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Survival of inoculated Salmonella on the shell of hens' eggs and its potential significance
Food Control 28 (2012) 463e469
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Survival of inoculated Salmonella on the shell of hens’ eggs and its potential significance Pilar Botey-Saló, Amara Anyogu, Alan H. Varnam, Jane P. Sutherland* Microbiology Research Unit, School of Human Sciences, Faculty of Life Sciences, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2011 Received in revised form 8 May 2012 Accepted 15 May 2012
The aim of this research was to evaluate the risk of inoculated Salmonella persisting on the outside of the shell of hens’ eggs. Hens’ eggs were surface inoculated with a cocktail of Salmonella strains and stored for up to 54 days at 4, 10 and 20 C and at 80 and 90% relative humidity. Salmonella survival showed an irregular pattern, with extremes of high recovery and no recovery. However, salmonellae were always recovered after resuscitation. Monte Carlo simulation of different scenarios using relevant assumptions indicated that the distribution of surviving Salmonella was skewed towards low numbers, suggesting higher chances of Salmonella persisting on the eggs in low numbers (<104 cfu egg1). Although numbers were low, the research demonstrated the ability of salmonellae to survive on the shells of eggs following contamination and this clearly has safety implications for handling of eggs in the food industry and the domestic environment. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Eggs Survival Monte Carlo simulation
1. Introduction Cases of salmonellosis increased dramatically worldwide between 1980 and the end of the 1990s. In England and Wales, cases of salmonellosis peaked in 1997 with 31,480 cases (Anon, 2008), but since implementation of preventative and control measures in 1997, the number of cases decreased in Europe (Nygård et al., 2004). Additionally, during the same period there was a large increase in egg-associated salmonellosis worldwide caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) which has an increased ability to invade the reproductive system of the hens and be present within an eggshell (FAO/WHO, 2002). Moreover, an EFSA report (2012) states that “Salmonella was only found in a very low proportion of table eggs, at levels of 0.3%”, a 0.5% reduction from 2009, attributable to implementation of new EU regulations. External contamination of the shell of eggs due to exposure to a contaminated environment is important, because not only can salmonellae penetrate the shell and contaminate the contents of the egg (horizontal transmission; De Reu et al., 2006), but also the eggs themselves can contaminate other surfaces. External contamination of the egg also occurs through contamination of the oviduct and faecal deposition on the shell (Gantois et al., 2009). The shell, rather than the interior, may have the higher incidence of the bacterium (Schoeni, Glass, McDermott, & Wong, 1995), thereby presenting a hazard for anyone handling the eggs, due not only to the risk of * Corresponding author. Tel.: þ44 0 20 7133 4576. E-mail address: [email protected] (J.P. Sutherland). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.033
having Salmonella on the hands, but also to the risk of Salmonella contaminating the contents of the egg during breaking. Incidence of Salmonella contamination of flocks is 0e0.5% (Hopper & Mawer,1988; Humphrey, Greenwood, Gilbert, Rowe, & Chapman, 1989; Radkowski, 2001). However, the incidence of S. Enteritidis contamination of eggs (in USA) has been estimated at about 0.005% (Gast, Guard-Bouldin, & Holt, 2004). Although this number is relatively small, the quantity of eggs entering the human food chain is extremely large, so it is difficult to estimate the level of consumer exposure or degree of risk. Published data concerning behaviour of Salmonella on the shell surface are limited. Schoeni et al. (1995) reported rapid death, although survival was promoted by high relative humidity and storage at low temperature. Survival can also be influenced by strain characteristics (Humphrey & Whitehead, 1993). The physiological condition of Salmonella and of the eggshell also affects survival. Monte Carlo simulation is a technique based on random numbers and probability statistics used to solve mathematical problems. It is used in processes that contain random elements where conventional statistics may not be appropriate (Latimer, Jaykus, Morales, Cowen, & Crawford-Brown, 2002). The basis of the technique is to select random values from within a predetermined fixed range to create different scenarios of a specific problem. The technique is of particular value when there is a substantial degree of uncertainty affecting these values, e.g. factors determining survival of Salmonella on shells of eggs. The purpose of this research was to assess the extent to which contamination of the shells of eggs with Salmonella, in particular S. Enteritidis, may represent a potential risk to the egg handler.
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2. Materials and methods 2.1. Strains of salmonellae The bacterial strains used in this study were S. Enteritidis 2C, S. Enteritidis I, S. Enteritidis P36099, and S. Enteritidis 32410 kindly provided by Professor T.J. Humphrey (University of Liverpool School of Veterinary Science, Leahurst, Neston CH64 7TE, UK). These strains were isolated from outbreaks of egg-associated food poisoning. A fifth strain, Salmonella enterica serovar Typhimurium DT124 (S. Typhimurium) originated from the culture collection of the Microbiology Research Unit, London Metropolitan University. 2.2. Inoculum preparation for experiments on survival of salmonellae on shell of eggs The inoculum was a cocktail of four strains of S. Enteritidis and one strain of S. Typhimurium. It was prepared by inoculating Salmonella strains separately into 10 ml Tryptone Soya Broth (TSB; Oxoid CM131, Basingstoke, UK) and incubating for 24 h at 37 C, followed by serial subculture into TSB at the same time each day on two subsequent days. The third and final subculture of each strain required inoculation of 500 ml TSB at the appointed time and incubation for 24 h at 37 C. Cells from the final subculture were harvested by centrifugation (4000 rpm for 10 min) and resuspended in 500 ml quarter-strength Ringer’s solution (Oxoid BR52). Viable counts were estimated by decimal dilution in quarterstrength Ringer’s solution and surface plating on Tryptone Soya Agar (TSA; Oxoid CM131) incubated at 37 C for 24 h. Each of the five salmonellae culture was added (10 ml) to a suspension of Ringer’s solution (450 ml) containing sterile, antibiotic-free chicken faeces (2% w/v) to make a total volume of 500 ml inoculum batches at 107 cfu ml1. This constituted the inoculum. 2.3. Inoculation of eggs used for survival experiments at different temperatures and relative humidities Eggs purchased from a local supermarket (therefore likely to have originated from different laying flocks) were examined carefully by candling and those with cracks, pinholes and deposition were discarded. Selected eggs were inoculated by dipping for 30 s in the suspension of mixed Salmonella strains to confer an initial concentration of ca.104e105 cfu egg1. To obtain this level of shell contamination, the Salmonella concentration of the dipping solution was about 108 cfu ml1. Up to 15 eggs were dipped in a particular suspension before exchanging the suspension for a fresh one. These parameters were determined from preliminary experiments. Inoculated eggs were air-dried at ambient temperature and stored at 4, 10 and 20 C in conditions of 80 and 90% relative humidity (RH), i.e. six combinations of conditions. Storage time was 16 days in early experiments but was extended to up to 54 days in later experiments. Inoculation of salmonellae using each combination of conditions was carried out twice in most cases, but three times for the 20 C/90% RH combination and just once for the 4 C/90% RH combination. Eggs were always tested on days 1, 2 and 3 and then every 3 days. During longer experiments eggs were sampled on days 1, 2 and 3 and then every 7 days. 2.4. Detection and enumeration of surviving Salmonella on eggs Eggs stored in each environmental condition were sampled in triplicate at intervals during a 54 day storage period by placing them individually in 100 ml buffered peptone water (BPW; Oxoid CM509) and dislodging Salmonella cells by treating with low power ultrasound for 1 min. Volumes of 3.7 ml from each of the three individual
