Egg Penetration by Salmonella Typhimurium in Washed and Unwashed Eggs

Food borne illness costs Australia an estimated $1.2 billion per year (Hall et al., 2005). The annual report of the OzFoodnet network (2009) reported 9,533 cases of Salmonella infection. Although the eggs produced in Australia are of good quality, the egg industry is often blamed for cases of food poisoning due to Salmonellosis. Salmonellosis can be acquired by the ingestion of raw or undercooked eggs. As well, cross contamination of ready to eat meals with Salmonella also plays major role in food poisoning cases. Intact eggs can be contaminated by Salmonella by either vertical (which is common in case of Salmonella Enteritidis) or horizontal transmission. Salmonella Enteritidis, which is of major concern for the food industry worldwide, is not prevalent in Australian layer flocks. According to Humphrey (1994), horizontal transmission is the most common route for Salmonellae other than Salmonella Enteritidis. The eggshell can be contaminated by any surface with which the egg comes in contact such as faeces, water, caging material, nesting material, insects, hands, broken eggs, dust on egg belt, blood and soil (Board and Tranter, 1995; Davies and Breslin, 2003). Egg washing is used to reduce eggshell contamination in many countries such as the United States, Australia and Japan (Hutchison et al., 2004). Egg washing can reduce the microbial load on the eggshell surface (Messens et al., 2011) and thus may lower the rate of horizontal transmission of Salmonella across the eggshell. However, egg washing chemicals may affect the cuticle of the eggshell (Wang and Slavik, 1998). Hence, benefits and losses due to egg washing are under debate. As ST is the most common serovar notified in Salmonellosis food poisoning casesin Australia (Oz Foodnet, 2009), the objectives of current study were to examine the effect of egg washing on the survival of Salmonella on the eggshell surface, to investigate the penetration ability of different ST PTs and to study the effect of egg washing on bacterial eggshell penetration. All PT used in this study have been reported in egg product related Salmonella poisoning cases in Australia. 

In the present study, the eggshell penetration ability of four isolates ST, each of a different PT (PT 9, 44, 170 and 193) was investigated. All ofthe isolates were from Australian layer farms and were obtained from the Australian Salmonella Reference Centre, Adelaide. For each PT, 90 eggs were collected from hens in early lay. These eggs were divided into two groups: washed (30 eggs) and unwashed (60 eggs). For egg washing, a commercial detergent (a hydroxide and hypochlorite based washer used at concentration of 0.45% (v/v) which equates to a pH of ~12 and ~200 ppm hypochlorite in the working solution) and the compatible sanitiser (at concentration of 0.16% (v/v) which equates to ~200 ppm hypochlorite in the working solution) were used. The pressure of the sprays was 3 pounds per square inch (psi) without brushes. Eggs (90) were divided into 9 different treatment groups based on washing, dose of infection and incubation temperature. All eggs were dipped in 70% ethanol for 30 sec in order to kill bacteria present on the eggshell surface. After drying, each egg was dipped in a solution containing PBS (control), 10³ Colony forming units (CFU)/ml or 105 CFU/ml dose of ST for 90 sec followed by incubation at either 20° C or 37° C for 21 days. After incubation, the survivals of Salmonellae on the eggshells were studied; egg was put in a whirl-pak bag containing 10 ml of BPW (Oxoid, Australia) and was massaged for 1 min. A 10 ul aliquot of the BPW was plated onto XLD plates and the plates were incubated at 37° C overnight. Next day, the colonies on the plate were observed for Salmonella. Similarly, to investigate the penetration and survival of Salmonella in internal contents, a 2 ml aliquot of the internal contents wastransferred into 8 ml of BPW and 10 ul of this BPW was plated on XLD plates and the plates were incubated at 37° C overnight. After incubation, the colonies on the plate were observed for red colonies with a black centre of Salmonella. Finally, statistical analysis was conducted using Fisher exact test using Graph pad software. 

