Egg-borne Salmonella Outbreak Strain Survival

When it comes to minimizing consumer risk, the underlying factors that contribute to Salmonella contamination matter. In the case of Australian egg-associated outbreaks, the serovar Salmonella Typhimurium contaminates externally but the precise mechanisms remain unclear.

To evaluate these, McAuley et al. (2015) used three strains of Salmonella (outbreak strain S. Typhimurium PT135, laboratory reference S. Typhimurium ATCC 14028, and non-egg associated S. Sofia 1296a) to test growth in egg contents, attachment to egg shells, and survival on shell.¹ They performed these experiments at temperatures common to the egg production industry: 4°C (common refrigeration), 15°C (recommended storage temperature in Australia), 22°C (ambient air), 37°C (optimal growth for Salmonella), and 42°C (chicken body temperature and within recommended range for egg wash).

Growth in Egg Contents

First, the team grew Salmonella strains in Tryptone Soya Broth and diluted cultures using Maximum Recovery Diluent (both Thermo Scientific) to produce an inoculum close to the limit of detection: 5×10² CFU/mL for egg yolk and whole eggs; 5×10⁴ CFU/mL for egg whites.The team serially diluted and plated the samples on Tryptone Soya Agar (TSA, Thermo Scientific) for counting, enriching as needed. After counting, they used a Salmonella latex test kit (Thermo Scientific) to confirm.

The team found no significant difference in Salmonella growth for any isolate, sample type, or temperature. This indicates that multiple strains grow readily in egg products under suitable conditions. They report that the highest growth rates occurred at higher temperatures and that growth was significantly greater in egg yolk compared to whole egg or egg white.

Table: Mean Maximum Growth Rates (CFU/mL/h) of Combined Salmonella Isolates

temperature	egg yolk	whole egg	egg white
15°C	0.114	0.095	−0.001
22°C	0.325	0.228	0.028
37°C	0.842	0.612	0.060
combined	0.427	0.312	0.029

The team further reports that all isolates reached stationary phase (10⁸-10⁹ CFU/mL) in egg yolk and whole egg samples. However, only some (6 of 24) of the higher inoculum egg white samples reached stationary phase with significant variation.

Table: Time to Stationary Phase

temperature	egg yolk	whole egg	egg white
15°C	3 days	4 days	34 days*
22°C	26 hours	34 hours	3 to >30 days
37°C	9 to 10 hours	12 to 16 hours	levels unchanged for 16 hours then declined for both S. typhimurium strains by 6 days

*only one isolate, one replicate, higher inoculum

Attachment to Shells

Next, the team dipped whole eggs (sourced from retail market or farm gate) in inoculum (8.66 +-0.16 CFU/mL) for one or 20 minutes in buffered peptone water (BPW, Thermo Scientific) held at three temperatures (4°, 22°, and 42°C). They rinsed the eggs before massaging them in a stomacher to detach the bacteria. After plating on TSA and counting, they confirmed using a Salmonella latex test kit. The team also used Xylose Lysine Deoxycholate agar (XLD, Thermo Scientific) to plate and count controls.

The team reported no Salmonella detected on controls. All total viable counts were above 410 CFU/mL, and all strains attached at similar levels. The highest attachment (5.85 log₁₀ CFU/mL) occurred on farm eggs using the reference strain at 22°C after 20 minutes of exposure. The lowest attachment (3.77 log₁₀ CFU/mL) occurred on farm eggs using S. Sofia at 4°C after 1 minute of exposure; with the same egg source, temperature, and exposure level, the outbreak strain and reference strain attached more significantly.

 Table: Attachment Levels (CFU/mL) for Retail Eggs

Temperature	S. Sofia 1 minute 20 minutes	Outbreak Strain 1 minute 20 minutes	Reference Strain 1 minute 20 minutes
4°C	4.43 ± 0.36 4.78 ± 0.29	5.01 ± 0.08 5.14 ± 0.34	4.96 ± 0.31 5.36 ± 0.24
22°C	4.78 ± 0.80 5.24 ± 0.18	4.88 ± 0.35 5.19 ± 0.23	4.99 ± 0.23 5.25 ± 0.46
42°C	5.26 ± 0.28 5.32 ± 0.15	4.82 ± 0.26 5.1 ± 0.15	4.85 ± 0.28 5.28 ± 0.42

Table Attachment Levels (CFU/mL) for Farm Eggs

Temperature	S. Sofia 1 minute 20 minutes	Outbreak Strain 1 minute 20 minutes	Reference Strain 1 minute 20 minutes
4°C	4.14 ± 0.25 4.64 ± 0.19	4.72 ± 0.39 5.11 ± 0.15	5.10 ± 0.16 5.23 ± 0.16
22°C	4.16 ± 0.18 4.85 ± 0.12	4.60 ± 0.19 5.00 ± 0.20	4.83 ± 0.14 5.75 ± 0.10
42°C	4.68 ± 0.19 4.81 ± 0.24	5.06 ± 0.65 5.09 ± 0.26	5.04 ± 0.12 5.56 ± 0.21

Survival on Shells

Finally, the team tested Salmonella survival on egg shells under retail conditions, using only outbreak S. Typhimurium and S. Sofia strains. They grew cultures in Heart Infusion Broth (Thermo Scientific) and diluted before inoculation (6 log₁₀ CFU) via surface swabbing. They held these samples at 4° and 22°C, testing (weeks 0, 1, 2, and 4) using Most Probable Number (MPN) or selective enrichment.

Neither strain persisted beyond 4 weeks under refrigerated or ambient conditions. The team was able to isolate S. Sofia at both storage temperatures at week two (4 out of 5 eggs). For the outbreak strain, they only recovered isolates at week one (2 out of 5 eggs) at 22°C.

Table: Survival on Egg Shells Over Time (log10MPN/cm2)

Time (weeks)	S. sofia 4°C 22°C	outbreak strain 4°C 22°C
0	3.89 3.55	3.39 2.88
1	1.52 0.56	<−0.05 0.25
2	0.48 0.10	<−0.05 <−0.05
4	<−0.05 <−0.05	<−0.05 <−0.05

Overall, the team observed no significant differences in the outbreak strain’s growth in egg contents, attachment to egg surfaces, or survival abilities when compared to either S. Sofia or the reference strain. This indicates that Australian outbreaks of Salmonella linked to eggs occurred for reasons other than those examined here. They recommend a retail shelf-life of 6 weeks with maintenance of refrigeration conditions at 4°C to limit consumer risk.

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Reference

1 McAuley, C.M. et al. (2015) ‘Salmonella Typhimurium and Salmonella Sofia: Growth in and Persistence on Eggs under Production and Retail Conditions.’ BioMed Research International, Article ID 914987, in press.