Prevalence and Antimicrobial Resistance of Bacterial Foodborne Pathogens Isolated from Raw Bivalve Molluscs Subjected to Consumption in Poland during a Ten-Year Period

The aim of this study was to evaluate the microbiological contamination of raw bivalve molluscan shellfish (BMS) available on the Polish market and determinate the antimicrobial resistance of the obtained isolates. A total of 1000 mollusc samples were tested for the presence of Salmonella spp., L. monocytogenes, V. parahaemolyticus, and S. aureus using the ISO standard methods. Additionally, the bacterial isolates’ susceptibility to antimicrobials was determined using the minimum inhibitory concentration (MIC) method. The obtained results showed that Salmonella spp. was detected in 31 (3.1%) samples, and 51.6% of the bacterial isolates were classified as Salmonella Typhimurium. A total of 74.2% of the Salmonella isolates were sensitive to all antimicrobial agents, whereas three isolates were multiresistant. L. monocytogenes was isolated from 18 (1.8%) BMS, and the isolates belonged to serogroups IIa, IIb, and IVb. Most of them were resistant to ceftriaxone (77.8%) and oxacillin (55.6%). V. parahaemolyticus was present in 24.2% BMS. These isolates were mainly resistant to ampicillin (77.3%) and streptomycin (64.0%). Moreover, 15.2% of the bivalve molluscs were contaminated with S. aureus. Most isolates belonging to this species were resistant to penicillin (84.9%). A total of 60 (6.0%) bivalve molluscs were contaminated with more than one pathogen simultaneously. In addition, the tested bacteria were more likely to be identified during the warmer period (53.9%) compared to the samples analyzed in colder months (35.7%). The obtained results indicate that raw bivalve molluscs from the Polish market are frequently contaminated with bacterial foodborne pathogens, which may be resistant to antimicrobials.


Introduction
Bivalve molluscs play an important role in the human diet, mainly due to their health benefits, providing a rich source of animal protein, omega-3 fatty acids, vitamin D, selenium, and iodine [1]. The consumption of bivalve molluscs has significantly increased worldwide in recent years, which has led to a high demand for their production and import in various parts of the world [2,3].
BMS can be contaminated with pathogenic microorganisms for humans, including bacteria that are naturally occurring in marine environments, such as Vibrio spp., and those associated with water pollution, such as Salmonella spp., but also pathogens transmitted to food through contaminated processing environment (Listeria spp.) and through poor hand hygiene (coagulase-positive staphylococci, CPS) [3,4]. The presence of bacterial pathogens is often closely correlated with the quality of the waters in which bivalve molluscs live. Household and agricultural wastewater, the uncontrolled storage of organic waste, and municipal and industrial pollution may be sources of the microbiological contamination of foods of marine origin [5,6]. Environmental factors also have a strong influence on the degree of BMS contamination. The variability of temperature, oxygen concentration, and water salinity during that year have a significant impact on the microbial populations in the aquatic environment, as well as on the physiological condition of bivalve molluscs and their ability to filter and accumulate bacteria [7][8][9]. Increases in the water temperature of seas and oceans have been observed as a result of global climate warming, which favors the appearance of pathogenic bacteria in seafood [9,10]. Moreover, bivalve molluscs can be contaminated with some microorganisms after they have been harvested, often due to cross-contamination during processing, distribution, and selling [11]. The greatest risk associated with the consumption of seafood contaminated with bacteria occurs when the bivalve molluscs are consumed raw or after only a short heat treatment. According to the recent EFSA/ECDC report, in European countries in 2020, crustaceans, including raw bivalve molluscs, were the source of 15.3% of confirmed foodborne outbreaks [12].
Bivalve molluscs may also serve as reservoirs of bacteria that are resistant to antimicrobials. The acquisition of a resistance to antimicrobial agents among bacterial pathogens may be a result of their common use in human and veterinary medicine, as well as in agriculture and aquaculture [13][14][15]. An aquatic environment can be a source of antimicrobial-resistant bacteria that can be transmitted directly or with food and water and cause infections in humans, including those that are difficult to treat [13,15]. Therefore, it is very important to conduct studies on the identification of various bacterial pathogens in seafood and their antimicrobial resistance to assess the risk of infection for consumers.
The aim of this study was to evaluate the microbiological contamination of raw bivalve molluscs available on the Polish market and to determine the antimicrobial resistance of the obtained isolates.

Sample Collection
A total of 1000 raw bivalve molluscs were collected during 2009-2018 (100 samples per year) according to the monitoring plan for the bacterial contamination of bivalve molluscs developed by the National Reference Laboratory in Poland. Different species of BMS were examined: clams (n = 437), mussels (n = 269), oysters (n = 225), and scallops (n = 69). All samples, from various countries, were obtained from the wholesale companies and retail shops located all over Poland (Table 1). After collection, the molluscs were immediately transported to the laboratory at a refrigerated temperature and tested within 24 h.

Isolation and Identification of Bacterial Pathogens
The bivalve molluscs (with flesh and intravalvular liquid) were prepared for bacteriological examinations according to the ISO 6887-3:2003 standard [16]. The samples were then homogenized in a blender for 2 min and subjected to specific analyses.

