarily from the contents of intact eggs (Saeed, 1998). Improper handling of eggs can cause secondary and cross-contamination of minimally processed foods; therefore, consumption of egg-containing products (ice cream, cream cakes, mayonnaise, etc.) and undercooked eggs may be dangerous.

Salmonella enterica serovar Virchow (referred to henceforth as Salmonella Virchow) emerged during the 1980s as one of the most virulent human Salmonellae in distinct places such as England, Scotland, Australia, Spain, and Israel (Bignardi and Khong, 1989; Usera et al., 1996, 1998; Martin et al., 2001; Weinberger et al., 2006). In Israel, serovar Virchow is 1 of the 3 Salmonella serovars most frequently isolated from human patients in the last 15 yr, accounting for 16 to 20% of nontyphoidal Salmonella illnesses and all stool isolates, and 27% of all blood isolates (Weinberger and Keller, 2005; Weinberger et al., 2006), and it is highly invasive in children (Weinberger and Keller, 2005). Since 1986, when it was first detected in food imported into Israel (E. Berman, Israeli Veterinary Services, Bet Dagan; personal communication), Salmonella Virchow’s prevalence has increased dramatically, characterized by parallel increases in humans and poultry (Figure 1Go). In poultry, its prevalence rose from less than 1% of all Salmonella isolations (approximately 1,500 cases) in 1986, to 15 to 25% of all isolations (approximately 2,700 cases) in 1993 and later, when it became one of the dominant serovars isolated in chickens and turkeys.


Figure 1
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  		Figure 1. Prevalence of Salmonella enterica serovar Virchow relative to all serovars in human and poultry in Israel from 1981 to 2001. Data gathered from annual reports of the Israeli Ministry of Health and the Veterinary Services (E. Berman, Israeli Veterinary Services; personal communication). Numbers represent percentages of all reported cases of Salmonella.

 
There is a lack of information on the zoonotic potential of Salmonella Virchow but several reports have documented its presence in eggs (Mrden et al., 1988; Suryana and Istiana, 1997) and the potential of eggs to disseminate Salmonella to humans (Maris et al., 1998; Coughlin et al., 2003).

In light of the detection of a similar increasing trend in the incidence of salmonellosis caused by Salmonella Virchow in both poultry and humans in Israel during the last 2 decades (Figure 1Go), it is possible that eggs are involved in the spread of this emerging pathogen in Israel also. Although considerable information is available regarding the capacity of Salmonella Enteritidis to survive in table eggs (Saeed, 1998; Gast and Holt, 2000; Cogan et al., 2001; Chen et al., 2005), nothing is known regarding the fate of Salmonella Virchow in eggs. The objective of the present study, therefore, was to determine the fate of Salmonella Virchow in eggs stored at room temperature (25°C) and in cold storage (6°C).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Salmonella Inoculation of Eggs

The Salmonella enterica serovars Enteritidis (phage type 4) and Virchow were clinical isolates received from the Israeli Salmonella Reference Center (Ministry of Health, Central Laboratories, Jerusalem, Israel). Salmonella serovars were cultured at 37°C in Oxoid Nutrient Broth No. 2, pH 7.3 (Oxoid Ltd., Basingstoke, UK) for 18 h and the number of colony-forming units was determined. The inoculation experiments for Salmonella included 3 groups of eggs treated as follows: 1) surface inoculation, 2) injection into egg yolk, 3) control (i.e., no inoculation). Fresh, unfertilized shell eggs, graded large, were purchased from a retailer and used in the experiments as is (i.e., unwashed). We intended to use 30 eggs for each serovar, but because of breaking of some eggs in the Salmonella Virchow experiment, we examined only 20 eggs with this serovar. Surface inoculation was performed by spreading 105 cfu in 50 µL of suspension over a premarked area (about 1 cm2) that had been disinfected with 70% ethanol, on the wide side of each egg. The surface inoculation was performed at average room temperature and relative humidity (approximately 25°C, 60 to 65% RH). Intraegg inoculation was performed by injecting 100 µL of culture containing 105 cfu into the egg yolk through a hole drilled in a disinfected area of the shell. After injection, the hole was sealed with contact glue. The inoculum dose that was used, 105 cfu/egg, was equivalent to approximately 2 x 103 cfu/mL of egg content.

Egg Storage and Sampling

In each experiment, half of the eggs were stored in a refrigerator (6°C, 90% RH) and half at room temperature (25°C, 63% RH). At each time point, starting immediately after inoculation (time 0) and at 2, 4, 6, and 8 wk postinoculation, 2 or 3 eggs per treatment were withdrawn for detection of Salmonella on the inoculated areas of the shell and in the egg contents.