samples were combined. Ten ml of this combined undiluted suspension and 10 ml of a -1 dilution of the combinate were each filtered through 0.45 mm membrane filters and the membranes placed on Salmonella Identification agar plates (SMID; Biomerieux, Basingstoke, UK). In addition, duplicate 0.1 ml volumes of the concentrated and diluted combinates were spread on the surfaces of SMID plates. All SMID plates were incubated at 37 C for 18e24 h before counting typical colonies. The remainder of the three original suspensions in BPW (with the eggs) was incubated at 37 C for 18 h (resuscitation). If no colonies were recovered on the SMID plates after 18e24 h (i.e. <27 salmonellae egg1) then 1 ml of the resuscitated suspensions were transferred to 9 ml selenite-cysteine broth (Oxoid CM699) with addition of 0.4% sodium biselenite (Oxoid L121) for 24 h at 37 C (enrichment) and streaked on Xylose Lysine Deoxycholate agar (XLD; Oxoid CM469) followed by incubation at 37 C for 24 h. Colonies on XLD agar were subjected to confirmation as Salmonella using the Oxoid Biochemical Identification System test (O.B.I.S test, Oxoid ID0570.) and Salmonella latex test (Oxoid FT0203). 2.5. Monte Carlo simulation Based on data obtained from previous experiments, Monte Carlo simulation using Crystal BallÔ risk analysis software (Oracle) as an add-on to Microsoft Excel was used. A number of “what if?” scenarios were constructed with associated assumptions (Table 1). The triangular distribution was selected for all assumptions since (from the post-resuscitation results) the minimum number of surviving salmonellae was known to be at least 1 cfu egg1 and the maximum number 106 cfu egg1. The most likely number will fall between the minimum and the maximum, resulting in a triangularshaped distribution. This distribution is usually used when limited information is known about a variable, as is the case for the initial contamination load. Simulations were carried out using 10,000 iterations. For investigation of survival, a two-stage system was used, variables being (i) initial load on egg (IL), (ii) reduction of initial load by a given order of magnitude after storage (RAS). Initial load (IL) assumptions were intended to reflect the likelihood of natural contamination of eggs while assumptions of RAS were based on inoculated eggs from survival experiments. Surviving salmonellae after storage (SSAS) were predicted using the following formula:
SSAS ¼ IL ðIL RASÞ
(1)
A further assumption was that experimental values were true. Table 1 Scenarios and assumptions made during Monte Carlo simulation (triangular distribution). Scenario
Lowest value
1. Very high load, very low survival 103 Load cfu egg1 Reduction during storage 102 2. Very high load, high survival 103 Load cfu egg1 Reduction during storage 10 3. High load, low survival 10 Load cfu egg1 Reduction during storage 9 4. High load, high survival 10 Load cfu egg1 Reduction during storage 1 5. Low load, low survival 1 Load cfu egg1 Reduction during storage 9 6. Low load, high survival 1 Load cfu egg1 Reduction during storage 10
Highest value
Most likely
106 106
104 103
106 103
104 102
105 104
103 102
105 103
103 102
103 104
102 102
103 103
102 102
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3. Results 3.1. Survival of salmonellae on shell of eggs at different temperatures and relative humidities The initial inoculum concentration in each experiment was intended to be of the order of 105e106 cfu egg1, but in most cases a lower value of 104e105 cfu egg1 was recorded. However, in two experiments at 20 C (Fig. 1c; 90% RH and Fig. 2a; 80% RH) the initial recovery approached 107 cfu egg1 and in one experiment at 4 C and 80% RH (Fig. 6a) it was <102 cfu egg1. Fig. 1 shows the results of three trials of the survival of a cocktail of Salmonella inoculated onto eggshells and stored at 20 C and 90% RH. The results observed in the first trial (Fig. 1a) were very different from the second and third trials (Fig. 1b and c). The first trial demonstrated immense variability of numbers of salmonellae recovered, alternating between 102 and 106 cfu egg1, during 16 days storage. The second and third trials were continued for longer (26 and 49 days respectively) and showed considerable variation, but not the pulsatile pattern of the first trial. Salmonellae were not always recovered directly on SMID agar (i.e. sometimes only after resuscitation, indicated on Figs. 1e6 by baseline recovery points) on a number of sampling occasions. Moreover, they showed abrupt, unexpected recoveries of 106 cfu egg1 after 21 days (Fig. 1b) and 104 cfu egg1 after 41 days (Fig. 1c). It is noteworthy that salmonellae were always recovered after resuscitation and enrichment, indicating its invariable presence on eggs, even when not recoverable by direct plating.
Fig. 1. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 20 C and 90% RH from three trials: a, b and c.
Fig. 2. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 20 C and 80% RH from two trials: a and b.
Recovery of salmonellae from eggs stored at 20 C and 80% RH (Fig. 2) also showed differences between the first and second trials. During the first trial (Fig. 2a), after an initially high recovery, low and very low (i.e. recovery only after resuscitation) numbers of salmonellae were recovered alternately, while the second trial (Fig. 2b) revealed pulsatile recoveries, with up to 106 cfu egg1 recovered on days 8 and 20 of storage. When inoculated eggs were stored at 10 C and 90% and 80% RH (Figs. 3 and 4), numbers of salmonellae surviving on the shells were very low and showed the same pulsatile variability, although the individual pulses were not as extreme as at 20 C. Frequently salmonellae were not recovered directly on SMID agar (Figs. 3b and 4a) but nevertheless, they were always recovered after resuscitation and enrichment.
Fig. 3. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 10 C and 90% RH from two trials: a and b.
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a
1.00E+08
cfu/egg
1.00E+06 1.00E+04 1.00E+02 1.00E+00 0
10
20
30
40
50
60
da y s
b
1.00E+08
cfu/egg
1.00E+06 1.00E+04 1.00E+02 1.00E+00 0
5
10
15
20
25
30
35
da y s Fig. 4. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 10 C and 80% RH from two trials: a and b.
At 4 C and 90% and 80% RH (Figs. 5 and 6) the survival pattern during storage was different from that recorded at higher temperatures. Numbers of inoculated salmonellae tended to show less variability. Salmonellae were consistently recovered at a level between 102 and 104 cfu egg1 with only two incidences of failure to recover directly on SMID; at 90% RH on days 19 and 34 (Fig. 5a) and at 80% RH on day 23 (Fig. 6b). Overall, survival profiles were highly variable, showing pulsed recoveries, particularly at 10 and 20 C. Most importantly, however, Salmonella was always recovered following resuscitation and enrichment, even when numbers were below the limit of detection using direct plating. Regression analysis (results not shown) failed to demonstrate a consistent pattern of death of Salmonella on inoculated eggs in relation to storage conditions. However, the results showed that inoculated Salmonella could survive for at least 54 days on the surface of eggs. 3.2. Monte Carlo simulation Results obtained during survival experiments provided the basis to define the scenarios presented in Table 1 and the overall results
Fig. 5. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at a) 4 C and 90% RH.
