Detection of Listeria monocytogenes while testing food raw materials and products of animal origin for microbiological contamination

INTRODUCTION

Listeriosis is an infectious disease affecting most farm and domestic species (pigs, horses, cattle, sheep, goats, rabbits, chickens, and ducks) as well as humans. The disease-related economic damage includes reduced animal productivity, expenses for medical treatment and prevention, and quarantine control measures. In Russia and other countries worldwide, the incidence of foodborne diseases, including listeriosis, shows no signs of decline [1][2][3]. Listeriosis ranks fifth among the most frequently reported zoonoses in humans in the European Union and is one of the most significant foodborne disea­ses [4]. Infection caused by Listeria monocytogenes is highly dangerous for pregnant women (causing miscarriages), infants (resulting in newborn fatalities) and immunocompromised individuals [5]. Listeriosis in pregnant women and their newborns have been reported in the Republic of Dagestan [6]. In 2021, a Listeria meningoencephalitis case was diagnosed in a COVID-19 patient in the Voronezh Oblast [7]. In 2022, a case of neonatal listeriosis was registered in the Tula Oblast [8].

In the Russian Federation, animal listeriosis has been documented since 1956, while human listeriosis was officially recognized as a distinct nosological phenomenon and included in health records by the Russian Ministry of Health in 1992 [9][10]. Pursuant to the State Report of the Rospotrebnadzor, 100 cases of listeriosis (18 deaths) were registered in the Rusian Federation in 2023. Most cases were reported: in Moscow (32 cases) and St. Petersburg (19 cases) [11].

A key feature of Listeria is its wide growth temperature range, i.e. from 4 to 45 °C (optimum 36–38 °C) and pH range of 5–11 [12]. It has been established that L. monocytogenes is killed when heated to 100 °C for 3–5 minutes or to 75–90 °C for 20 minutes [13]. Listeria demonstrates environmental persistence [14][15], grows in high concentrations of sodium chloride and carbon dioxide, and can survive freezing and drying. They can survive in an oxygen-free environment and are capable of intracellular parasitism [16].

Listeria infection usually occurs when eating contaminated products of animal origin, including fish and seafood that have not been properly heat-treated, as well as vegetables and fruit [17]. A large number of listeriosis cases (15–20%) are associated with consumption of contaminated meat from domestic animals and poultry (15–80%) [18]. In Ethiopia, according to X. Wei et al., L. monocytogenes was detected in raw and pasteurized milk [19]. As the US Centers for Disease Control and Prevention (CDC) reports, there was a listeriosis human case in 2022 caused by cheese produced by Old Europe Cheese, Inc. In 2022, Big Olaf ice cream was the reason behind listeriosis outbreaks in the States of Florida and Ohio. In 2023, CDC reported listeriosis outbreaks associa­ted with leafy greens, as well as peaches, nectarines, and plums [20]. In 2024, in multiple U. S. States, deli meats and liver sausage products under the Boar’s Head brand were contaminated with Listeria, resulting in human disease and death cases [21][22]. According to several studies, the infection may develop even at relatively low bacterial concentrations in food products (10² CFU/g) [23].

According to Food Safety News, in 2022 Federal Office of Public Health and Federal Office for Food Safety and Veterinary Affairs of Switzerland reported a listeriosis outbreak associated with smoked trout [24].

Thus, detection of pathogenic Listeria in the products of animal origin, food raw materials, and ready-to-eat products remains a critical task.

The objective of this research is to detect L. monocytogenes contamination in products of animal origin (meat, fish, dairy) manufactured and marketed in the Nizhny Novgorod Oblast from 2023 to 2024.

MATERIALS AND METHODS

Samples of animal products, including food raw materials and ready-to-eat food products were submitted to the testing laboratory of the Nizhny Novgorod Branch of Federal Centre for Animal Health for the required tests.

Test materials. Totally, 3,650 samples from livestock and aquaculture were analyzed. The samples were collected between 2023 and 2024 from commercial poultry farms and retail chains in the Nizhny Novgorod Oblast of the Russian Federation.

Sampling. Product samples were collected at different storage time points (within the shelf-life period) in accordance with established sampling requirements for microbiological testing. They were delivered to the laboratory in a cooler bag. The delivery time did not exceed an hour.

Culture media: Fraser broth (State Research Center for Applied Biotechnology and Microbiology, Russia); ALOA – Agar Listeria according to Ottaviani and Agosti (Merck, Germany); Listeria Identification Agar Base, PALCAM agar (HiMedia Laboratories Pvt Ltd., India); blood agar (Sredoff, Russia).

Methods. The samples were analyzed and the pure microbial culture was identified based on a set of morphological and biochemical characteristics confirming they belong to L. monocytogenes, in accordance with GOST 32031-2022 “Food products. Methods for detection of Listeria monocytogenes and other Listeria (Listeria spp.)”¹.

