The importance and necessity of controlling food microbiological safety in the Republic of Armenia

Introduction

Improperly managed food can become a breeding ground for various pathogenic bacteria, which pose significant challenges to public health by causing a high number of illnesses. Bacterial food poisoning is most commonly associated with Salmonella spp., Campylobacter spp., Clostridium perfringens, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes, among others (Hernández-Cortez et al., 2017). These bacteria possess characteristics that enable them to evade the host immune system, leading to various health disorders.

Cooking food before consumption reduces the risk of foodborne illness by killing harmful bacteria. However, some bacterial genera, such as Staphylococcus, Bacillus, and Clostridium, produce heat-stable toxins that can remain harmful even after cooking (Hernández-Cortez et al., 2017,Merry, 1997). Outbreaks of foodborne illness have been linked to a variety of food sources, including meat, dairy products, vegetables, fish, cereals, and legumes.

Poultry, in particular, is often contaminated with Salmonella spp., Staphylococcus aureus, and occasionally Bacillus cereus, as well as psychrotrophic pathogens such as Listeria monocytogenes. In the European Union, eggs and egg products are major reservoirs of human salmonellosis (Mor-Mur et al., 2010). The levels of certain pathogenic species, such as Listeria monocytogenes and Staphylococcus aureus, found in raw milk are generally lower than disease-causing concentrations.

Between the 1950s and 1970s, there were several incidents involving pathogenic microbes in processed dairy products, including Salmonella species in dried milk products and Staphylococcus aureus in cheese (Collins et al., 1968;Hendricks et al., 1959). Ready-to-eat foods may be contaminated by various bacteria, including species of Bacillus, coagulase-negative staphylococci, Escherichia coli, Listeria monocytogenes, Pseudomonas spp., and Staphylococcus aureus (Annan-Prah, 2011; Festus and Damilola, 2018;Gdoura-Ben Amor et al., 2018). Therefore, it is crucial to analyse data related to foodborne illness incidents that have occurred in the Republic of Armenia.

Materials and methods

Sample collection

The material for microbiological analysis consisted of food products submitted for examination to the National Bureau of Expertise of the Republic of Armenia.he material for microbiological studies was composed of food products forwarded for the examination to the National Bureau of Expertise of the Republic of Armenia. These included vegetable and meat salads, cheese and meat appetisers, pork, lamb, fish dishes, and other food products. A total of 23 forensic investigations were conducted as part of the cases related to food poisoning incidents that occurred in various institutions throughout the Republic of Armenia, such as hotels, restaurants, and kindergartens.

State Standard Specifications (GOST in Russian) applied for the study

The bacteriological study of the material involved the isolation and identification of microbial cultures. These procedures were performed using specific methodologies to determine each group of microorganisms, in accordance with GOST standards. For isolation and determination of coliforms, QMAFAnM, yeasts and fungi, sulfite-reducing clostridia, Staphylococcus aureus, Proteus spp., and Pseudomonas aeruginosa were appliedGOST 31747-2012,GOST 10444.15-94,GOST 10444.12-2013,GOST 29185-2014,GOST 31746-2012,GOST 7702.2.7-2013, andGOST R 54755-2011, respectively . The collected data were compared with the permissible units outlined inTR CU 021/2011 ("Technical Regulation of the Customs Union on Food Safety") (Table 1).

Table 1. The measurement units and permissible standards of microbial indicators as outlined in TR CU 021/2011 (“Technical Regulation of the Customs Union on Food Safety”)

Data processing

Data evaluation was performed using the appropriate function in Microsoft Excel 2016. Each procedure throughout the analysis has been carried out in duplicate. The statistical analysis of the results included the standard error (m) and the confidence interval (CI) with a 95% probability.

Results and discussion

The National Bureau of Expertise of the National Academy of Sciences of the Republic of Armenia received over 23 cases for examination related to food poisoning. The examination of food products primarily revealed the presence of coliform bacteria (particularly Escherichia coli), Staphylococcus aureus, and other bacterial species at levels exceeding the permissible limits set by regulatory documents (TR CU 021/2011).

A total of 16 types of food products and drinking water samples were tested to evaluate their compliance with current regulatory requirements. These included nine thermally processed food products and seven non-thermally processed products. It is important to note that the same product could be tested for multiple parameters at the same time.

The total number of tested samples is 205, with 138 (67.3%) not meeting the permissible units specified in the regulatory documents of the Republic of Armenia (Table 2) corresponding to 242 non-compliant indicators, as some samples failed multiple criteria simultaneously (Table 3).

It is important to note that soups (8 samples, 100%), which are typically cooked at high temperatures, can still become contaminated with harmful microbes, especially coliforms (refer to Table 3, Supplementary 1), due to secondary contamination (Valero et al., 2016). Coliforms are known to be sensitive to elevated temperatures and generally do not survive temperatures exceeding 60 °C (Denis et al., 2006).

Regarding meat products, including thermally treated ones (40 samples, 93%), the failure to comply with permissible limits in both thermally non-processed and thermally processed samples reflects inadequate hygienic and thermal conditions during preparation or storage (Orta-Ramirez and Smith, 2002).

