Prevalence and antimicrobial resistance of Enterobacterales bacteria isolated from retail food in Poland

Enterobacterales are Gram-negative, mostly non-pathogenic or opportunistic bacteria that are part of the gastrointestinal microflora of humans and animals (19). They can include pathogenic species (e.g. of the Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella and Citrobacter genera) responsible for foodborne infections which are some of the most serious health problems, especially in persons with compromised immunity and in the elderly, children or pregnant women (1, 15, 16, 22). Furthermore, extraintestinal infections linked to enterobacterial species are also often observed, mainly as a result of bacterial translocation from the gastrointestinal tract to the blood or lymph nodes. These infections can trigger generalised disease processes such as bloodstream infections, chronic kidney disease, haemolytic uraemic syndrome, spleen infections, osteomyelitis and meningitis (16, 25). Infections with Enterobacterales are usually treated with β-lactam antibiotics including penicillins, carbapenems and cephalosporins, and antimicrobial resistance to these antibiotics is a major current problem in medicine (19, 26), threatening public health and impacting the economy negatively. Antimicrobial agents are not always effective in the treatment of infections because in some instances bacteria have acquired resistance to all available antibiotics (5, 9). Acquisition of resistance genes is likely for Enterobacterales with their wide environmental dispersion, and these bacteria can quickly transmit them to other microorganisms (39).

Recently, a rapid increase in the number of microorganisms resistant to antimicrobial agents has been observed, which is related to the common use of antimicrobials, especially in human and veterinary medicine, livestock farming and crop production (9, 33). Antimicrobial-resistant bacteria originating from the environment and animals may be the source of resistance mechanisms for components of the human microflora. In addition, food such as meat and meat products, milk and dairy products, fish, fruit and vegetables is an important reservoir of bacteria with antimicrobial resistance genes (5, 24, 37). Important mechanisms of Enterobacterales’ resistance to antimicrobials involve the enzymes they produce such as β-lactamases with an extended spectrum of action, metallo-β-lactamases and cephalosporinases, which are the main defence mechanisms of microorganisms against β-lactam antibiotics (23, 26). The ability of Enterobacterales to accumulate, disseminate and express antimicrobial resistance mechanisms is also a very important public health issue. Countering the risk of the rapid spread of antimicrobial-resistant bacteria, especially multidrug-resistant (MDR) strains in the human environment, requires the implementation of monitoring and control procedures.

Moreover, a major risk for public health constitutes ready-to-eat food, including retail food of animal origin intended for direct human consumption. In this type of food pathogenic microorganisms such as Listeria monocytogenes, Staphylococcus aureus but also various species of Enterobacterales can easily survive and cause foodborne illness (29). Food of this type is susceptible to microbiological hazards to an extent depending on the hygienic conditions, food preparation method and the conditions of distribution and storage of the products (38). In recent years, the consumption of this kind of food has significantly increased, especially in developed countries as a result of lifestyle changes and its greater availability. Consumption carries the risk of foodborne diseases, which poses a serious threat to consumer health (29, 38).

The aim of the present study was to investigate the prevalence and antimicrobial resistance of selected species of Enterobacterales isolated from retail food of animal origin produced in Poland. This study does not mainly focus on Salmonella, Klebsiella or Shiga toxinproducing E. coli species, which are the Enterobacterales with widely reported human pathogenicity in the literature. It also examines non-pathogenic members of the Enterobacterales order, which serve as useful indicator organisms providing information on general food hygiene.

Food may be a source of bacterial pathogens, including antimicrobial resistant strains which pose a serious threat to consumer health. For this reason, studies on the prevalence of potentially pathogenic microorganisms in different foods are performed in many countries. In the present study, the prevalence and antimicrobial resistance of Enterobacterales bacteria isolated from food products of animal origin available in retail in Poland were described. A high prevalence of Enterobacterales was observed in all tested samples. These results are similar to those reported by other authors, where Enterobacteriaceae were detected at a level of 87.2% in Portuguese deli meats and in 68.0%–100% of Egyptian fish samples, with the percentage depending on the species of fish tested (2, 28). The dairy product results of previous research are also consistent with our results: in Egyptian raw milk Enterobacteriaceae were identified in 84% of samples, and in Spanish unpasteurised San Simón cheese they were found in 96.1% (31, 34), which confirms that cheeses produced from raw milk may be contaminated by Enterobacteriaceae (20). On the other hand, lower ranges of Enterobacteriaceae prevalence were observed by Sobeih et al. (31) in Egyptian yogurt (32%–40%) and ice cream (20%–64%).