Direct Enumeration of Salmonella

Salmonella deposited on the eggshell surface were counted by aseptically cutting a piece of contaminated shell about 1 cm2 in area and immersing it in 2 mL of buffered peptone water (BPW), pH 7.2 (Difco, Becton, Dickinson & Co., Franklin Lakes, NJ). The shell piece was vortexed at maximum speed in a Stir-Mixer (Tuttnauer Co., Jerusalem, Israel) for 2 min and stored at 4°C overnight to facilitate the release of attached bacteria. Serial decimal dilutions in saline were plated on brilliant green agar plates (BGA, CM329, Oxoid Ltd.) and incubated for 24 h at 37°C. Red colonies on BGA were suspected to be Salmonella, and the identity of putative Salmonella colonies was confirmed by the application of standard biochemical analysis and serogrouping to representative colonies from each plate.

Salmonella within the egg contents were determined by pouring the contents (i.e., yolk and albumen) into a sterile 50-mL beaker, and hand mixing thoroughly for 30 s. A 1-mL aliquot of the homogenized egg contents was serially diluted in saline and spread-plated on BGA to enumerate Salmonella, as described above.

Enrichment of Salmonella

Samples derived from shell-containing BPW as well as mixed egg contents were diluted 1:10 in 10 mL of tetrathionate-iodine brilliant green medium (Difco) and incubated for 18 h at 37°C. Parallel samples were also diluted 1:100 in 10 mL of Rappaport-Vassiliadis medium (Oxoid Ltd.) and incubated for 48 h at 42°C. Following incubation, samples from each of the enrichment media (tetrathionate-iodine brilliant green and Rappaport-Vassiliadis) were plated on BGA that had been incubated for 18 h at 37°C, and the presence of red colonies was recorded. In most cases, both enrichment media exhibited the same results. Biochemical and serogrouping tests were used to confirm the presence of Salmonella Enteritidis (serogroup D) or Virchow (serogroup C).

Statistical Analysis

Statistical analysis was performed by using SAS software (Alice, 1985). Statistical estimations included ANOVA to determine the influence of each of the independent variables (serovar, holding temperature, time after inoculation) on the log10 bacterial count, and to obtain the inter-holding temperature differences in each time point for each serovar, and the interserovar differences at each time point for each temperature. Groups were considered significantly different at P < 0.05.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Survival on Eggshells

The surfaces of eggs that were inoculated with Salmonella at a dosage of 105 cfu/egg had the survival of the pathogen evaluated subsequently at several time points. Storage conditions were 6 or 25°C. After storage for 2 wk at 25°C, Salmonella Enteritidis was recovered from only 1 eggshell of 3 eggs; the number of recovered Salmonella was 3.6 x 103 cfu/egg, and no further Salmonella cells were detected on eggshells during a follow-up period of 4 to 8 wk. No salmonellae were recovered from the eggshell at 6°C or in the control group at any of the time points. There was also no detection of Salmonella following the enrichment procedure. Salmonella Virchow was not detected on any of the eggshells at any of the time points, at either 6 or 25°C, nor was it detected in the control group or following enrichment.

Survival of S. enterica on eggshells was previously shown to be rare because the pathogen died rapidly during storage (Messens et al., 2005). Our present findings, that Salmonella Enteritidis was found just on one eggshell at 25°C after 2 wk, whereas Salmonella Virchow did not survive at all, suggest that both serovars are limited in their capacity to survive on dry eggshells. It should be noted, however, that Salmonella Enteritidis might survive for longer periods, at least 4 wk, on shells contaminated with feces, from which the pathogens obtain their required nutrients (Schoeni et al., 1995; Braun et al., 1999; Little et al., 2007); however, this was not the situation in our research.

Survival Inside Eggs

It is generally accepted that Salmonella contamination occurs either via trans-ovarian transmission (Humphrey et al., 1991; Guard-Petter, 2001) or by penetration of a surface-contaminated eggshell (Padron, 1990; Chen et al., 1996). To simulate yolk contamination, 105 cfu of Salmonella Enteritidis were injected directly into the yolk, which is the major site of bacterial growth within the egg (Cogan et al., 2001), and the eggs were stored at 6 or 25°C. Survival of Salmonella was determined by enumerating the Salmonella cells found in the egg content. The inoculated dose of 105 cfu per egg (i.e., approximately 2 x 103 cells/mL) is a model for environmental contamination of the eggs via penetration of bacteria after laying in the poultry house or during storage and is not intended to represent natural contamination of intact eggs from the layer, which occurs at lower levels. In previous studies, Neill et al. (1985) immersed eggs in suspensions of Salmonella Virchow cells at 107 to 108 cfu/mL to study penetration into the egg, Gast and Holt (2001) inoculated Salmonella Enteritidis cells at 105 cfu/mL into egg yolk, and Howard et al. (2006) inoculated Salmonella Typhimurium cells at 108 cfu/mL onto the vitelline membrane of eggs; in all 3 cases, multiplication within the egg was demonstrated.

