• Christian Nehme

INTRODUCTION

An investigation of a food poisoning outbreak requires some knowledge into what food poisoning is and some common culprit bacteria which trigger such outbreaks. Food poisoning, also known as foodborne illness, is the name for the range of illnesses caused by eating or drinking contaminated food or drink. Food poisoning occurs in two main ways: poisoning by toxic agent or by infectious agent. Food intoxication is when the food contains toxins, usually occurring when the organism that produced the toxin is no longer present or able to cause infection. Food infection, on the other hand, is when the food contains bacteria which infect the body after it is eaten. Foodborne illness is quite common, affecting almost 5.5 million Australians each year.

Two common food poisoning causing bacteria are B.cereus vs S. aureus. Bacillus species are Gram positive, aerobic heterotrophs, with the ability to form resistant spore coats.

Do they have similar symptoms, toxins?

cases in Australia.

Description of Scenario

As a special treat paid for by the Department of Health and Aging, 435 pensioners were taken on a catered summer’s day harbour cruise. Later that day, a number of the participants became very ill.

The food was prepared on shore and brought onto the boat that day, The boat left Circular Quay at around 10am and cruised around Sydney Harbour past Watsons Bay, into Darling Harbour and disembarking at Circular Quay at 3pm.. The water was rather calm and there was a medium breeze.

Local hospitals and ambulances were mobilized to respond to the outbreak. It also came to the attention of the local PHU and their personnel were able to retrieve some remnant food and patient specimens from the scene and hospitals.

Questionnaires were distributed to the guest list once this was obtained by the local PHU and the NSW Food Authority. Of the questionnaires sent out, 339 were returned providing the following information.

Symptoms

Of the total number who were sick, 153 suffered severe diarrhoea; 139 vomited; 122 experienced abdominal cramps; 117 said that they felt nauseous; 11 complained of numbness in the arms and legs; and 3 developed haematuria. Thankfully no deaths were recorded as a consequence of this outbreak.

Reported details on illness, and what was eaten and not eaten were compiled into Food Attack Tables.

RESULTS

Number of individuals who attended the cruise:435

Number of questionnaires returned:339

Number of individuals who suffered diarrhea: 153

Number of individuals who suffered vomiting: 139

Number of individuals who suffered abdominal cramps: 122

Number of individuals who suffered nausea: 117

Number of individuals who suffered numbness in arms / legs: 139

Number of individuals who suffered haematuria: 139

Number of deaths: 0

The incubation periods of the sick individuals (number of cases) are displayed in Figure 1. The food-specific attack rates for foods thought to be implicated are shown in Table 1.

Figure 1: Epidemic curve presenting incubation periods of sick pensioners aboard the harbour cruise. Results obtained from responses to a questionnaire. Onset times appear as two distinct peaks, one occurring between 2-3 hours and the other between 6-15 hours, after consuming lunch.

Data Calculated

Percentage morbidity: (219 x 100) / 339 = 64.6%

Percentage mortality: (0 x 100) / 339= 0.0%

Percentage case fatality rate: (0 x 100) / 219= 0.0%

Average incubation period: = 9.9 Hours

Table 1: Food-specific Attack rates using data from questionnaire and other calculations including the Odds Ratio, Chi squared and Confidence Interval for each food type.

Odds Ratio: An odds ratio greater than 1 indicates a higher risk of becoming risk on eating that particular food type. In Table 1 above, all meats have an odds ratio less than 1, and only Rice Pudding with Custard has an odds ratio greater than 1.

Confidence Interval: the Odds Ratio is within a 95% confidence level if the CI does not include 1. From Table 1, Roast Lamb is the only food not within a 95% confidence level, whilst the 3 other food types are within a 95% confidence level.