The rates of non-compliance with QMAFAnM, coliform bacteria, E. coli, S. aureus, Proteus spp., sulfite-reducing clostridia (Cl. perfringens), P. aeruginosa, yeast count, fungal count, total yeast and fungal count, and B. cereus were 25.6% (95% CI: 20.1–31.1), 42.1% (95% CI: 35.8–48.4), 9.5% (95% CI: 5.8–13.2), 5.8% (95% CI: 2.8–8.7), 3.3% (95% CI: 1.1–5.5), 1.2%, 1.7% (95% CI: 0.1–3.3), 2.5% (95% CI: 0.5–4.4), 5.8% (95% CI: 1.3–10.3), 2.1% (95% CI: 0.3–3.9), and 0.4%, respectively.A typical representative of coliform bacteria is E. coli, which is associated with sanitary conditions. The presence of E. coli in food samples indicates recent fecal contamination. In our analysis, E. coli was isolated from the following food items: three samples of soups, five samples of thermally processed meat products, two samples of thermally non-processed meat products, six samples of sausages, five samples of salads containing vegetables and meat, and one sample each of thermally processed and thermally non-processed egg products. Since the samples submitted to our laboratory are linked to forensic cases, we have access to comprehensive information about each case, including details about other laboratory experiments conducted. It is important to note that personnel involved in handling these food items, as well as associated surfaces, were also examined.

Table 2. The overview of the microbial contaminates in analysed food and beverage samples

Table 3. Indicators of microbial contamination in the analyzed food and beverage samples not complying with regulatory standards

It is important to note that the staff handling these food items, as well as the surfaces associated with them, have also been examined. Coliform bacteria were detected on the hands of the personnel.It was evident that coliform bacteria were present on their hands. As anticipated, coliform bacteria were also detected on food-contact surfaces and related items. The isolation of E. coli from ready-to-eat food samples, such as soups, barbecue, pilafs, and salads, highlights its significant importance (Table 4). The presence of coliforms in these items indicates potential secondary contamination (Oh et al., 2018,Herawati et al., 2023). Furthermore, the prevalence of E. coli in industrially produced foods, including sausages, basturma, and sujukh, suggests that inadequate hygienic conditions during production may be responsible (Shaltout et al., 2022). It is worth noting that while non-pathogenic strains of E. coli are generally harmless, they can still lead to gastrointestinal issues in individuals with a weakened immune response (Kashima et al., 2021). The presence of Bacillus cereus bacteria was detected in a canned poultry product sample. Therefore, among various harmful bacteria, Bacillus cereus can survive in sterilized canned food (Bergey, 1994).

A higher prevalence of Staphylococcus aureus was found in salads with addition of mayonnaises. This was followed by ready-to-eat meals, confectioneries, raw chicken meat, and sausages. One of the most severe cases of food poisoning occurred at a wedding, where more than 50 guests became ill, including the bride and groom, who were hospitalized on their wedding day at the Nork Infectious Diseases Hospital. Medical assistance was provided based on the severity of their symptoms. Individuals began to feel unwell 1.5 to 2 hours after consuming the food, which is characteristic of Staphylococcus aureus poisoning. These cases included one sample each from bakery and meat products, as well as five salad samples (Figure 1).

These samples did not meet regulatory standards. The bacteria were primarily detected in salads prepared with homemade mayonnaise, including Olivier salad, Caesar salad, chicken salad, and salad with boiled beef tongue, among others.

During the epidemiological investigation, it was determined that the individual preparing the mayonnaise was a carrier of Staphylococcus aureus. Additionally, non-compliance was identified in terms of QMAFAnM (21 out of 25 samples) and coliform bacteria (23 out of 25 samples). The most affected individuals were children and women who had not consumed alcohol. Individuals who consumed alcohol together with the contaminated food exhibited less severe signs of intoxication. Individuals who consumed alcohol together with the contaminated food exhibited less severe signs of intoxication.

As discussed in one research, commercial mayonnaise has been reported to prevent growth of Staphylococcus aureus (Doyle et al., 1982). In many countries, mayonnaise is mainly made at home and has higher pH values compared to commercial varieties. Additionally, mayonnaise and similar foods may be stored at inappropriate temperatures (Gomez-Lucia et al., 1987).

Proteus species were detected in six raw meat samples and two thermally processed meat products (Figure 1).Wang et al. (2010) reported a case of food poisoning at a restaurant in Beijing, China, caused by Proteus mirabilis. Genotyping and the Dienes method identified the same bacterial clone in the food consumed, as well as in stool samples from patrons, the cook, and the waiter. It is likely that the lack of hygiene among the staff facilitated the transmission of the bacteria to the meal.

Sulfite-reducing clostridia were detected in 3 of 138 samples, including one thermally processed meat product and two canned food samples . These bacteria were primarily found in samples of ready-to-eat meat. This finding is consistent with data from other studies indicating that storing cooked meat at room temperature can promote the germination and growth of Clostridia spores, potentially leading to foodborne illness (Bataeva et al., 2020).

Figure 1. The prevalence of S.aureus, Proteus spp., P.aeruginosa, and Sulfite-reducing clostridia bacteria in various food products examined in our research.

Pseudomonas aeruginosa was predominantly detected in three water samples, including both tap and bottled water. It was also detected in one sample of thermally processed meat product. The highest prevalence of P. aeruginosa in drinking water samples were recorded in Croatia, Serbia and Montenegro (Vukić Lušić et al., 2021).

Conclusion

The examination of food products and beverages showed that each tested sample could fail to meet regulatory standards based on multiple criteria. Overall, the findings of this study indicate that the samples submitted for examination were associated with food poisoning incidents, which explains the high prevalence of microorganisms detected in the analysed food products and beverages.

Author Contributions: AT and SN conceived and designed the study. SN and AT performed the experiments. NA and SM provided reagents and analytical tools. SN and AT wrote the manuscript. NA and SM edited the manuscript and gave recommendations for the experiment. All authors read and approved the final manuscript.

Funding: This work was supported by the National Academy of Sciences (NAS RA) and the Government of the Republic of Armenia.

Acknowledgments: The authors thank the staff of the National Bureau of Expertise of the Republic of Armenia and their colleagues for their assistance and support.

Conflicts of Interest: The authors come out with no conflicts of interest.

Literature