The ANOVA revealed highly significant effects (P < 0.001) for each of the independent variables; that is, serovar, holding temperature, and time after inoculation. There was also a significant effect for the interactions of serovar x temperature x time, meaning that the combined effect of serovar and temperature on bacterial count varies according to postinoculation time.

The concentration of Salmonella Enteritidis increased by 1 order of magnitude (decimal log) during the first 2 wk of postinoculation storage at 6°C, after which it remained constant around 4 decimal logs for up to 8 wk. At 25°C, the bacterial concentration increased by 5 orders of magnitude by 4 wk postinoculation and remained at 8 to 9 logs until the end of the experiment (Figure 2aGo). This observation is in accordance with a report of Chen et al. (2005), who found that the Salmonella Enteritidis population on eggs inoculated with 106 cfu multiplied to a level of up to 1010 cfu/egg within a holding period of 4 wk at 22°C, and a report of Little et al. (2007), who found that eggs stored above 8°C were contaminated more frequently. The differences between concentrations of Salmonella Enteritidis at the 2 storage temperatures were significant between 4 and 8 wk postinoculation (see Table 1Go).


Figure 2
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  		Figure 2. Concentration (cfu/mL, log10 values) of Salmonella enterica serovars Enteritidis (a) and Virchow (b) in contents of eggs stored at 6 and 25°C following intraegg inoculation of broth (105 cfu/egg), up to 8 wk postinoculation. The data presented reflect the mean concentration ± standard deviation (log10 cfu/mL) found in each time point in 3 (Salmonella Enteritidis) or 2 (Salmonella Virchow) eggs. Noninoculated eggs remained free of Salmonella during the whole experiment.

 

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  		Table 1. Concentration of Salmonella (cfu/mL, log10 values) in contents of eggs stored at 6°C and 25°C following intraegg inoculation of broth (105 cfu/egg) up to 8 wk postinoculation, and statistical analyses of the effects of holding temperature (6 or 25°C) and serovar (Enteritidis, Virchow) on the bacterial count at each time point1
 
In contrast to Salmonella Enteritidis, the number of viable Salmonella Virchow cells decreased below 1 decimal log by 2 wk postinoculation at 6°C, and remained constant at this concentration until wk 6; thereafter, the number of viable Salmonella Virchow cells declined below the detection level (Figure 2bGo). At 8 wk, no Salmonella could be detected in eggs, even following enrichment. At 25°C, Salmonella Virchow concentration increased by 5 orders of magnitude immediately during the first 2 wk postinoculation, and then remained almost constant at a level of 8 to 9 log10 cfu up to 8 wk (Figure 2bGo). During the sampling period, similarly to Salmonella Enteritidis, the concentrations of Salmonella Virchow differed significantly between the 2 storage temperatures from 2 wk postinoculation onward (see Table 1Go). All uninfected control groups were free of Salmonella at any of the holding temperatures and at all time points postinoculation, indicating that the eggs were not contaminated naturally with Salmonella (data not shown). The P-values between each serovar and the uninfected control group were always P ≤ 0.001, except for serovar Enteritidis with P ≤ 0.01 for 6°C in wk 2 and 6, and P ≤ 0.05 for 6°C in wk 8, and serovar Virchow with P ≤ 0.01 for 25°C in wk 4, and nonsignificant differences for 6°C in wk 2, 6, and 8.

Comparison of the number of viable cells between the 2 serovars demonstrated different behavior in the 2 holding temperatures. At 25°C, the number of viable Salmonella Virchow cells was slightly but significantly greater than that of Salmonella Enteritidis at most time points. Conversely, at 6°C, the number of viable Salmonella Enteritidis cells was greater by 3 to 4 log10 cfu than that of Salmonella Virchow during the period between 2 and 8 wk postinoculation (Table 1Go).