In the first experiment, all the controls worked well as all the eggshell surfaces and internal contents of theses control group eggs were found negative for Salmonella. Results indicated that ST PT 44 (at 10⁵ CFU) penetration in washed eggs was significantly higher compared to unwashed eggs (Table 1). However, in the cases of other ST PTs (PT 9, 170 and 193), there were no significant difference in the Salmonella penetration of washed and unwashed eggs. At 20° C, in total, 20% (16/80) of the washed eggs and 10% (8/80) of the unwashed eggs were penetrated. Our results also indicated that the internal contents of whole eggs were contaminated by ST PT 44 (23.33%) followed by PT 170 (20%), PT 9 (10 %) and PT 193 (10 %). As the penetration of bacteria across the eggshell is dependent on the survival of bacteria on the eggshell, we compared the survival of ST PT on the eggshell surface of washed and unwashed eggs; however, there was no significant difference in Salmonella survival on the egg shell surface of washed and unwashed eggs (data not shown). Survival rate on eggshell surface was highest for ST PT 9 (83.33%) followed by ST PT 44 (53.33%), ST PT 193 (43.33%) and ST PT 170 (43.33%). It was also found that there was no significant effect of incubation temperature (20° C and 37° C) on the penetration ability of any of the ST PT used in this study (data not shown). 

In Australia, there is limited information available regarding the survival ability of different ST PTs on the eggshell surfaces and egg penetration ability of these phage types and also the effect of egg washing on Salmonella penetration. The present study showed that; if eggs are stored at 20°C or 37°C, ST can survive on the eggshells for three weeks following contamination. This finding underlines the importance of proper storage, careful handling of eggs in the food industry and the domestic environment. These findings are in agreement with De Reu et al. (2006) who found a high survival rate of Salmonella Enteritidis on the eggshell surfaces 21 days after infection. They also reported that cuticle deposition and specific gravity may have an impact on the survival of bacteria on the eggshell surfaces. In order to study the egg penetration ability of ST PT, a more holistic approach was used in the current study as compared to some other studies where Salmonella culture was artificially inoculated into the albumen using a syringe. Results indicated that all phage types used in the present study are capable of penetrating the eggshells and are also able to survive in the egg albumen which is considered to be a hostile environment for the survival of bacteria. In the current study, it was found that ST PT 44 penetration wassignificantly higher in washed eggs than unwashed eggs, which could be due to damage to the cuticle by egg washing chemicals. However, to draw concrete conclusions, it is essential to conduct more experiments to investigate the effect of egg washing on the ultrastructure of the cuticle. Findings in the present study are in agreement with earlier findings by Wang and Slavik (1998) who reported that bacterial invasion was increased as a result of alteration in the eggshell surface due to the washing chemical alkaline sodium carbonate. Although Leleu et al. (2011) reported that egg washing had no effect on cuticle quality, eggs used in their study were from old hens and cuticle thickness decreases with increasing hen age (EFSA 2005). Other ST PTs (PT 9, 170 and 193) penetrated washed and unwashed eggs equally. There was no significant effect of storage temperature (20° C and 37° C) on penetration of ST across the eggshell. A good quality eggshell protects the internal contents from bacterial penetration. A cracked or damaged egg encourages bacteria to move across the eggshell and into the contents which is likely to increase the risk of food poisoning. Hence, in order to study the effect of eggshell quality on bacterial penetration, an agar penetration approach will be used for all of these ST PT in further studies. In the present study, only one isolate per PT was used, hence further investigation using multiple isolates of same PT is essential in order to confirm the variation in penetration ability of different phage types.

Table 1 - Whole egg penetration by different SalmonellaTyphimurium phage types: comparison between washed and unwashed egg at 20° C

Figure 1 - Percent eggshell surface survival and egg internal content contamination by various Salmonella Typhimurium phage types

 

ACKNOWLEDGEMENT: This research was conducted within the Poultry CRC, established and supported under the Australian Government's Cooperative Research Centres Program. Salmonella isolates were obtained from Ms. Diane Davos, Australian Salmonella Reference Centre, Adelaide. 

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