Chi-Squared:

H_o = the sickness is not a result of any of the food consumed

H₁ = the sickness is a result of any of the food consumed

When the P-Value is less than 0.05 (non-significant), the null hypothesis can be rejected. Since the P-Value is less than 0.05 for the Chicken, Ham & Rice Pudding, the null hypothesis can be rejected, indicating that the case of the sickness was due to the consumption of either one of, or a combination of these foods. Since the P-Value of the Roast Lamb is much greater than 0.05, the null hypothesis cannot be rejected, signifying that the consumption of the Roast Lamb was most probably not a cause of the sickness.

% Ill (Attack Rate): this is a measurement of correlation of the percentage of passengers who got sick and the total number who ate or did not eat a specific type of food. The correlation for Rice Pudding was the one to stand out, where only 20% of the passengers who did not eat rice pudding actually reported that they got sick. This is dramatically less than the other food types, which were above 60% each. Also, Rice Pudding had the highest attack rate of 57.8%.

Flowchart of Experiment

Food Swabs

Cook’s Nose Swab Faecal Swab

Results of Experiment

DISCUSSION

As evident from Figure 1, the number of cases are spread out from 2-21 hours after consuming lunch. There are 2 distinct peaks, occurring at 2-3 and 6-15 hours after consuming lunch. At this stage, a pathogen can possibly be the causative agent as it has shown to portray such symptoms on passing from the Upper Gastrointestinal tract (GIT) to the Lower GIT(Kho et al. 2011). These peaks can be linked with vomiting and diarrhoea, respectively. Of the commonly known pathogens, Bacillus cereus, a spore-forming pathogen, causes two distinct forms of foodborne effects: an emetic syndrome (vomiting-typified by an incubation period of 1–6 hours) and a diarrheal illness typified by an incubation period of 6–24 hours (Benenson AS, ed).

Table 1 yields useful results which play a critical role in identifying the trigger organism for the outbreak. The attack rate for people who ate the different food types was the highest for Rice Pudding & Custard (57.8%), although the other food types were closely behind, Roast Lamb being the closest at 56%. This is not enough information to be able to conclude the causative food type since the attack rate only ranges by 7%. This close range could be due to the fact that these food types were consumed in varying combinations, which makes it difficult to pinpoint the exact, single food at fault. This attack rate, however, can be combined with the attack rate of the pensioners who did not eat a particular food type, giving a clearer picture as to what the causative food source was. Rice Pudding & Custard, having the lowest attack rate at just 20%, suggests that if it were not eaten, the chance of becoming ill is heavily less compared to the nearest food type, Roast Lamb, with an attack rate of 62.2%. This, combined with the high attack rate of pensioners who ate the Rice Pudding & Custard, signals it to be the culprit source of pathogens.

The odds ratio of Rice Pudding & Custard, being 5.47, is significantly greater than other food types (which are all less than 1), suggesting that the risk of becoming ill upon consumption is 5.47 times greater than if Rice Pudding weren’t consumed. Furthermore, since the P-Value is less than 0.05 for the Chicken, Ham & Rice Pudding, the null hypothesis can be rejected (that the sickness is not a result of any of the food consumed). This indicates that the case of the sickness was due to the consumption of either one of, or a combination of these foods. Since the P-Value of the Roast Lamb is much greater than 0.05, the null hypothesis cannot be rejected, signifying that the consumption of the Roast Lamb was most probably not a cause of the outbreak. However, the Confidence Interval for Rice Pudding & Custard lies within a 95% confidence level, further supporting the claim that the Rice Pudding & Custard was the causative food source.

Knowing the possible food source which triggered the outbreak, and having an idea that the causative organism behind the outbreak was B. cereus (due to the two peaks correlating to the typical symptoms), laboratory investigations were then conducted to conclude what the exact organism was. The first test conducted was inoculating each food sample onto a PEMBA plate. The results varied as to the PEMBA count (cfu/g), possibly due to experimental error. However, Rice Pudding had the highest consistent overall count, with 8.5 x10⁶ and 7.9 x 10⁶ cfu/g. The colonies that grew on the PEMBA plates for the Chicken, Ham and Roast Lamb were small, yellow colonies, measuring approximately 1-2mm each, indicating the possibility of Gram Positive Cocci. The Rice Pudding, however, displayed large (4-5mm), matt blue colonies with a halo of precipitate. The PEMBA isolate for the Rice Pudding were Gram Positive Rods, occurring in chains with an oval central spore. The lipid granule test was positive and catalase positive, with motile cells. Since none of the other food types PEMBA isolates showed GPR, no further testing was conducted on Chicken, Ham and Roast Lamb.