Our results suggest that at 6°C Salmonella Enteritidis is more adapted to the egg yolk environment than Salmonella Virchow (Table 1Go). On the other hand, both serovars seem to be well adapted to this milieu at 25°C. Multiplication of Salmonella Enteritidis inside eggs has been reported by many researchers (Humphrey et al., 1991; Saeed and Koons, 1993; Gast and Holt, 2000, 2001; Cogan et al., 2001; Chen et al., 2005; Howard et al., 2006), but, as far as we know, this is the first report on multiplication of Salmonella Virchow inside eggs. Neill et al. (1985) demonstrated penetration of Salmonella Virchow into eggs through the eggshell, but they did not look for multiplication of the bacteria in the egg. At 2 wk postinoculation, Salmonella Virchow reached its maximal concentration of 109 cfu/mL and remained almost constant up to 8 wk postinoculation (Figure 2bGo), whereas serovar Enteritidis reached that concentration only after 4 wk (Figure 2aGo). The concentration of serovar Virchow inside the eggs stored at 25°C, except for 4 wk postinoculation, was slightly greater than that of Salmonella Enteritidis (Table 1Go). It seems that both serovars flourished in the egg yolk environment at 25°C. Similarly, others have also documented multiplication of various Salmonella serovars, including Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Heidelberg (Schoeni et al., 1995). Yang et al. (2001) inoculated several Salmonella serovars into steamed and scrambled eggs and held them at temperatures between 5 and 60°C: they found that Salmonella multiplied within 36 h at 22°C, but not at 5°C.

Penetration of Salmonella into Eggs

Salmonella serovars Enteritidis and Virchow were deposited on the surface of eggs and their presence in the egg contents was monitored at various periods at each of the 2 holding temperatures. No penetration of Salmonella cells through Salmonella Virchow-infected eggshells or into control (uninfected) eggs was observed up to 8 wk, in contrast to the work of Neill et al. (1985). In contrast to Salmonella Virchow, when the shells were infected with Salmonella Enteritidis among eggs stored at 25°C but not in those stored at 6°C, 1 egg out of 3 was found to harbor Salmonella Enteritidis after 2 wk of postinoculation storage, but subsequently no Salmonella cells could be found within the eggs. Our finding might suggest, with caution because of the low number of eggs per treatment, that the penetration of Salmonella Enteritidis into eggs is sporadic in nature. It should be noted that other factors such as rate of change of temperature and cleanliness of the eggs might influence pathogen penetration through the shell. Furthermore, contamination of egg contents with Salmonella enteritidis can occur via other routes such as the trans-ovarian route. Presently, it is not known if the same applies to Salmonella Virchow.

Himathongkham et al. (1999) showed that experimental contamination of the surface of eggshells by dipping the eggs in a culture of Salmonella Enteritidis did not result in the presence of Salmonella Enteritidis in the egg contents. In contrast to that, Schoeni et al. (1995) demonstrated penetration of Salmonella Enteritidis through shells and their membranes at a storage temperature of 25°C. Thus, our present finding of penetration in one egg may be consistent with those of Schoeni et al. (1995) and Braun et al. (1999), who demonstrated increased penetration of Salmonella with increasing temperature; however, this may be coincidental.

Our findings extend the knowledge regarding the fate of the emergent serovar Virchow in table eggs, and raise a safety concern regarding the current national health regulations in Israel (from 1994) for storage conditions of table eggs. According to these regulations, eggs may be stored for up to 3 mo in a refrigerator and for up to 16 d at room temperature. However, the Advisory Committee on the Microbiological Safety of Foods in the United Kingdom limits the storage period before table eggs are consumed to 3 wk at 8°C (Kinderlerer, 1994). In the present study, we found that Salmonella Virchow persisted for at least 6 wk under refrigeration at 6°C, but it should be remembered that at lower inocula such as those occurring in naturally contaminated eggs the persistence of Salmonella might be much shorter. In the case of Salmonella Enteritidis, we found that cell numbers remained constant throughout 8 wk of storage at 6°C following a slight increase in the first 2 wk, and similar findings were reported by Gast and Holt (2000).

In conclusion, serovar Virchow, like serovar Enteritidis, is capable of multiplication to large numbers in table eggs stored at room temperature. However, in cold storage (and unlike serovar Enteritidis) serovar Virchow can survive for an extended period up to 6 wk after which its concentration decreases below the detection level. Our results support the recommendation to keep eggs at a low holding temperature as a simple means to control eggborne salmonellosis.


   ACKNOWLEDGMENTS
 
We thank the Israeli Ministry of Health, Central Laboratories, Salmonella Reference Laboratory for providing the S. enterica serovars. This study was partially supported by a BARD grant (US-3949–06) awarded to SS.

Received for publication April 14, 2008. Accepted for publication June 16, 2008.


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 RESULTS AND DISCUSSION
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