Scientific Achievements in the Study of the Occurrence and Antimicrobial Susceptibility Profile of Major Foodborne Pathogenic Bacteria in Foods and Food Processing Environments in Romania: Review of the Last Decade

Pathogenic bacteria are recognized as a major cause of foodborne diseases in humans, globally, with negative impact on the economy of each country. The aim of the present review was to obtain a comprehensive understanding about the frequency of isolation, diversity, and antimicrobial susceptibility profile of the six major foodborne pathogenic bacteria in food matrices and food processing environment, in Romania. In this regard, results of relevant epidemiological studies, published during the last decade and retrieved from the Web of Science Core Collection database, were analyzed, with special emphasis on scientific achievements, main knowledge gaps, and future perspectives. The summarized and harmonized results offer useful insight, especially for public health authorities and researchers, having a reference effect in stimulating further opportunities for studies to be carried out to address some of the limitations of the current status.


Introduction
Bacterial foodborne pathogens are considered the most frequently implicated biological agents in food poisoning syndrome in humans, often called foodborne illness (FBI). FBI is usually characterized by acute health problems, with gastrointestinal (e.g., diarrhea, vomiting, nausea, or abdominal cramps), or neurological (e.g., headaches, paralysis, or paresthesia) manifestations. In "sensu stricto", two types of FBI are known, namely infection, which is the consequence of ingesting food or water contaminated with pathogenic bacteria, and intoxication, meaning the situation when the toxin produced by the pathogen causes the illness [1][2][3][4].
Currently, due to the existence of several contamination sources with harmful bacteria via the food chain (e.g., animals, soil, water, air, food handlers during production and storage), the obtaining of safe and nutritious food products for the consumer is considered to be a great challenge for the food industry, worldwide. However, adequate cold preservation (e.g., refrigeration or freezing), associated with proper thermal processing of foods can prevent FBI. In case of foodborne diseases, the effectiveness of public health interventions is frequently hampered by the involvement of antibiotic-resistant bacteria. Thus, the continuous monitoring of the antimicrobial resistance phenomenon must be considered a priority for the public health sector [1,4,5].
In each year, the European Food Safety Authority (EFSA), together with the European Center for Disease Prevention and Control (ECDC) and with the contribution of each member state, publishes an open access summarized report about the occurrence of foodborne pathogen bacteria and their antimicrobial resistance profile in the humananimal-food chain [1,4]. Accordingly, year by year, the most frequently registered top five pathogens in descending order, causing severe infections in humans and economic losses are Campylobacter spp., Salmonella serovars, Yersinia spp., shiga toxin-producing Escherichia coli, and Listeria monocytogenes. The data reported by member states are the result of a harmonized interinstitutional action, which encompasses positive findings from clinical cases, official and national control programs, as well as from the self-control process of the production units [4]. The resulted synopsis from member states, including Romania, does not include results from scientific publications, neither provides exact information about the origin and type of the tested food matrix, in direct association with the detailed antibiotic resistance profile of the implicated bacterial species and/or serotypes. In this regard, summarizing the current understanding, together with the awareness of knowledge gaps in a timely manner, can be the cornerstone of an appropriate control plan of the main bacterial foodborne diseases.
With these considerations, the present review aimed to obtain an overview of the baseline data on the occurrence of major food-borne pathogenic bacteria (Salmonella spp., Listeria monocytogenes, E. coli, Staphylococcus spp., Campylobacter spp., and Yersinia spp.), and their antimicrobial susceptibility profile (where data is available) in a food matrix and/or food processing environment, processing results of relevant epidemiological studies retrieved in the Web of Science Core Collection database and published during the last decade. In the foreground, scientific achievements, main knowledge gaps, and future perspectives are approached.

Materials and Methods
A search was conducted in the Web of Science Core Collection database, in order to identify papers published in international scientific journals, which have undergone a rigorous peer review process, containing research data generated by Romanian researchers at country-level, between January 1st, 2010 and March 31th, 2020. The search strategy consisted of simultaneous use of three search terms, specifically, the name of one of the targeted foodborne bacteria, including "Salmonella", "Listeria", "E. coli", "Staphylococcus" "Campylobacter" and "Yersinia", and other two basic search terms namely "Romania" and "food".
At first, the inclusion criteria were based on the individual screening of the title and abstracts in search of resulting publications. Subsequently, if the basic information was deemed appropriate to meet the goal of the study, it was then included in the review and subjected to further, in-depth analysis.