Fig. 6. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at a) 4 C and 80% RH from two trials: a and b.
of the simulations are summarized in Table 2. The highest number of salmonellae recovered from eggs was 106 salmonellae egg1, while the lowest recovery was at least one Salmonella cell egg1 (based on the invariably positive results for recovery after enrichment). The intermediate values were considered to be reasonable estimates. Application of Monte Carlo simulation to the scenarios shown in Table 1 demonstrated that under any given circumstances, the highest predicted values of surviving Salmonella occurred at a low frequency. When the assumption was made that the eggshell was very highly contaminated at the point of collection, i.e. after laying; equivalent in these experiments to the inoculation stage, the Monte Carlo distribution was slightly skewed towards lower numbers with a mean of 2.2 104 cfu egg1 (Table 2, scenarios 1 and 2). Immediately after inoculation, the certainty of finding less than 104 Salmonella cfu egg1 was 33.9% when very low survival of salmonellae was considered and 33.3% when high survival was taken into account. After storage, the percentage certainty of finding less that 104 cfu egg1 was 73.3% when very low survival was considered and 98.7% when high survival was assumed. On assumption of a high contamination level by salmonellae at the egg collection stage (Table 2, scenarios 3 and 4, Figures not shown), Monte Carlo simulation results showed a normal distribution with a mean Salmonella load of 1.0 103 cfu egg1 at the collection stage and with a 73.2% certainty of finding less than 104 cfu egg1 if survival was considered low (Table 2, scenario 3) and 82.5% certainty of finding less that 104 cfu egg1 if survival was considered high (Table 2, scenario 4). However, in reality, the most probable scenarios are 5 and 6 (Figs. 7 and 8), where eggs may be contaminated with a low load of salmonellae. As the survival experiments showed, (Figs. 1e6), the circumstances in which the surviving number will be high or low cannot be identified. Where the assumption of low initial load was made, a mean Salmonella load of 1.0 102 cfu egg1 was obtained. After storage there was a 34.6% certainty that <102 cfu egg1
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Table 2 Summary of results from Monte Carlo simulation of Salmonella survival on eggshells. Scenario
1. 2. 3. 4. 5. 6.
Very high load, very low survival Very high load, high survival High load, low survival High load, high survival Low load, low survival Low load, high survival
a
Certainty (%) of finding < 102 cfu/egg.
Initial load of Salmonella on eggs
Remaining load of Salmonella on eggs after storage
Mean of log cfu egg1
Frequency (%)
Certainty (%) of <104 cfu egg1
Mean of log cfu egg1
Frequency (%)
Certainty (%) of <104 cfu egg1
4.34 4.34 3.0 3.0 2.00 1.99
3.4 3.5 3.8 3.8 3.5 4.2
33.9 33.3 87.3 87.3 50.3a 50.7a
0.68 2.33 0.66 1.00 0.34 0.01
4.2 4.20 4.00 4.20 4.00 4.3
73.3 98.7 73.2 82.5 34.6a 49.5a
salmonellae could be found on eggs in case of low survival (Fig. 7), and 49.5% certainty when survival was assumed to be high (Fig. 8).
There has been substantial research concerning internal contamination of eggs by Salmonella, and S. Enteritidis in particular. Gantois et al. (2009) summarized studies analysing internal egg contamination with Salmonella. Moreover, although a number of authors discuss penetration of Salmonella through the eggshell to the interior of the egg (De Reu et al., 2006; Schoeni et al., 1995), relatively little research exists regarding survival of salmonellae on the eggshell (Radkowski, 2002). Our research focused on survival of a cocktail of salmonellae inoculated onto shells of eggs and subsequently stored at 20, 10 and 4 C at relative humidities of 80 and 90%. The results obtained in
this research are in agreement with those of Radkowski (2002) who reported gradual death of salmonellae on shells at 30, 20 and 2 C, with the slowest decline at 2 C. However, in contrast to our results, Radkowski (2002) observed no recovery of salmonellae on eggs stored at 20 and 30 C 7 days after inoculation. Results obtained in our experiments also correspond with those of Baker (1990), who stored inoculated eggs at room temperature and 7 C and reported that salmonellae survived longer at 7 C, and Simons, Ayres, and Kraft (1970), who found better survival of salmonellae at 10 C than at 15 and 23 C. Radkowski (2002) suggested that the longer survival of salmonellae at lower temperatures was due to a slower metabolic rate, induced by adverse conditions on the egg surface (i.e. dryness, shell quality). Occasional “spikes” of Salmonella recovery after periods of no or low recovery (as herein reported) were also observed by Baker (1990), who showed that S. Enteritidis was recovered from shells
Fig. 7. Representative forecast chart obtained from Monte Carlo simulation of scenario 5 (low load, low survival) considering the survival of salmonellae on the shells a) just after egg inoculation (initial load) and b) after storage. Black area indicates % certainty of finding <102 cfu egg1.
Fig. 8. Representative forecast chart obtained from Monte Carlo simulation of scenario 6 (low load, high survival) considering the survival of salmonellae on the shells a) just after egg inoculation (initial load) and b) after storage. Black area indicates % certainty of finding <102 cfu egg1.
4. Discussion and conclusions
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between days one to five at 7 C. No recovery was found after day five, except on days nine and 12. Jones, Curtis, Anderson, and Jones (2004) recorded that the average S. Enteritidis counts on shells declined by nearly 1 log cycle when eggs were stored for 2 weeks at 26 C, but increased by 2 log cycles when eggs were stored for 4 weeks. De Reu et al. (2006) studied different factors that could affect eggshell penetration and whole egg contamination by different bacteria (including S. Enteritidis) and found that the main factors affecting survival on eggshells were cuticle composition and shell quality (measured as specific gravity). In that research, agar-filled and whole eggs were immersed in suspensions of different bacteria (including S. Enteritidis) followed by storage at 20 C and 60% RH for 21 days. Significantly, all 45 inoculated whole eggs and 51 agar-filled eggs were still shell-contaminated at 21 days. According to Gantois et al. (2009), in 2006 the overall prevalence of Salmonella in table eggs in the EU was 0.8%. More than 90% of all egg isolates were S. Enteritidis. This is unsurprising, considering that in the European Union 30.8% of large scale laying hen holdings were positive for Salmonella, based on analyses of faeces and dust samples (EFSA, 2007). Messens, Grijspeerdt, and Herman (2005) reported that 7.8% of shells were contaminated when Salmonella was isolated from 72% of environmental samples from a laying house, but in contrast Hara-Kudo et al. (2001) did not find naturally-occurring Salmonella on the shells in any of 80 eggs tested when studying the effects of laying seasons on growth of S. Enteritidis on shells. Murchie et al. (2007) studied prevalence of Salmonella in grade A whole shell eggs in the island of Ireland (i.e. both the Republic of Ireland and Northern Ireland). Of 5031 eggs sampled, only 2 (0.04%) were positive for Salmonella. Interestingly however, those two positives were isolated from shell samples. It should be noted that when separating shell from contents, shell membranes were included with shell analyses. The low prevalence may be due to testing being geographically restricted to a single small island. Little et al. (2007) found that of 1744 samples of six pooled eggs, Salmonella was found in 157 samples (9%). All 157 isolations were from the shell of the eggs, and of these 157, 10 had also Salmonella in the contents. In that procedure, eggs were cracked and shells and contents separated for analysis; inner membranes were analysed with the shell. Eight Salmonella serotypes were found, with S. Enteritidis the most prevalent (84.9%). These studies highlight the fact that Salmonella can be isolated more readily from shells of eggs than the interior, with consequent potential risk to food-handlers. Contamination of the shell is more likely than contamination of the egg interior, due to higher chances of faecal or environmental contamination (FSA, 2004). Use of Monte Carlo simulation to assess risk to the population posed by eggs contaminated with salmonellae, and S. Enteritidis in particular, has been reported by FAO/WHO (2002) and Schroeder et al. (2006). Monte Carlo simulation was also used during this research. Our model presents a simplistic (parsimonious) approach, where the main assumptions were that eggs may carry an initial load of salmonellae on their shells (high or low) and that during storage this initial load would decrease according to the survival capability of the salmonellae. Six scenarios were described (Table 1). Based on published data (EFSA, 2007; FAO, 2002; Winfield & Groisman 2003), it was considered that scenarios 5 and 6 (low load, high/low survival) would be the most realistic events. The Monte Carlo simulation in the current research was based on two main assumptions to give a general overview of the risk associated with salmonellae on eggshells. Future simulations may consider more complex scenarios, where handler storage time, consumer storage time, retail storage time and reduction during consumer storage should be considered.