Preparations from pure microbial cultures were fixed, Gram-stained, examined microscopically, and identified based on their ability to grow at 25 °C, β-hemolysis, catalase production, Voges – Proskauer test, fermentation of xylose and rhamnose, and lecithinase activity. API Listeria identification system (bioMérieux, France), which includes 10 biochemical tests, was used for identification.

Automatic miniVidas analyzer and a Vidas Listeria test kit (bioMérieux, France) were used to detect L. monocytogenes in food samples.

For statistical data processing and graph construction, Microsoft Excel and standard statistical data analysis methods were used.

RESULTS AND DISCUSSION

Between 2023 and 2024, the testing laboratory of the Nizhny Novgorod Branch of Federal Centre for Animal Health tested 3,650 samples of animal-derived products for L. monocytogenes, i.e. 680 samples of dairy products, 615 samples of fishery products, and samples of 2,355 meat products. Non-heat treated meat products included 323 beef samples, 834 poultry meat samples, 326 combined semi-finished meat products, and 288 pork samples. Moreover 584 samples of finished meat products were also tested, including 187 samples of poultry meat and 397 samples of ready-to-eat meat products, except for poultry meat products. Ready-to-eat meat products included jellied products, sausages, pâtés, as well as heat-treated meat and meat-based convenience products, excluding sausages.

The conducted tests revealed that the bacterial isolates exhibited cell morphology characteristic of Listeria, stained Gram-positive, were catalase-positive, were Voges – Proskauer positive, motile at (25 ± 1) °C, fermented rhamnose but did not ferment xylose, showed lecithinase activity on charcoal agar, and formed a zone of β-hemolysis on blood agar. Thus, identification of pure bacterial cultures isolated from the contaminated samples of the tested products demonstrated that all cultures belonged to L. monocytogenes species.

The test results for animal products produced and marketed in the Nizhny Novgorod Oblast from 2023 to 2024 are given in Table 1.

In 2023, tests revealed 41 L. monocytogenes-contaminated samples, which accounted for 2.1% of all the tested samples (N = 1,970). The proportion of Listeria detected in frozen and chilled products did not differ statistically and amounted to 1.1 and 1.0%, respectively.

Testing of food products produced in 2024 revealed 16 L. monocytogenes-contaminated samples, which accounted for 1.0% of all the samples tested (N = 1,680). In frozen and chilled products, this type of Listeria was detected in 0.4 and 0.6% of cases, respectively.

Figure 1 shows results for food samples tested in 2023. The maximum detection rate of Listeria was recorded in combined semi-finished meat products – 6.3%, beef products – 5.1% and poultry meat – 3.8%. The bacteria rate detected in fishery products was 1.0% of the total number of the tested samples in this category.

Figure 2 shows results for products of animal origin tested in 2024. It was established, that 2.0% of positive samples accounted for combined semi-finished meat products and beef products. Listeria detections in fishery products was 1.5% of the total number of such products tested; in poultry products Listeria was detected in 1.7%; the lowest percentage of detections was noted in pork products – 0.6%.

No L. monocytogenes were detected in dairy products and ready-to-eat meat products that we tested in 2023 and 2024. In 2023, no pathogenic microorganisms were detected in pork products either.

Table 2 gives data on Listeria detected in poultry meat products produced from 2023 to 2024. The maximum L. monocytogenes detection rate was reported in poultry products (N = 834), i.e. in chopped semi-finished poultry meat products (including minced meat) – 1.2%, and the minimum detection rate was reported in mechanically deboned poultry meat – 0.4%.

When testing poultry products, namely poultry meat (carcasses, half-carcasses, wing, leg, thigh), L. monocytogenes were detected in 1.6% of the tes­ted samples of this product category (total number N = 385), and in frozen and chilled products the detection number was equal – 0.8% for each category.

Listeria contamination in the tested chopped semi-finished poultry meat products, such as cutlets, kupati (i.e. spicy Georgian sausages), minced meat, etc., amounted to 4.2% of the tested samples belonging to this product group (N = 236), while 0.4% of detections were reported in frozen products and 3.8% of detections were reported in chilled products.

Listeria was recorded in 9.3% of samples of dough-wrapped semi-finished poultry meat products (frozen dumplings, N = 43), and in 7.1% of tested samples of mechanically deboned poultry meat (only in frozen products, N = 42).

No L. monocytogenes were detected in samples of poultry offal (heart, stomach, liver, raw fat), skin, uncooked convenience semi-finished poultry meat products, and cut-style poultry meat semi-finished products (including the ones soaked in marinade).