Results and Discussion
Initial search in the database generated a total of 61 publications. Notwithstanding of that total, only 21 studies contained data that met our inclusion criteria and were subsequently included and processed in this review. The Table 1 summary presents the harmonized results of the included papers.
3.1. Salmonella spp. Salmonella spp. is a widely distributed pathogen, causing one of the most feared infections in humans, the so-called salmonellosis. Due to this fact, their presence is closely monitored within the food chain steps. For the finished foodstuffs, the absence of Salmonella spp. represents a safety criterion. In agreement with these considerations, the search results revealed that Salmonella spp. has been the most studied foodborne pathogen in Romania within the last decade. The processing of the data from the 11 publications dedicated to the Salmonella topic has revealed the isolation of a total of 435 strains. Out of them, 402 (92.4%) have been serotyped.
Among the analyzed categories of nonfood matrices, Salmonella was recovered from absorbent food pads from packages with raw chicken meat [12], scald water sludge, and detritus from hair removal [7] with different detection rates, as highlighted in Table 1.
Antimicrobial resistance was recorded for at least one of the 37 antimicrobial agents reported as tested. The exhibited resistance profile of the tested Salmonella strains varied widely from one study to another ( Table 1). The registered differences can be sustained by the use of different testing methodologies and lack of uniformity of the drugs enrolled in the investigations. Nevertheless, the overuse of some antimicrobials (e.g., tetracycline, azithromycin, nalidixic acid, sulfamethoxazole, streptomycin) in Romanian veterinary medicine is noteworthy. Contrary, five studies [3,4,[7][8][9] reported complete susceptibility for ceftazidime, which can 2 BioMed Research International Table  1: Overview about the occurrence and diversity of major foodborne pathogenic bacteria and their antimicrobial susceptibility profile in foods and food processing environments, in Romania.

Listeria
Monocytogenes. L. monocytogenes is the causative agent of listeriosis in humans, a severe illness with a high fatality rate and atypical evolution. Like in the case of other foodborne diseases, consumers such as young children, elderly people, and pregnant women are more susceptible to foodborne listeriosis than healthy adults [13]. The readyto-eat (RTE) products are considered high-risk foods, because they support the growth of L. monocytogenes and do not require heat treatment before consumption. Also, the persistence of L. monocytogenes in the food processing environments is well documented [14]. Therefore, the control of this pathogen for producers of RTE foods is a major challenge. Over the last decade, in Romania, there was a total of nine studies that published data on the isolation frequency of L. monocytogenes from a food matrix or food processing environment. The pathogen was recovered from a variety of food products, including raw (pork, beef, sheep, poultry, and snail) and mechanically processed meat, RTE, milk and dairy products, chicken carcasses, fish, and fresh salads [5,6,10,[14][15][16], with different prevalence values as highlighted in Table 1. Two studies were designed to identify L. monocytogenes predominantly in the food processing environment, including food contact and nonfood contact surfaces [14,17]. These investigations offered baseline information for food safety managers from the surveyed production units, in order to improve their hazard analysis critical control point plans and sanitation programs. Also, the successful isolation of L. monocytogenes from household refrigerators draws the attention about the risk that is posed, especially for vulnerable consumers, in case of inadequate cooling practices [13].
In the context of the importance of the global fight against antimicrobial resistance, only two studies approaching this problem have been published [5,17]. The results indicate a high prevalence of multidrug-resistant strains in the pork and poultry meat industries. Encouraging results were obtained regarding the total susceptibility of some tested antimicrobials (Table 1), but caution should be taken in interpreting these results, considering the limited number of the tested L. monocytogenes isolates.

Escherichia Coli.
Intestinal and extraintestinal pathogenic variants of E. coli, which is considered a commensal bacterium of the large intestinal tract of healthy hosts, are able to produce infections in humans and animals, with a wide range of manifestations (e.g., mild to severe diarrhea, hemorrhagic colitis, meningitis, septicemia, hemolyticuremic syndrome, or urinary tract infections). Thus, according to the pathogenic mechanism and virulence traits, the diarrheagenic E. coli strains causing diarrhea syndromes have been classified into six subpathotypes including enterohemorrhagic (EHEC, also known as Shiga toxin-producing E. coli [STEC]), enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroaggregative (EAEC), enteroinvasive (EIEC), and attaching and effacing (A/EEC) groups. Human extraintestinal E. coli infections are strongly related by the involvement of the uropathogenic E. coli (UPEC) strains. As member of the fecal coliform group, E. coli is the most commonly used hygienic indicator in the food industry. Presently, within the bacterial genetics' investigations, it is one of the most intensely studied microorganisms, due to its ability to adapt and acquire new genofunds, especially antimicrobial resistance genes [18].
Despite these considerations, a very limited number of studies providing data on the occurrence of E. coli strains in different food products have been published over the last decade in Romania. Likewise, three out of five available surveys do not provide differentiation data between pathogenic and nonpathogenic strains [5,12,19]. The results published by another study, based on biotyping tools, which investigate a large number of samples [6], tried to cover this gap. Accordingly, only the presence of the biotype I (126/11048, 1.14%) has been demonstrated and none of the verocytotoxin producing E. coli O157:H7 (0/454) isolates were identified [6]. Nevertheless, the fact that Romania regularly reports the occurrence of VTEC/STEC infections in humans within the European Centre for Disease Prevention and Control (ECDC) surveillance system [20] opens the opportunity for large scale studies to be carried out, which address some of the limitations of the currently available investigations, with special emphasis on the identification of the O157:H7 E. coli serotype within the food chain.
The antimicrobial susceptibility testing profile of a total of 77 chicken meat origin E. coli isolates showed variable results, from one study to another [5,12,19]. It is noteworthy that the registered high resistance pattern towards some antimicrobials (e.g., tetracycline, ciprofloxacin, sulfamethoxazole) suggests their overuse in the Romanian poultry industry.