Results from our research indicate that it is not possible to make any definitive statement concerning death of Salmonella cells on any egg. From the viewpoint of consumer protection, the assumption must be made that there is no change in numbers of Salmonella on eggs between the stages of contamination and use. However, to put this into perspective, the real risk determinant is whether or not the egg is contaminated with Salmonella, and our results show that this was invariably the case. On this basis, overall risk from Salmonella, taking into account the probable low numbers of naturally occurring contaminants, can be assessed as very low, whether by inspection of data or by Monte Carlo simulation. However, if an externally contaminated egg enters a food-handling environment, a finite hazard exists. The risk of this hazard being realised depends on the number of Salmonella on the egg and the effectiveness of food handling procedures in preventing crosscontamination to other foods. The overall conclusion is that a contaminated egg is a potential hazard at every stage of handling subsequent to contamination. While the probability of any egg being contaminated is low, there is no way of knowing whether or not Salmonella is present and thus precautions must be consistently applied. Guidelines on egghandling by food-handlers and consumers should consider the risk of exposure to Salmonella present on the shells and also the fact that survival of salmonellae is promoted by storage at low temperatures. Acknowledgements We wish to thank the UK Food Standards Agency for funding this work. We are also grateful to Professor TJ Humphrey (University of Liverpool Veterinary School) for provision of S. Enteritidis strains, Dr. Peter Cripps (University of Liverpool Veterinary School) for statistical input and Professor M-L Delignette-Muller for valuable advice concerning Monte Carlo simulation. Dr. Alan Varnam unfortunately died during the course of this work, but we wish to acknowledge his substantial contribution to the initiation, planning and management of this research by including him as an author. References Anonymous. (2008). Salmonella in humans (excluding S. Typhi and S. Paratyphi). Faecal and lower gastrointestinal isolates reported to the Health Protection Agency Centre for Infections England and Wales, 1981e2008. Available at http://www. hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1195733760280? p¼1191942172078 Accessed 16.03.11. Baker, R. C. (1990). Survival of Salmonella enteritidis on and in shelled eggs, liquid eggs and cooked egg products. Dairy, Food and Environmental Sanitation, 10, 273e275. De Reu, K., Grijspeerdt, K., Messens, W., Heyndrickx, M., Uyttendaele, M., Bebevere, J., et al. (2006). Eggshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella enteritidis. International Journal of Food Microbiology, 112, 253e260. EFSA. (2007). Report of the task force on zoonoses data collection on the analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus. The EFSA Journal, 130, 34e117. EFSA. (2012). EU summary report on zoonoses, zoonotic agents and food-borne outbreaks 2010. The EFSA Journal, 10(3), 2597. FAO/WHO (Food and Agricultural Organization of the United Nations/World Health Organization). (2002). Risk assessments of Salmonella in eggs and broiler chickens: Interpretive summary. Available at www.fao.org/docrep/005/y4393e/ y4393e00.htm Accessed 20.05.09. FSA. (2004). Report of the survey of Salmonella contamination of UK produced shell eggs on retail sale. Available at http://www.food.gov.uk/multimedia/pdfs/ fsis5004report.pdf Accessed 03.06.09. Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Gast, R., Humphrey, T. J., et al. (2009). Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews, 33, 1e21. Gast, R. K., Guard-Bouldin, J., & Holt, P. S. (2004). Colonization of reproductive organs and internal contamination of eggs after experimental infection of laying hens with Salmonella Heidelberg and Salmonella Enteritidis. Avian Diseases, 48(4), 863e869.
P. Botey-Saló et al. / Food Control 28 (2012) 463e469 Hara-Kudo, Y., Kumagai, S., Masuda, T., Goto, K., Ohtsuka, K., Masaki, H., et al. (2001). Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating. International Journal of Food Microbiology, 64, 395e399. Hopper, S. A., & Mawer, S. (1988). Salmonella enteritidis in a commercial layer flock. Veterinary Record, 123, 351. Humphrey, T. J., Greenwood, M., Gilbert, R. J., Rowe, B., & Chapman, P. A. (1989). The survival of Salmonella in shell eggs cooked under simulated domestic conditions. Epidemiology and Infection, 103, 35e45. Humphrey, T. J., & Whitehead, A. (1993). Egg age and the growth of Salmonella Enteritidis PT4 in egg contents. Epidemiology and Infection, 111, 209e219. Jones, D. R., Curtis, P. A., Anderson, K. E., & Jones, F. T. (2004). Microbial contamination in inoculated shell eggs: II effects of layer strain and egg storage. Poultry Science, 83, 95e100. Latimer, H. K., Jaykus, L. A., Morales, R. A., Cowen, P., & Crawford-Brown, D. (2002). Sensitivity analysis of Salmonella enteritidis levels in contaminated shell eggs using a biphasic growth model. International Journal of Food Microbiology, 75, 71e87. Little, C. L., Walsh, S., Hucklesby, L., Surman-Lee, S., Pathak, K., Gatty, Y., et al. (2007). Survey of Salmonella contamination of Non-United Kingdom produced raw shell eggs on retail sale in the Northwest of England and London, 2005 to 2006. Journal of Food Protection, 70, 2259e2265. Messens, W., Grijspeerdt, K., & Herman, L. (2005). Eggshell penetration by Salmonella: a review. World’s Poultry Science Association, 61, 71e82.
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Murchie, L., Whyte, P., Xia, B., Horrigan, S., Kelly, L., & Madden, R. H. (2007). Prevalence of Salmonella in grade A whole shell eggs in the island of Ireland. Journal of Food Protection, 70, 1238e1240. Nygård, K., de Jong, B., Guerin, P. J., Andersson, Y., Olsson, A., & Giesecke, J. (2004). Emergence of new Salmonella Enteritidis phage types in Europe? Surveillance of infections in returning travelers. BMC Medicine, Available at http://www. biomedcentral.com/1741-7015/2/32 Accessed on 07.07.08. Radkowski, M. (2001). Occurrence of Salmonella spp. in consumption eggs in Poland. International Journal of Food Microbiology, 64, 189e191. Radkowski, M. (2002). Effect of moisture and temperature on survival of Salmonella Enteritidis on shell eggs. Archiv fur Geflugelkunde, 66, 119e123. Schoeni, J. L., Glass, K. A., McDermott, J. L., & Wong, A. C. L. (1995). Growth and penetration of Salmonella enteritidis, Salmonella heidelberg and Salmonella typhimurium in eggs. International Journal of Food Microbiology, 24, 385e396. Schroeder, C. M., Latimer, H. K., Schlosser, W. D., Golden, N. J., Marks, H. M., Coleman, M. E., et al. (2006). Overview and summary of the Food safety and inspection service risk assessment for Salmonella Enteritidis in shell eggs, October 2005. Foodborne Pathogens and Disease, 3, 403e413. Simons, E. R., Ayres, J. C., & Kraft, A. A. (1970). Effect of moisture and temperature on ability of Salmonella to infect shell eggs. Poultry Science, 49, 761e768. Winfield, M. D., & Groisman, E. A. (2003). Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli. Applied and Environmental Microbiology, 69, 3687e3694.
