Introduction

Staphylococcus aureus is a gram-positive, facultative, anaerobic, nonmotile, nonsporing, catalase, and coagulase-positive bacteria (Decastelli et al., 2023). This commensal and opportunistic pathogen can cause a wide range of infections, from superficial skin infections to severe and potentially fatal invasive disease (Kadariya et al., 2014). It produces numerous toxins, including staphylococcal enterotoxins (SEs), specifically named SEA to SEE, SEG to SEI, and SER to SET, with demonstrated emetic activity. The enterotoxins are among the most common foodborne causative agents of food poisoning worldwide (Balaban and Rasooly, 2000; Hennekinne, 2012). SEs are a major cause of food poisoning, which typically occurs after ingestion of foods, particularly processed meat and dairy products, contaminated with S. aureus due to improper handling and inadequate storage at elevated temperature (Argudín et al., 2010). Staphylococcal food poisoning is usually associated with the consumption of protein-rich processed foods, with neutral pH and without high background microflora (e.g. meat preparations, creams, and dairy products) (De Buyser et al., 2001). These food items are vehicles of amino acids and low-molecular-weight peptides that support the survival and growth of S. aureus (Peles et al., 2007).

In general, bacterial toxins are among the most frequent causes of foodborne outbreaks (FBOs) in western countries. The European Food Safety Authority (EFSA) (The European Union, 2022) reported that in 2021 FBOs were caused by bacteria (28.5%), bacterial toxins (17%), viruses (6.8%), parasites, and other causative agents (2%).

Toxigenic S. aureus strains can synthesize SEs during the logarithmic phase or during the transition from the exponential to the stationary phase.

The molecular weight of SEs ranges between 20 and 30 kDa. Their mechanism seems to disrupt intestinal activity and induce food poisoning, with nausea, vomiting, abdominal pain, and diarrhea, but usually without fever or hypotension (Castro et al., 2018; Otto, 2014; Hu et al., 2021). Based on their antigenic heterogeneity, more than 20 SEs (SEA–SElV) have been identified. Of the 24 SEs reported in literature, five (SEA, SEB, SEC, SED, SEE, the so-called classic enterotoxins) are well described and can be detected by commercially available assays or in-house methods (Nia et al., 2016; Grispoldi et al., 2021).

The effective dose of SEs causing symptoms has not been defined in humans: estimates range from 6.1 ng SEA (Guillier et al., 2016) to 0.1 μg (Le Loir et al., 2003 ; Asao et al., 2003). In monkeys, all SEs can induce an emetic reaction at a dose of 100 μg/kg (10 μg/kg for SEA) (Omoe et al., 2013).

Regulation (EC) 2073/2005 regulates food microbiological safety in Europe (EU, 2005) based on microbiological criteria for foodstuff. The Regulation expounds the criteria for food safety and process hygiene. If test results for either type of criteria are unsatisfactory, food businesses must take specific action in compliance with the Regulation. For food categories, such as cheese, milk, and whey powder, SEs are listed under food safety criteria applicable to products placed on the market during their shelf life; SEs must not be detectable in 25 g.

Coagulase-positive staphylococci (CPS) are listed under the criterion of process hygiene for a variety of dairy products (made from raw milk, ripened cheese, unripened soft cheese, etc.), with limits ranging from 10 CFU/g to 10⁵ CFU/g depending on the type of heat treatment that they have undergone. In any case, if the number of CPS is >10⁵ CFU/g, the cheese batch is to be tested for SEs.

An FBO is suspected when two or more cases of a similar disease seek medical attention at a hospital or from their family physician after ingestion of a common food. The Foodborne Disease Outbreaks Guidelines for Investigation and Control (WHO, 2008) specify the actions to be undertaken in the event of an FBO, as well as delineate the roles, objectives, operative procedures, and management of investigations for communicating information that the competent agencies can and must release.

Here, we report a multidisciplinary investigation that food competent authorities conducted in collaboration with veterinary and health and hygiene service, after a notification of suspected symptoms in consumers of a common meal.

Materials and Methods

Case Presentation

A family (two grandparents, two daughters, four young grandchildren) were having a short vacation in the Alps in Piedmont, northern Italy, in the summer of 2022. On 9 August, four of them presented with gastrointestinal symptoms (vomiting and diarrhea) or headache at around 6.30 p.m. No fever was reported by any of the patients (Table 1). On this day, the family had breakfast at their place of stay and lunch at a restaurant during a mountain hike (Table 2). The family received medical attention for symptoms of suspected food poisoning. Food safety competent authority inspectors were notified for collecting food samples. Due to self-limiting illness, biological fluid samples were not collected.

Food Sample Collection

On 10 August, food safety agency inspectors were asked to make an inspection in a small restaurant on the Alps where the family (eight members) had lunch the day before and after which one adult and three children showed gastrointestinal symptoms. The food inspectors collected food samples at the cafeteria where the family consumed sandwiches. Five portions of ham and five portions of raw milk cheese, both used for preparing sandwiches, were collected with sterile devices (scissor, laboratory plastic bags, cutter, and scalpel) and transported refrigerated to the Food Safety and Quality Laboratory, Istituto Zooprofilattico Sperimentale del Piemonte Liguria and Valle d’Aosta, Turin. The food business operators were informed that a microbiological investigation for suspected food poisoning was being conducted; they were summoned to appear in person or send their legal representative to the laboratory at 11 a.m. on 11 August. From the moment of collection to the moment of analysis, samples were always kept refrigerated at 4±2 °C for 24 h.