Staphylococcus
Aureus. Staphylococcus aureus is considered one of the main causes of foodborne intoxications, being widespread bacteria throughout nature. Its food poisoning effect is related to the ingestion of the preformed enterotoxin produced by strains developed in a food matrix. Additionally, it is well known that S. aureus is able to produce nosocomial and invasive infections in humans (e.g., septicemia, osteomyelitis, skin infections, pneumonia, or infections of the central nervous system) [18,21].
In Romania, during the last decade, despite the existence of a limited number of studies concerning the occurrence of S. aureus within the food chain, the monitoring of this 7 BioMed Research International pathogen in illegally sold food products has been attracting increased attention. In this regard, two studies confirmed the occurrence of the methicillin-resistant S. aureus (MRSA) strains, reporting positive findings in pork lard samples [21], as well as in different fish, dairy and raw meat products [22] ( Table 1). Nowadays, the globally distributed MRSA strains constitute a major concern for the public health, due to their ability to easily gain novel antimicrobial resistance mechanisms [21]. In these studies, successful Spa typing of a total of 16 isolates has resulted in the expression of nine profiles, including t304 (3 isolates), t524 (2), t011, t091, t398, t449 (4), t1606 (2), t3625, and t803, respectively. The first five enumerated types have been frequently reported as being implicated in human infections. Other studies have demonstrated the presence of S. aureus in drinking milk which resulted in a food poisoning outbreak [23], or its absence in raw and ground meat [6].
The antimicrobial susceptibility profile of the tested Staphylococcus strains, presented in two studies [12,21], shows different resistance levels towards different drugs, and complete susceptibility to others (Table 1), but caution should be taken in drawing valid conclusions, considering the low number of tested samples.
3.5. Campylobacter spp. and Yersinia spp. From the species belonging to the Campylobacter genus, which cause campylobacteriosis, C. jejuni and C. coli have the most frequent implication. Campylobacteriosis is the most common cause of human bacterial gastroenteritis among European countries, on a yearly basis [24]. Most food poisonings are caused by the consumption of undercooked poultry meat, and the clinical signs include fever, nausea, vomiting, and intestinal disorders.
The review data showed the availability of only three studies. These investigations have been focused on Campylobacter monitoring mainly in raw chicken meat [4,5,22]. As expected, the overall dominance of C. jejuni has been observed, but the isolation of C. coli has been also demonstrated in a reduced number of samples ( Table 1).
The exhibited multidrug resistance patterns of the isolates highlighted a public health risk, but, at the same time, the total susceptibility against gentamicin, observed in both studies, can constitute an alternative for pathogen control (Table 1). Nonetheless, further investigations, on a larger scale, are still necessary to strengthen this observation.
Human yersiniosis is a foodborne disease, most commonly caused by Yersinia enterocolitica, which is a widely distributed pathogen throughout the environment. Despite the fact that yersiniosis is the third most commonly reported bacterial foodborne gastroenteritis within the European Union in the last years, there is no published evidence of the occurrence of Yersinia spp. in the Romanian food chain appearing in the database. A single study attempted to fulfill this purpose, but all of the investigated chicken carcass samples were negative.
As has been highlighted in other review reports achieved in the United States [26] and China [27], our summarized data can represent a realistic starting and orientation point for applying small meta-analysis models, individually con-structed for pathogen and sample type subgroups, in order to generate hypotheses for future research and risk assessment for food safety [28].

Conclusion Remarks and Perspectives
Analysis of data obtained from a total of 21 relevant studies showed the common occurrence of the targeted major bacterial pathogens within the food chain, differing broadly in terms of examined matrices and depth of analysis. The resulted overall worrying antimicrobial resistance profile of the tested strains strengthens the urgent need of an integrated surveillance system of the dispersion and transmission of drug-resistant bacteria, within the entire food chain. This action requires an excellent collaboration between the environment, veterinary, and public health sectors, according to the One Health approach.
Although the scientific achievements and progresses are remarkable, it is obvious that the number of published papers is limited, precluding meaningful observations in trends of the pathogen isolation frequency. In addition, the large disparity observed between available investigations, according to the targeted pathogen (e.g., Salmonella vs. Campylobacter and Yersinia), and the lack of homogenous reports between some studies restrict the provision of an accurate and straightforward overview of the available data. These limitations may be related to the existence of routine pathogen surveillance within the self-control process of each production unit in accordance with the EU legislation in force, together with the official controls organized by authorities. In this regard, the highly attractive positive findings that were obtained are not disclosed to the knowledge of experts in order to be compared with research-derived data or integrated in a harmonized preventive risk-based program. Nevertheless, enhancing the involvement of Romanian researchers in international collaborations and successful access to financial support from European or national funding agencies can increase the international visibility of Romanian research in the field of food safety.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.