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Survival of inoculated Salmonella on the shell of hens’ eggs and its potential significance Pilar Botey-Saló, Amara Anyogu, Alan H. Varnam, Jane P. Sutherland* Microbiology Research Unit, School of Human Sciences, Faculty of Life Sciences, London Metropolitan University, 166-220 Holloway Road, London N7 8DB, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2011 Received in revised form 8 May 2012 Accepted 15 May 2012
The aim of this research was to evaluate the risk of inoculated Salmonella persisting on the outside of the shell of hens’ eggs. Hens’ eggs were surface inoculated with a cocktail of Salmonella strains and stored for up to 54 days at 4, 10 and 20 C and at 80 and 90% relative humidity. Salmonella survival showed an irregular pattern, with extremes of high recovery and no recovery. However, salmonellae were always recovered after resuscitation. Monte Carlo simulation of different scenarios using relevant assumptions indicated that the distribution of surviving Salmonella was skewed towards low numbers, suggesting higher chances of Salmonella persisting on the eggs in low numbers (<104 cfu egg1). Although numbers were low, the research demonstrated the ability of salmonellae to survive on the shells of eggs following contamination and this clearly has safety implications for handling of eggs in the food industry and the domestic environment. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Eggs Survival Monte Carlo simulation
1. Introduction Cases of salmonellosis increased dramatically worldwide between 1980 and the end of the 1990s. In England and Wales, cases of salmonellosis peaked in 1997 with 31,480 cases (Anon, 2008), but since implementation of preventative and control measures in 1997, the number of cases decreased in Europe (Nygård et al., 2004). Additionally, during the same period there was a large increase in egg-associated salmonellosis worldwide caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) which has an increased ability to invade the reproductive system of the hens and be present within an eggshell (FAO/WHO, 2002). Moreover, an EFSA report (2012) states that “Salmonella was only found in a very low proportion of table eggs, at levels of 0.3%”, a 0.5% reduction from 2009, attributable to implementation of new EU regulations. External contamination of the shell of eggs due to exposure to a contaminated environment is important, because not only can salmonellae penetrate the shell and contaminate the contents of the egg (horizontal transmission; De Reu et al., 2006), but also the eggs themselves can contaminate other surfaces. External contamination of the egg also occurs through contamination of the oviduct and faecal deposition on the shell (Gantois et al., 2009). The shell, rather than the interior, may have the higher incidence of the bacterium (Schoeni, Glass, McDermott, & Wong, 1995), thereby presenting a hazard for anyone handling the eggs, due not only to the risk of * Corresponding author. Tel.: þ44 0 20 7133 4576. E-mail address: [email protected] (J.P. Sutherland). 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2012.05.033
having Salmonella on the hands, but also to the risk of Salmonella contaminating the contents of the egg during breaking. Incidence of Salmonella contamination of flocks is 0e0.5% (Hopper & Mawer,1988; Humphrey, Greenwood, Gilbert, Rowe, & Chapman, 1989; Radkowski, 2001). However, the incidence of S. Enteritidis contamination of eggs (in USA) has been estimated at about 0.005% (Gast, Guard-Bouldin, & Holt, 2004). Although this number is relatively small, the quantity of eggs entering the human food chain is extremely large, so it is difficult to estimate the level of consumer exposure or degree of risk. Published data concerning behaviour of Salmonella on the shell surface are limited. Schoeni et al. (1995) reported rapid death, although survival was promoted by high relative humidity and storage at low temperature. Survival can also be influenced by strain characteristics (Humphrey & Whitehead, 1993). The physiological condition of Salmonella and of the eggshell also affects survival. Monte Carlo simulation is a technique based on random numbers and probability statistics used to solve mathematical problems. It is used in processes that contain random elements where conventional statistics may not be appropriate (Latimer, Jaykus, Morales, Cowen, & Crawford-Brown, 2002). The basis of the technique is to select random values from within a predetermined fixed range to create different scenarios of a specific problem. The technique is of particular value when there is a substantial degree of uncertainty affecting these values, e.g. factors determining survival of Salmonella on shells of eggs. The purpose of this research was to assess the extent to which contamination of the shells of eggs with Salmonella, in particular S. Enteritidis, may represent a potential risk to the egg handler.
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2. Materials and methods 2.1. Strains of salmonellae The bacterial strains used in this study were S. Enteritidis 2C, S. Enteritidis I, S. Enteritidis P36099, and S. Enteritidis 32410 kindly provided by Professor T.J. Humphrey (University of Liverpool School of Veterinary Science, Leahurst, Neston CH64 7TE, UK). These strains were isolated from outbreaks of egg-associated food poisoning. A fifth strain, Salmonella enterica serovar Typhimurium DT124 (S. Typhimurium) originated from the culture collection of the Microbiology Research Unit, London Metropolitan University. 2.2. Inoculum preparation for experiments on survival of salmonellae on shell of eggs The inoculum was a cocktail of four strains of S. Enteritidis and one strain of S. Typhimurium. It was prepared by inoculating Salmonella strains separately into 10 ml Tryptone Soya Broth (TSB; Oxoid CM131, Basingstoke, UK) and incubating for 24 h at 37 C, followed by serial subculture into TSB at the same time each day on two subsequent days. The third and final subculture of each strain required inoculation of 500 ml TSB at the appointed time and incubation for 24 h at 37 C. Cells from the final subculture were harvested by centrifugation (4000 rpm for 10 min) and resuspended in 500 ml quarter-strength Ringer’s solution (Oxoid BR52). Viable counts were estimated by decimal dilution in quarterstrength Ringer’s solution and surface plating on Tryptone Soya Agar (TSA; Oxoid CM131) incubated at 37 C for 24 h. Each of the five salmonellae culture was added (10 ml) to a suspension of Ringer’s solution (450 ml) containing sterile, antibiotic-free chicken faeces (2% w/v) to make a total volume of 500 ml inoculum batches at 107 cfu ml1. This constituted the inoculum. 2.3. Inoculation of eggs used for survival experiments at different temperatures and relative humidities Eggs purchased from a local supermarket (therefore likely to have originated from different laying flocks) were examined carefully by candling and those with cracks, pinholes and deposition were discarded. Selected eggs were inoculated by dipping for 30 s in the suspension of mixed Salmonella strains to confer an initial concentration of ca.104e105 cfu egg1. To obtain this level of shell contamination, the Salmonella concentration of the dipping solution was about 108 cfu ml1. Up to 15 eggs were dipped in a particular suspension before exchanging the suspension for a fresh one. These parameters were determined from preliminary experiments. Inoculated eggs were air-dried at ambient temperature and stored at 4, 10 and 20 C in conditions of 80 and 90% relative humidity (RH), i.e. six combinations of conditions. Storage time was 16 days in early experiments but was extended to up to 54 days in later experiments. Inoculation of salmonellae using each combination of conditions was carried out twice in most cases, but three times for the 20 C/90% RH combination and just once for the 4 C/90% RH combination. Eggs were always tested on days 1, 2 and 3 and then every 3 days. During longer experiments eggs were sampled on days 1, 2 and 3 and then every 7 days. 2.4. Detection and enumeration of surviving Salmonella on eggs Eggs stored in each environmental condition were sampled in triplicate at intervals during a 54 day storage period by placing them individually in 100 ml buffered peptone water (BPW; Oxoid CM509) and dislodging Salmonella cells by treating with low power ultrasound for 1 min. Volumes of 3.7 ml from each of the three individual