Percentage of contaminated samples from all the beef products tested in 2023–2024 (N = 323) (by product type): large-cut semi-finished products – 1.9% (0.6% – frozen; 1.2% – chilled products), minced beef – 0.9% (chilled products), offal, small-cut semi-finished products and semi-finished products wrapped in dough – 0.3% each. No L. monocytogenes were detected in chopped semi-finished meat products (cutlets, meatballs and etc.). The relevant tests results are given in Table 3.

From 2023 to 2024 L. monocytogenes was found in the following types of combined semi-finished meat products (N = 326): in chopped semi-finished products – 2.76% (1.23% – in frozen and 1.53% – in chilled products), in minced meat (chilled only) – in 0.6% of samples, in dough-wrapped semi-finished products – 0.9% (in frozen).

Listeria contamination was also detected in fishery products, namely in freshly frozen shrimps and semi-finished shrimp products containing a flour component (breaded cutlets).

Contamination with L. monocytogenes was found in one of the samples of frozen minced pork, which accounted for 0.3% of the total number of pork products samples (N = 288). The remaining pork processed products (large and small-cut semi-finished products, chopped products, products wrapped in dough, offal) met safety requirements and did not contain Listeria.

The work conducted between 2023 and 2024 resulted in detection of 57 L. monocytogenes contaminated samples, which accounted for 1.6% of the tested animal products. The percentage of Listeria detected in frozen and chilled meat products did not differ statistically – 0.7 and 0.8% of the total number of the samples tested, respectively. Listeria contamination in various food products ranged from 0.6% (samples of pork products) to 6.3% (samples of combined semi-finished meat products). L. monocytogenes detection in fishery products accounted for 1.1% of the total number of tested samples of these products (N = 615), in pork products – 0.3%.

The results we obtained align with the published scientific data from other researchers. Thus, according to the results of food monitoring in 14 EU countries, the total number of L. monocytogenes detections in beef products intended for human consumption in 2019 was 4.2%, in 2020 the number increased to 7.4%, and in 2021, decreased to 3.9% [4]. According to the foreign data, in 2023, number of samples contaminated with L. monocytogenes in category “fish” was 1.1% [3], which correlates with the results of our tests.

As foreign sources report, L. monocytogenes has been detected in milk, different types of cheese, butter, cream, and ice cream [4][20][25][26], as well as in ready-to-eat meat products [21][22][27]. Absence of L. monocytogenes in ready-to-eat meat and dairy products, as confirmed in tests conduc­ted in the Nizhny Novgorod Branch of Federal Centre for Animal Health, may suggest that the establishments where these food products have been produced comply with technological and sanitary standards, the products are properly heat-treated, production hygiene is maintained, quality control of raw materials is well-organized, an effective quality control system is in place, product storage and transportation of conditions meet relevant regulatory requirements. However, regardless of the good current results, continuous monitoring of products shall be in place.

The data obtained indicate that contamination of meat products with L. monocytogenes does not depend on their storage temperature (frozen or chilled). This confirms the cold stress adaptation mechanisms of this pathogen described by foreign researchers [28][29][30]

CONCLUSION

Tests of products of animal origin manufactured and sold in the Nizhny Novgorod Oblast conduc­еted in 2023–2024 show that 1.6% of samples were L. monocytogenes-contaminated. The highest level of contamination was found in combined semi-finished meat products (6.3% in 2023 and 2.0% in 2024), as well as in beef products (5.1 and 2.0%, respectively) and poultry meat (3.8 and 1.7%). At the same time, no L. monocytogenes was detected in dairy products and ready-to-eat meat products, which may indicate compliance of production with technological and sanitary standards.

As for poultry meat products, the highest level of contamination was detected in semi-finished poultry meat products wrapped in dough (9.3%), in mechanically deboned poultry meat samples (7.1%) and chopped products (4.2%), while in whole poultry carcasses the level was lower (1.6%). Among beef products were minced beef (10.7%) and large-cut semi-finished beef products (4.6%). As for fishery products, L. monocytogenes was found in 1.1% (in shrimps and fish cutlets).

Storage temperature (refrigeration/freezing) did not have a significant impact on the level of L. monocytogenes contamination, indicating the cold tolerance of this pathogenic microorganism.

Contribution of the authors: Logatskaya L. N. – review concept, research and analysis, manuscript preparation and writing; Pruntova O. V. –supervision, scientific advice, review concept, manuscript preparation and writing; Zhbanova T. V. – scientific advice on research and analysis, manuscript preparation and writing.

Вклад авторов: Логацкая Л. Н. – концепция обзора, проведение поисково-аналитической работы и исследований, подготовка и написание статьи; Прунтова О. В. – курирование, научное консультирование, концепция обзора, подготовка и написание статьи; Жбанова Т. В. – научное консультирование по проведению поисково-аналитической работы, подготовка и написание статьи.