Salmonella, Enterococcus and Shigella may have been possible pathogenic causing bacteria which contributed to the outbreak. In order to eliminate or support this possibility, a Xylose Lysine Deoxycholate (XLD) plate was used. Faecal swabs of the sick pensioners who had eaten a particular food type were inoculated onto the XLD media. The only differentiating outcome was the appearance of the colonies of the faecal sample from people who consumed the Rice Pudding & Custard, were slightly larger than those for the other food types. No definitive conclusion can be made from this. Faecal swabs were also plated on PEMBA plates and inoculated into the XLD media. The PEMBA plates displayed small, yellow Gram Positive Cocci colonies for the Chicken, Ham & Roast Lamb specimens. The Rice Pudding & Custard however, displayed large, Gram Positive Rods, which appeared as large, blue, matte colonies when sub-cultured onto the PEMBA plates. This further supports the finding that B.cereus was the causative organism.

The next step is to eliminate any possibility that the pathogen Staphylococci spp had any part to play, or to confirm that it did. To do this, the Cook’s nose swab was inoculated on Blood Agar (BA) to stablish haemolysis and on Mannitol Salt Agar (MSA), as a selective mechanism for salt-tolerant microorganisms such as Staphylococcus or Enterococcus. After testing, small, round, flat, γ – haemolytic white colonies were seen on the Blood Agar, with the BA Isolate being Gram Positive Cocci, occurring in clusters and being catalase positive. Although this suggests the possible presence of a Staphylococci species, the MSA plate rendered small, white colonies with pink agar throughout. The pink agar confirms that the Mannitol was not fermented, which is an indicator that no Staphylococci species was present. Further proof for the non-existence of S.aureus was seen with the Latex test displaying no clumping.

In order to confirm that B.cereus was the causative organism for the outbreak, further tests were conducted. These include gelatin positive, starch positive, casein negative, nitrate positive (no gas formed). The sample tested positive for Glucose VP. This means that glucose was broken down by the Rice Pudding specimen to form acetoin, and was evident through the dark red colour formed. B.cereus tested negative for the indole test, meaning it was unable to split indole from tryptophan (Wong, Chang & Fan 1988). All these test results correspond with the presence of B. cereus and at this point, we are more confident that this is the causative organism. To test for purity, and to confirm that no other specimen influenced these results, a BHIA purity test was performed, which resulted with a pure result, meaning the listed test results were solely caused by the Rice Pudding & Custard specimen.

Through the results of the media growth and the results of the confirmatory tests conducted, it was determined that the causative food source was the Rice Pudding & Custard, which contained a strain of B.cereus. This resulted through the Cook not cooking the Rice at the ideal temperature, and since the temperature used was hot enough to trigger the B.cereus to enter their vegetative state, but was not high enough to kill the B.cereus, this resulted in the widespread growth of the specimen. Accompanied with the fact that the rice was prepared the night before and the warmer environment in which the rice was kept for the period of time overnight were suitable conditions and promoted its growth, further accounts for the growth of B.cereus (Jesen et al. 2003).