samples were combined. Ten ml of this combined undiluted suspension and 10 ml of a -1 dilution of the combinate were each filtered through 0.45 mm membrane filters and the membranes placed on Salmonella Identification agar plates (SMID; Biomerieux, Basingstoke, UK). In addition, duplicate 0.1 ml volumes of the concentrated and diluted combinates were spread on the surfaces of SMID plates. All SMID plates were incubated at 37 C for 18e24 h before counting typical colonies. The remainder of the three original suspensions in BPW (with the eggs) was incubated at 37 C for 18 h (resuscitation). If no colonies were recovered on the SMID plates after 18e24 h (i.e. <27 salmonellae egg1) then 1 ml of the resuscitated suspensions were transferred to 9 ml selenite-cysteine broth (Oxoid CM699) with addition of 0.4% sodium biselenite (Oxoid L121) for 24 h at 37 C (enrichment) and streaked on Xylose Lysine Deoxycholate agar (XLD; Oxoid CM469) followed by incubation at 37 C for 24 h. Colonies on XLD agar were subjected to confirmation as Salmonella using the Oxoid Biochemical Identification System test (O.B.I.S test, Oxoid ID0570.) and Salmonella latex test (Oxoid FT0203). 2.5. Monte Carlo simulation Based on data obtained from previous experiments, Monte Carlo simulation using Crystal BallÔ risk analysis software (Oracle) as an add-on to Microsoft Excel was used. A number of “what if?” scenarios were constructed with associated assumptions (Table 1). The triangular distribution was selected for all assumptions since (from the post-resuscitation results) the minimum number of surviving salmonellae was known to be at least 1 cfu egg1 and the maximum number 106 cfu egg1. The most likely number will fall between the minimum and the maximum, resulting in a triangularshaped distribution. This distribution is usually used when limited information is known about a variable, as is the case for the initial contamination load. Simulations were carried out using 10,000 iterations. For investigation of survival, a two-stage system was used, variables being (i) initial load on egg (IL), (ii) reduction of initial load by a given order of magnitude after storage (RAS). Initial load (IL) assumptions were intended to reflect the likelihood of natural contamination of eggs while assumptions of RAS were based on inoculated eggs from survival experiments. Surviving salmonellae after storage (SSAS) were predicted using the following formula:
SSAS ¼ IL ðIL RASÞ
(1)
A further assumption was that experimental values were true. Table 1 Scenarios and assumptions made during Monte Carlo simulation (triangular distribution). Scenario
Lowest value
1. Very high load, very low survival 103 Load cfu egg1 Reduction during storage 102 2. Very high load, high survival 103 Load cfu egg1 Reduction during storage 10 3. High load, low survival 10 Load cfu egg1 Reduction during storage 9 4. High load, high survival 10 Load cfu egg1 Reduction during storage 1 5. Low load, low survival 1 Load cfu egg1 Reduction during storage 9 6. Low load, high survival 1 Load cfu egg1 Reduction during storage 10
Highest value
Most likely
106 106
104 103
106 103
104 102
105 104
103 102
105 103
103 102
103 104
102 102
103 103
102 102
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3. Results 3.1. Survival of salmonellae on shell of eggs at different temperatures and relative humidities The initial inoculum concentration in each experiment was intended to be of the order of 105e106 cfu egg1, but in most cases a lower value of 104e105 cfu egg1 was recorded. However, in two experiments at 20 C (Fig. 1c; 90% RH and Fig. 2a; 80% RH) the initial recovery approached 107 cfu egg1 and in one experiment at 4 C and 80% RH (Fig. 6a) it was <102 cfu egg1. Fig. 1 shows the results of three trials of the survival of a cocktail of Salmonella inoculated onto eggshells and stored at 20 C and 90% RH. The results observed in the first trial (Fig. 1a) were very different from the second and third trials (Fig. 1b and c). The first trial demonstrated immense variability of numbers of salmonellae recovered, alternating between 102 and 106 cfu egg1, during 16 days storage. The second and third trials were continued for longer (26 and 49 days respectively) and showed considerable variation, but not the pulsatile pattern of the first trial. Salmonellae were not always recovered directly on SMID agar (i.e. sometimes only after resuscitation, indicated on Figs. 1e6 by baseline recovery points) on a number of sampling occasions. Moreover, they showed abrupt, unexpected recoveries of 106 cfu egg1 after 21 days (Fig. 1b) and 104 cfu egg1 after 41 days (Fig. 1c). It is noteworthy that salmonellae were always recovered after resuscitation and enrichment, indicating its invariable presence on eggs, even when not recoverable by direct plating.
Fig. 1. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 20 C and 90% RH from three trials: a, b and c.
Fig. 2. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 20 C and 80% RH from two trials: a and b.
Recovery of salmonellae from eggs stored at 20 C and 80% RH (Fig. 2) also showed differences between the first and second trials. During the first trial (Fig. 2a), after an initially high recovery, low and very low (i.e. recovery only after resuscitation) numbers of salmonellae were recovered alternately, while the second trial (Fig. 2b) revealed pulsatile recoveries, with up to 106 cfu egg1 recovered on days 8 and 20 of storage. When inoculated eggs were stored at 10 C and 90% and 80% RH (Figs. 3 and 4), numbers of salmonellae surviving on the shells were very low and showed the same pulsatile variability, although the individual pulses were not as extreme as at 20 C. Frequently salmonellae were not recovered directly on SMID agar (Figs. 3b and 4a) but nevertheless, they were always recovered after resuscitation and enrichment.
Fig. 3. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 10 C and 90% RH from two trials: a and b.
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a
1.00E+08
cfu/egg
1.00E+06 1.00E+04 1.00E+02 1.00E+00 0
10
20
30
40
50
60
da y s
b
1.00E+08
cfu/egg
1.00E+06 1.00E+04 1.00E+02 1.00E+00 0
5
10
15
20
25
30
35
da y s Fig. 4. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at 10 C and 80% RH from two trials: a and b.
At 4 C and 90% and 80% RH (Figs. 5 and 6) the survival pattern during storage was different from that recorded at higher temperatures. Numbers of inoculated salmonellae tended to show less variability. Salmonellae were consistently recovered at a level between 102 and 104 cfu egg1 with only two incidences of failure to recover directly on SMID; at 90% RH on days 19 and 34 (Fig. 5a) and at 80% RH on day 23 (Fig. 6b). Overall, survival profiles were highly variable, showing pulsed recoveries, particularly at 10 and 20 C. Most importantly, however, Salmonella was always recovered following resuscitation and enrichment, even when numbers were below the limit of detection using direct plating. Regression analysis (results not shown) failed to demonstrate a consistent pattern of death of Salmonella on inoculated eggs in relation to storage conditions. However, the results showed that inoculated Salmonella could survive for at least 54 days on the surface of eggs. 3.2. Monte Carlo simulation Results obtained during survival experiments provided the basis to define the scenarios presented in Table 1 and the overall results
Fig. 5. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at a) 4 C and 90% RH.