In the current investigation, E. coli, E. cloacae, H. alvei, Citrobacter spp., S. liquefaciens and K. oxytoca were the most frequently isolated bacteria. Similar results were obtained by other authors who observed a significant contamination of different food products with such bacteria (19, 31, 32). In the present study it was also noted that the presence of specific Enterobacterales microorganisms was related to the type of samples tested. H. alvei, E. cloacae, and S. liquefaciens were most frequently detected both in cold cuts and cold-smoked fish, whereas the majority of bacterial isolates from cheeses were recognised as E. coli. A large species diversity of bacteria isolated from various products similar to that observed in this study was also described by other authors. A previous study conducted in Poland (32) showed that Enterobacter spp., Proteus spp., Hafnia spp., Serratia spp., Klebsiella spp. and E. coli were the most frequently detected microorganisms in cold cuts. Furthermore, E. coli, M. morganii, K. oxytoca and K. pneumoniae were isolated from deli meat in Portugal (2). In addition, Jansen et al. (17) found E. coli in meat products imported into the European Union, and in the large-scale studies of Schwaiger et al. (30), Enterobacter, Citrobacter, Serratia and Klebsiella were the most common microbial contamination identified in chicken and pork. However, these results are difficult to compare with our findings, because those studies were related to raw meat and not the retail meat products which were analysed in the current investigation. According to Mladenović et al. (20, 21), K. oxytoca, K. pneumoniae, K. ornithinolytica and E. coli were the most frequently identified bacterial species in raw milk cheese. Trmčić et al. (35) indicated that the milk type used for cheese production was also significantly associated with the species of Enterobacteriaceae detected in the finished product, which is consistent with our results. This may be related to the microbiological quality of the raw material used in the food production, the processing and technological processes and the distribution and storage conditions before consumption. Mostly dissonant species findings were presented in the investigations conducted on dairy products in Egypt, in which the most often isolated microorganisms from raw milk samples were Hafnia alive (31.0%), S. liquefaciens (25.0%) and K. pneumonia (15.5%), whereas E. coli was the predominant microorganism identified in yoghurt (44.4%) (31). In another study, 221 isolates from San Simón cheese in Spain were mainly identified as K. oxytoca, H. alvei, E. coli, E. cloacae and C. freundii (34). Some literature data which are similar to our current results indicate noticeable associations between the type of food samples and their contamination with specific Enterobacterales species. The Proteus, Hafnia, Escherichia and Enterobacter genera are usually associated with mammalian gastrointestinal systems, and their occurrence in meat, cold cuts or dairy products may be the result of food cross-contamination (16, 19, 31). However, the sources of microbiological contamination of food of animal origin intended for direct human consumption are usually not clear and may be related to contamination at various stages of food production.

The six most frequent Enterobacterales bacteria identified in the current study were then analysed for resistance to antimicrobials. It was shown that E. coli was mainly resistant to tetracycline, but also to a lesser extent to ampicillin, ciprofloxacin, nalidixic acid, sulphamethoxazole, trimethoprim and gentamicin. Similar results were obtained by the authors of other studies, which showed that E. coli isolates very often were resistant to many classes of antibacterial agents. In the study of Ryu et al. (27), resistance to tetracycline (30.7%), streptomycin (12.8%), cephalothin (11.7%), ampicillin (6.7%), trimethoprim/sulphamethoxazole (6.7%) and nalidixic acid (5.6%) was found in E. coli strains isolated from fish and seafood in Korea. The majority of isolates identified in our study as E. cloacae were resistant to ampicillin and azithromycin, whereas K. oxytoca was mainly resistant to ampicillin. It was consistent with the observations reported in other studies (2, 19, 39). However, in the investigation of Schwaiger et al. (30), E. cloacae isolated from meat products were mainly colistin-resistant (45.5%), and K. oxytoca and Citrobacter isolates were resistant to many antimicrobial agents from the β-lactam and aminoglycoside classes. In the present investigation, low resistance (especially to ampicillin, cefotaxime and ciprofloxacin) was observed among Citrobacter isolates, while a high level of resistance to colistin was identified in S. liquefaciens and in H. alvei, which were also resistant to β-lactams. These observations were in line with the results reported by Szewczyk et al. (32) and Mladenović et al. (19).

Our findings also showed that 8.8% of isolates tested were MDR, which is a lower percentage than those reported by other authors. Amador et al. (2, 3) observed MDR phenotypes among isolates from delicatessen meat (35.9%) and cheese (31.4%). In that study, MDR was often associated with E. coli and S. enterica. Multidrug resistant isolates of Enterobacter, Citrobacter and Klebsiella species were also described by Chauhan et al. (4) and Fakruddin et al. (8) in various food samples, including raw milk and dairy milk products. On the other hand, Schwaiger et al. (30) and Uzeh et al. (36) identified MDR strains in Enterobacter spp., Serratia spp., Klebsiella spp. and Citrobacter spp. isolated from chicken meat.

The results of the present investigation indicate that retail food can be contaminated with various species of Enterobacterales, including isolates resistant to commonly used antimicrobial agents. Some bacterial species diversity in different food types was identified. The obtained results revealed that most Enterobacterales isolates showed resistance to the ampicillin and colistin, and to a lesser extent to cefotaxime and ceftazidime of the third-generation cephalosporins, and meropenem belonging to carbapenems. The significant resistance to antimicrobial agents, including MDR strains, may be the result of the common use of antibiotics in agriculture and animal husbandry. For this reason, it is necessary to monitor microbial resistance in the environment, including bacteria belonging to the Enterobacterales order, which are commonly isolated from food. Enterobacterales have public health and economic importance, and because they are considered indicatory microorganisms for the microbiological quality of food and the hygiene standards of its production, their presence in large numbers may suggest improper processing or post-processing contamination of food.