In the present FBO, the district food safety agency conducting the investigation requested the laboratory to test for microbial pathogens with longer incubation times and hygiene indicator microbes. This was done to exclude other pathogens or causative agents and to provide the health inspectors with information about the level of hygiene at the food business where the cheese sandwiches were prepared.

Food Sample Analysis

The Food Safety and Quality Laboratory is accredited according to ISO17025:2017; all analyses performed on official food samples are validated and accredited. The laboratory was requested by the food safety agency to conduct microbiological analysis to detect bacterial pathogens and bacterial toxins. Table 3 presents the potential causative agents and the laboratory method applied.

Confirmatory quantitative analysis for quantification of SEs was performed at the Anses Maison-Alfort Laboratory for Food Safety European Union Reference Laboratory for CPS, including S. aureus (EURL-CPS).

Results and Conclusion

Laboratory analyses were performed on two food matrices (ham and cheese) collected at the restaurant and used for the preparation of lunch sandwiches. Each sample was divided into five subsamples. Laboratory analysis was negative for Listeria monocytogenes, Salmonella spp, and Campylobacter spp. Table 4 presents the results of testing for hygienic criteria and enumeration of food contaminant bacteria if >10 CFU/g. The cheese subsamples were all positive for CPS (range, 1.1*10³ CFU/g to 8.1*10³ CFU/g). Enzyme-linked fluorescence detected SEs A to E in all five cheese subsamples. A portion of the five subsamples was sent to EURL-CPS; the pooled sample tested positive for staphylococcal enterotoxin D (SED) (estimated concentration, 0.649 ng/g).

In this FBO, the time to symptom onset was about 3 hours after lunch, which suggests that the food contained preformed toxins. Laboratory analysis detected SEs (about 0.649 ng/g) in the cheese sandwiches. Even if diarrhea is not the most representative symptom for SEs intoxication, it is often mentioned among symptoms of SEs FBO (Argudín et al., 2010). The other presenting symptoms were indicative of the causal agent and the quantity of toxins in the food. The sandwiches were approximately made with a standard portion of cheese (about 30 g); the likely amount of SED each case consumed was approximately 20 ng.

From an analytical perspective, a CPS dose between 1.3*10³ and 8.1*10³ CFU/g may seem to contradict the published data (Wong and Bergdoll, 2002) and the European food safety criterion (EU, 2005). It is widely recognized that the production and the release of emetic toxins by S. aureus strains occur at a concentration of about 10⁵ CFU/g. This concentration is cited by food safety norms. Indeed, health agencies require that official laboratories detect preformed toxins as a safety criterion and as a hygiene criterion whenever the concentration is above the limit. The laboratory tests revealed low concentrations of Escherichia coli and Enterobacteriaceae, indicating a fecal contamination, being nevertheless compliant with food safety legislation (EU, 2005). Although the hazard analysis and critical control point (HACCP) manual indicated that good manufacturing protocols were followed and well described, the food safety agency inspectors asked the food business operator to strictly respect the food safety own-check program, as well as the procedures for good hygiene practices, namely, cleaning and disinfection. In addition, the operator was asked to conduct staff training on food hygiene and to meet safety standards.

On further investigation by the district food safety competent authority, it was noted that the food business operator was unable to provide accurate information of the supplier of the cheese, and no invoices of the cheese were available. This violates general food safety regulation (EU, 2002) that requires food business operators to maintain records that can allow for traceability and backward traceability of the products they sell. Furthermore, the lack of traceability of the product did not permit the competent authority to perform additional inspections at the farm/facility where the cheese was made and to know the characteristics of the milk used. Due to the preformed toxins detected in the cheese sample, a primary CPS contamination at the production step is suspected. A secondary contamination deriving from human handling during the preparation of the sandwiches at the cafeteria wouldn’t have allowed SEs synthesis, due to short time.

During an official investigation into a suspected case of food poisoning, the microbiological examination of collected samples is a one-time process, primarily due to the perishable nature of the product under investigation. In addition, the food business operator or their legal representative could ask to be present at the laboratory. Finally, to ensure a representative sample for the laboratory, acknowledging that the causative agents may not be uniformly distributed throughout the sample, the official samples are divided into multiple smaller subsamples. Also in the reported case, the subsampling was crucial for the detection of toxins and pathogenic bacteria in single subsamples, which would have permitted to declare the entire original sample as noncompliant with food safety standards.

Here, we report an instance of FBO in which doctors, food safety agency staff, and official laboratory staff collaborated. Following notification of suspected food poisoning, rapid response by the district food safety agency facilitated the collection of samples of the same lot of cheese used for preparing the sandwiches at the food business facility. There was no other report or notification of suspected FBO in the same period and in the same area, probably due to small-scale cheese production in the mountain area in the summer or because of underreporting of self-limited illness.

According to this report, the current limit of 10⁵ CFU/g CPS for the search for SE must also be questioned. In cases of FBO with symptoms compatible with the presence of enterotoxins, it may be necessary to consider lower S. aureus loads. This paper showed that this parameter for detecting staphylococcal toxins may be suboptimal for identifying microbial contamination potentially harmful for consumers.