Fig. 6. Numbers of salmonellae recovered from inoculated eggs (105e106 cfu egg1) during storage at a) 4 C and 80% RH from two trials: a and b.
of the simulations are summarized in Table 2. The highest number of salmonellae recovered from eggs was 106 salmonellae egg1, while the lowest recovery was at least one Salmonella cell egg1 (based on the invariably positive results for recovery after enrichment). The intermediate values were considered to be reasonable estimates. Application of Monte Carlo simulation to the scenarios shown in Table 1 demonstrated that under any given circumstances, the highest predicted values of surviving Salmonella occurred at a low frequency. When the assumption was made that the eggshell was very highly contaminated at the point of collection, i.e. after laying; equivalent in these experiments to the inoculation stage, the Monte Carlo distribution was slightly skewed towards lower numbers with a mean of 2.2 104 cfu egg1 (Table 2, scenarios 1 and 2). Immediately after inoculation, the certainty of finding less than 104 Salmonella cfu egg1 was 33.9% when very low survival of salmonellae was considered and 33.3% when high survival was taken into account. After storage, the percentage certainty of finding less that 104 cfu egg1 was 73.3% when very low survival was considered and 98.7% when high survival was assumed. On assumption of a high contamination level by salmonellae at the egg collection stage (Table 2, scenarios 3 and 4, Figures not shown), Monte Carlo simulation results showed a normal distribution with a mean Salmonella load of 1.0 103 cfu egg1 at the collection stage and with a 73.2% certainty of finding less than 104 cfu egg1 if survival was considered low (Table 2, scenario 3) and 82.5% certainty of finding less that 104 cfu egg1 if survival was considered high (Table 2, scenario 4). However, in reality, the most probable scenarios are 5 and 6 (Figs. 7 and 8), where eggs may be contaminated with a low load of salmonellae. As the survival experiments showed, (Figs. 1e6), the circumstances in which the surviving number will be high or low cannot be identified. Where the assumption of low initial load was made, a mean Salmonella load of 1.0 102 cfu egg1 was obtained. After storage there was a 34.6% certainty that <102 cfu egg1
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Table 2 Summary of results from Monte Carlo simulation of Salmonella survival on eggshells. Scenario
1. 2. 3. 4. 5. 6.
Very high load, very low survival Very high load, high survival High load, low survival High load, high survival Low load, low survival Low load, high survival
a
Certainty (%) of finding < 102 cfu/egg.
Initial load of Salmonella on eggs
Remaining load of Salmonella on eggs after storage
Mean of log cfu egg1
Frequency (%)
Certainty (%) of <104 cfu egg1
Mean of log cfu egg1
Frequency (%)
Certainty (%) of <104 cfu egg1
4.34 4.34 3.0 3.0 2.00 1.99
3.4 3.5 3.8 3.8 3.5 4.2
33.9 33.3 87.3 87.3 50.3a 50.7a
0.68 2.33 0.66 1.00 0.34 0.01
4.2 4.20 4.00 4.20 4.00 4.3
73.3 98.7 73.2 82.5 34.6a 49.5a
salmonellae could be found on eggs in case of low survival (Fig. 7), and 49.5% certainty when survival was assumed to be high (Fig. 8).
There has been substantial research concerning internal contamination of eggs by Salmonella, and S. Enteritidis in particular. Gantois et al. (2009) summarized studies analysing internal egg contamination with Salmonella. Moreover, although a number of authors discuss penetration of Salmonella through the eggshell to the interior of the egg (De Reu et al., 2006; Schoeni et al., 1995), relatively little research exists regarding survival of salmonellae on the eggshell (Radkowski, 2002). Our research focused on survival of a cocktail of salmonellae inoculated onto shells of eggs and subsequently stored at 20, 10 and 4 C at relative humidities of 80 and 90%. The results obtained in
this research are in agreement with those of Radkowski (2002) who reported gradual death of salmonellae on shells at 30, 20 and 2 C, with the slowest decline at 2 C. However, in contrast to our results, Radkowski (2002) observed no recovery of salmonellae on eggs stored at 20 and 30 C 7 days after inoculation. Results obtained in our experiments also correspond with those of Baker (1990), who stored inoculated eggs at room temperature and 7 C and reported that salmonellae survived longer at 7 C, and Simons, Ayres, and Kraft (1970), who found better survival of salmonellae at 10 C than at 15 and 23 C. Radkowski (2002) suggested that the longer survival of salmonellae at lower temperatures was due to a slower metabolic rate, induced by adverse conditions on the egg surface (i.e. dryness, shell quality). Occasional “spikes” of Salmonella recovery after periods of no or low recovery (as herein reported) were also observed by Baker (1990), who showed that S. Enteritidis was recovered from shells
Fig. 7. Representative forecast chart obtained from Monte Carlo simulation of scenario 5 (low load, low survival) considering the survival of salmonellae on the shells a) just after egg inoculation (initial load) and b) after storage. Black area indicates % certainty of finding <102 cfu egg1.
Fig. 8. Representative forecast chart obtained from Monte Carlo simulation of scenario 6 (low load, high survival) considering the survival of salmonellae on the shells a) just after egg inoculation (initial load) and b) after storage. Black area indicates % certainty of finding <102 cfu egg1.
4. Discussion and conclusions
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between days one to five at 7 C. No recovery was found after day five, except on days nine and 12. Jones, Curtis, Anderson, and Jones (2004) recorded that the average S. Enteritidis counts on shells declined by nearly 1 log cycle when eggs were stored for 2 weeks at 26 C, but increased by 2 log cycles when eggs were stored for 4 weeks. De Reu et al. (2006) studied different factors that could affect eggshell penetration and whole egg contamination by different bacteria (including S. Enteritidis) and found that the main factors affecting survival on eggshells were cuticle composition and shell quality (measured as specific gravity). In that research, agar-filled and whole eggs were immersed in suspensions of different bacteria (including S. Enteritidis) followed by storage at 20 C and 60% RH for 21 days. Significantly, all 45 inoculated whole eggs and 51 agar-filled eggs were still shell-contaminated at 21 days. According to Gantois et al. (2009), in 2006 the overall prevalence of Salmonella in table eggs in the EU was 0.8%. More than 90% of all egg isolates were S. Enteritidis. This is unsurprising, considering that in the European Union 30.8% of large scale laying hen holdings were positive for Salmonella, based on analyses of faeces and dust samples (EFSA, 2007). Messens, Grijspeerdt, and Herman (2005) reported that 7.8% of shells were contaminated when Salmonella was isolated from 72% of environmental samples from a laying house, but in contrast Hara-Kudo et al. (2001) did not find naturally-occurring Salmonella on the shells in any of 80 eggs tested when studying the effects of laying seasons on growth of S. Enteritidis on shells. Murchie et al. (2007) studied prevalence of Salmonella in grade A whole shell eggs in the island of Ireland (i.e. both the Republic of Ireland and Northern Ireland). Of 5031 eggs sampled, only 2 (0.04%) were positive for Salmonella. Interestingly however, those two positives were isolated from shell samples. It should be noted that when separating shell from contents, shell membranes were included with shell analyses. The low prevalence may be due to testing being geographically restricted to a single small island. Little et al. (2007) found that of 1744 samples of six pooled eggs, Salmonella was found in 157 samples (9%). All 157 isolations were from the shell of the eggs, and of these 157, 10 had also Salmonella in the contents. In that procedure, eggs were cracked and shells and contents separated for analysis; inner membranes were analysed with the shell. Eight Salmonella serotypes were found, with S. Enteritidis the most prevalent (84.9%). These studies highlight the fact that Salmonella can be isolated more readily from shells of eggs than the interior, with consequent potential risk to food-handlers. Contamination of the shell is more likely than contamination of the egg interior, due to higher chances of faecal or environmental contamination (FSA, 2004). Use of Monte Carlo simulation to assess risk to the population posed by eggs contaminated with salmonellae, and S. Enteritidis in particular, has been reported by FAO/WHO (2002) and Schroeder et al. (2006). Monte Carlo simulation was also used during this research. Our model presents a simplistic (parsimonious) approach, where the main assumptions were that eggs may carry an initial load of salmonellae on their shells (high or low) and that during storage this initial load would decrease according to the survival capability of the salmonellae. Six scenarios were described (Table 1). Based on published data (EFSA, 2007; FAO, 2002; Winfield & Groisman 2003), it was considered that scenarios 5 and 6 (low load, high/low survival) would be the most realistic events. The Monte Carlo simulation in the current research was based on two main assumptions to give a general overview of the risk associated with salmonellae on eggshells. Future simulations may consider more complex scenarios, where handler storage time, consumer storage time, retail storage time and reduction during consumer storage should be considered.