Outbreaks such as this could be avoided, and at least minimised if some precautions were taken by all those involved with the handling, making and consuming of foods. The following list outlines some main ways through which this can be achieved (Klietmann, W, 2002):

• Storage of hot food above 60⁰C and cold food in the fridge below 4⁰C
• Keeping cooked and raw food separated at all times, to avoid cross-contamination
• Using separate chopping boards and utensils for raw and cooked foods
• Improving hygiene such as washing hands before and after contact with food
• Utilising a heat process to destroy the vegetative cells and a rapid cooling process to prevent the spores from germinating
• Using appropriate cleaning and disinfection of contact surfaces of food, such as sodium hypochlorite based disinfectants
• Keep frozen foods frozen until used
• Cooked foods which won’t be consumed within an hour should be kept at temperatures below 10°C or above 50°C (Roberts, T.A)

Experimental Method

The main source of primary data was obtained from the questionaries. The accuracy of the answers to the questions asked has a direct influence on the results obtained, and on the findings extracted from the questionaries. Thus, if the questionnaires are completed accurately and in detail, the reliability of the results would increase and a higher potential in obtaining accurate leads as to which species were the triggers to the outbreak would be attained. However, out of the 435 pensioners who were on the cruise, only 339 questionnaires were completed and returned to the local PHU & the NSW Food Authority. This leaves 96 unaccounted for pensioners, which would have aided and fastened the search for the possible trigger. These 96 absences could have been a result of the pensioners not wanting to give out personal information, not having an easy method of returning the questionnaires, and some pensioners may not have received the questionaries in the first place. This absence of 96 responses has a direct effect on the accuracy of the collected data from these questionnaires, as the data is only a sample of the population, and is not a complete reflection of all pensioners involved. Furthermore, more detailed questions could have been asked on the questionnaires which may have sped the process of identifying the relationship between the symptoms caused and the time of onset.

Improvements with the data collection could have involved the collection of vomit and diarrhoea samples, which would have helped identify the pathogen early on in the investigation. API strips could have been used to aid in identifying the pathogen as fast as possible, as well as using advanced technological methods.

Testing the venue of where the food was prepared further and analysing all possible contributions to the outbreak at the source by further investigating the cook, the staff involved, whether other foods were being prepared at the same time, the overall hygiene of the kitchen, pest related influences, and previous occurrences and whether any have occurred since. Continuous monitoring of all food preparation methods and staff involved since the occurrence will aid in preventing a reoccurrence. A stricter enforcement of an increased hygiene standard could be rolled out onto not only similar cruise companies, but also all kitchens and restaurants throughout Australia. The most feasible and economical method would be to allocate fortnightly swabs of the kitchen, staff, floors, equipment and food samples at all these food-related location, and be sent to relevant laboratories to be analysed and reported. A safety-tick program could be implemented which takes these results and businesses can display them, notifying customers that they conduct regular hygiene tests and have passed all tests, giving the customer piece of mind that their chances of getting food poisoning is less likely.

REFERENCES

Jenson, I & Moir, C. J: In Foodborne Micro-organisms of Public Health Significance. A. D. Hocking et al. (editor) 6th edition. AIFST (NSW Branch) Waterloo NSW 2003.

SAA: Australian Standard. Food Microbiology. Method 2.6: Examination for specific organisms – AS 1766.2.6, pp. D8-D12, 1991.

Benenson AS, ed. Control of communicable diseases in man. 15th ed. Washington, DC: American

Public Health Association, 1990:177–8.

Kho, M.F., Bellier, A., Balasubramani, V., Hu, Y., Hsu, W., Nielsen-LeRoux, C., McGillivray, S.M., Nizet, V. & Aroian, R.V. 2011, ‘The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans’, PLoS One, vol. 6, no. 12, p. e29122.

Wong, H., Chang, M. & Fan, J. 1988, ‘Incidence and characterization of Bacillus cereus isolates contaminating dairy products’, Applied and environmental microbiology, vol. 54, pp. 699-702.

Jesen, G.B., Hansen, B.M., Eilenberg, J. & Mahillon, J. 2003, ‘The hidden lifestyles of Bacillus ceresus and relatives’, Environmental microbiology, vol. 5, pp. 631-40.

Roberts, T. A.; Baird-Parker, A. C.; Tompkin, R. B. (1996). Characteristics of microbial pathogens. London: Blackie Academic & Professional. p.24.

Klietmann, W. and Ruoff, K. 2002. Bioterrorism: Implications for the Clinical Microbiologist. Amer. Soc. Micro. 14(2):364-381.