Results from our research indicate that it is not possible to make any definitive statement concerning death of Salmonella cells on any egg. From the viewpoint of consumer protection, the assumption must be made that there is no change in numbers of Salmonella on eggs between the stages of contamination and use. However, to put this into perspective, the real risk determinant is whether or not the egg is contaminated with Salmonella, and our results show that this was invariably the case. On this basis, overall risk from Salmonella, taking into account the probable low numbers of naturally occurring contaminants, can be assessed as very low, whether by inspection of data or by Monte Carlo simulation. However, if an externally contaminated egg enters a food-handling environment, a finite hazard exists. The risk of this hazard being realised depends on the number of Salmonella on the egg and the effectiveness of food handling procedures in preventing crosscontamination to other foods. The overall conclusion is that a contaminated egg is a potential hazard at every stage of handling subsequent to contamination. While the probability of any egg being contaminated is low, there is no way of knowing whether or not Salmonella is present and thus precautions must be consistently applied. Guidelines on egghandling by food-handlers and consumers should consider the risk of exposure to Salmonella present on the shells and also the fact that survival of salmonellae is promoted by storage at low temperatures. Acknowledgements We wish to thank the UK Food Standards Agency for funding this work. We are also grateful to Professor TJ Humphrey (University of Liverpool Veterinary School) for provision of S. Enteritidis strains, Dr. Peter Cripps (University of Liverpool Veterinary School) for statistical input and Professor M-L Delignette-Muller for valuable advice concerning Monte Carlo simulation. Dr. Alan Varnam unfortunately died during the course of this work, but we wish to acknowledge his substantial contribution to the initiation, planning and management of this research by including him as an author. References Anonymous. (2008). Salmonella in humans (excluding S. Typhi and S. Paratyphi). Faecal and lower gastrointestinal isolates reported to the Health Protection Agency Centre for Infections England and Wales, 1981e2008. Available at http://www. hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1195733760280? p¼1191942172078 Accessed 16.03.11. Baker, R. C. (1990). Survival of Salmonella enteritidis on and in shelled eggs, liquid eggs and cooked egg products. Dairy, Food and Environmental Sanitation, 10, 273e275. De Reu, K., Grijspeerdt, K., Messens, W., Heyndrickx, M., Uyttendaele, M., Bebevere, J., et al. (2006). Eggshell factors influencing eggshell penetration and whole egg contamination by different bacteria, including Salmonella enteritidis. International Journal of Food Microbiology, 112, 253e260. EFSA. (2007). Report of the task force on zoonoses data collection on the analysis of the baseline study on the prevalence of Salmonella in holdings of laying hen flocks of Gallus gallus. The EFSA Journal, 130, 34e117. EFSA. (2012). EU summary report on zoonoses, zoonotic agents and food-borne outbreaks 2010. The EFSA Journal, 10(3), 2597. FAO/WHO (Food and Agricultural Organization of the United Nations/World Health Organization). (2002). Risk assessments of Salmonella in eggs and broiler chickens: Interpretive summary. Available at www.fao.org/docrep/005/y4393e/ y4393e00.htm Accessed 20.05.09. FSA. (2004). Report of the survey of Salmonella contamination of UK produced shell eggs on retail sale. Available at http://www.food.gov.uk/multimedia/pdfs/ fsis5004report.pdf Accessed 03.06.09. Gantois, I., Ducatelle, R., Pasmans, F., Haesebrouck, F., Gast, R., Humphrey, T. J., et al. (2009). Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiology Reviews, 33, 1e21. Gast, R. K., Guard-Bouldin, J., & Holt, P. S. (2004). Colonization of reproductive organs and internal contamination of eggs after experimental infection of laying hens with Salmonella Heidelberg and Salmonella Enteritidis. Avian Diseases, 48(4), 863e869.
P. Botey-Saló et al. / Food Control 28 (2012) 463e469 Hara-Kudo, Y., Kumagai, S., Masuda, T., Goto, K., Ohtsuka, K., Masaki, H., et al. (2001). Detection of Salmonella enteritidis in shell and liquid eggs using enrichment and plating. International Journal of Food Microbiology, 64, 395e399. Hopper, S. A., & Mawer, S. (1988). Salmonella enteritidis in a commercial layer flock. Veterinary Record, 123, 351. Humphrey, T. J., Greenwood, M., Gilbert, R. J., Rowe, B., & Chapman, P. A. (1989). The survival of Salmonella in shell eggs cooked under simulated domestic conditions. Epidemiology and Infection, 103, 35e45. Humphrey, T. J., & Whitehead, A. (1993). Egg age and the growth of Salmonella Enteritidis PT4 in egg contents. Epidemiology and Infection, 111, 209e219. Jones, D. R., Curtis, P. A., Anderson, K. E., & Jones, F. T. (2004). Microbial contamination in inoculated shell eggs: II effects of layer strain and egg storage. Poultry Science, 83, 95e100. Latimer, H. K., Jaykus, L. A., Morales, R. A., Cowen, P., & Crawford-Brown, D. (2002). Sensitivity analysis of Salmonella enteritidis levels in contaminated shell eggs using a biphasic growth model. International Journal of Food Microbiology, 75, 71e87. Little, C. L., Walsh, S., Hucklesby, L., Surman-Lee, S., Pathak, K., Gatty, Y., et al. (2007). Survey of Salmonella contamination of Non-United Kingdom produced raw shell eggs on retail sale in the Northwest of England and London, 2005 to 2006. Journal of Food Protection, 70, 2259e2265. Messens, W., Grijspeerdt, K., & Herman, L. (2005). Eggshell penetration by Salmonella: a review. World’s Poultry Science Association, 61, 71e82.
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Murchie, L., Whyte, P., Xia, B., Horrigan, S., Kelly, L., & Madden, R. H. (2007). Prevalence of Salmonella in grade A whole shell eggs in the island of Ireland. Journal of Food Protection, 70, 1238e1240. Nygård, K., de Jong, B., Guerin, P. J., Andersson, Y., Olsson, A., & Giesecke, J. (2004). Emergence of new Salmonella Enteritidis phage types in Europe? Surveillance of infections in returning travelers. BMC Medicine, Available at http://www. biomedcentral.com/1741-7015/2/32 Accessed on 07.07.08. Radkowski, M. (2001). Occurrence of Salmonella spp. in consumption eggs in Poland. International Journal of Food Microbiology, 64, 189e191. Radkowski, M. (2002). Effect of moisture and temperature on survival of Salmonella Enteritidis on shell eggs. Archiv fur Geflugelkunde, 66, 119e123. Schoeni, J. L., Glass, K. A., McDermott, J. L., & Wong, A. C. L. (1995). Growth and penetration of Salmonella enteritidis, Salmonella heidelberg and Salmonella typhimurium in eggs. International Journal of Food Microbiology, 24, 385e396. Schroeder, C. M., Latimer, H. K., Schlosser, W. D., Golden, N. J., Marks, H. M., Coleman, M. E., et al. (2006). Overview and summary of the Food safety and inspection service risk assessment for Salmonella Enteritidis in shell eggs, October 2005. Foodborne Pathogens and Disease, 3, 403e413. Simons, E. R., Ayres, J. C., & Kraft, A. A. (1970). Effect of moisture and temperature on ability of Salmonella to infect shell eggs. Poultry Science, 49, 761e768. Winfield, M. D., & Groisman, E. A. (2003). Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli. Applied and Environmental Microbiology, 69, 3687e3694.