L. monocytogenes
Twenty-five grams of bivalve molluscs sample were used for the detection of L. monocytogenes, according to the ISO 11290-1:1996 + A1:2004 standard [19]. The method involves selective enrichments in half-Fraser and Fraser broths (bioMérieux, Lyon, France), respectively, followed by the isolation of bacterial colonies on Agar Listeria, according to Ottaviani and Agosti (ALOA, bioMérieux, Lyon, France) and PALCAM agar (Bio-Rad, Hercules, CA, USA). The presumptive L. monocytogenes isolates were confirmed by biochemical and CAMP tests, as well as with the API Listeria and VITEC 2 Compact systems. Multiplex PCR was used to determine the main L. monocytogenes molecular serogroups (IIa, IIb, IIc, and IVb), as described previously [20]. For this purpose, DNA was extracted according to the GenomicMini protocol (A&A Biotechnology, Gdansk, Poland) with modifications by adding 15 µL of lysozyme (10 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and incubation at 37 • C for 30 min. The DNA amplification was carried out in a thermal cycler (Biometra, Jena, Germany) under the following conditions: initial DNA denaturation at 95 • C for 5 min, followed by 30 cycles of 94 • C for 1 min, 55 • C for 1 min, and 72 • C for 2 min. The final cycle was carried out at 55 • C for 2 min and 72 • C for 5 min.

V. parahaemolyticus
The isolation of V. parahaemolyticus was performed according to the ISO/TS 21872-1 +Cor1:2008 standard [21]. A 25-g sample was added to Alkaline Saline Peptone Water (ASPW, Bio-Rad, Hercules, CA, USA) used as the first and second enrichment broth. Then, the cultures were plated onto two selective media, Thiosulfate Citrate Bile and Sucrose agar (TCBS, Merck, Darmstadt, Germany) and Tryptone-Soya-Tetrazolium agar (TSAT, in-house, Poland), incubated at 37 • C for 24 ± 2 h. The suspected V. parahaemolyticus colonies were first confirmed by the API 32E, VITEC 2 Compact and a halotolerance test with different concentrations of NaCl. Then, PCR for the detection of the species-specific toxR gene, which is the recommended method to confirm the presence of V. parahaemolyticus, was used [ [23]. The samples were cultured in Giolitti-Cantoni broth (Bio-Rad, Hercules, CA, USA) with potassium tellurite, and the bacterial colonies were isolated on Baird-Parker agar with rabbit plasma and fibrinogen (BP-RPF, bioMérieux, Lyon, France). The obtained coagulase-positive Staphylococcus spp. isolates were then confirmed using the API Staph and VITEC 2 Compact for Gram-positive bacteria systems.

Antimicrobial Resistance Testing
One confirmed bacterial isolate from each positive sample was tested for resistance to the respective antimicrobial agents ( Table 2). A microbroth dilution method was used to establish the minimum inhibitory concentrations (MICs) of the antimicrobials contained in the appropriate Sensititer custom susceptibility plates (Thermo Fisher Scientific, Waltham, MA, USA) dedicated to each bacterium. Multidrug resistance (MDR) of the tested isolates was defined as resistance to at least three classes of the antimicrobials used in the study (Magiorakos et al. 2012).

Salmonella spp.
Salmonella isolates were subjected to antimicrobial susceptibility testing against antimicrobials present in the EUVSEC plate ( Table 2). The isolates were subcultured twice on nutrient agar, and then, the MIC values were determined with Sensititer Cation Adjusted/TES Mueller-Hinton Broth (Thermo Fisher Scientific) at 36 • C for 18-20 h using the Vision system (Trek Diagnostic System, East Grinstead, UK). The antimicrobials and cut-off values used for the interpretation of the MIC results were in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) recommendations based on the decision of the European Commission (EC) No. 652/2013 [24] (www.eucast.org; www.eurl-ar.eu; accessed on 20 September 2022).

L. monocytogenes
The antimicrobial resistance test for L. monocytogenes isolates was performed with the GPN3F plate containing a panel of 17 antimicrobials (Table 2), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [25,26], but the breakpoints for ampicillin, penicillin, and trimethoprim were defined by Lyon et al. [27] and Escolar et al. [28]. Moreover, for the purposes of the present study, isolates marked as intermediate were considered together with isolates sensitive to the antimicrobial agents.

V. parahaemolyticus
The MICs of the antimicrobials for the V. parahaemolyticus isolates were determined using Mueller-Hinton Broth with 1% NaCl (in-house, Poland) and antimicrobial plates (EUMVS2 and EUVSEC) containing antimicrobials used for the treatment of human infections ( Table 2). The obtained results were read using the Vision system and interpreted according to the CLSI guidelines [29]. The breakpoint for streptomycin, which was not established by the CLSI, was taken from earlier studies [30,31]. Table 2. Antimicrobials used for the determination of resistance of the tested bacteria.

Application for Resistance Testing
Salmonella spp.

L. monocytogenes
V. parahaemolyticus S. aureus antimicrobial was used; −, antimicrobial was not used.

S. aureus
The S. aureus isolates were cultured on Columbia agar supplemented with 5% sheep blood (bioMérieux), and the antimicrobial resistance was determined with Sensititer Cation Adjusted/TES Mueller-Hinton Broth and the DKVP and EUST plates. The antimicrobials and cut-off values used to determine the MICs were in accordance with the EUCAST and EURL-AR recommendations (www.eucast.org; www.eurl-ar.eu; accessed on 20 September 2022).

Statistical Analysis
The chi-square test with Yates's correction was used to the examine the differences in the prevalence of bacterial pathogens and type of bivalve molluscs tested, as well as the distribution of microorganisms using Statistica ver. 10 software (StatSoft, Krakow, Poland). p < 0.05 was considered significant.

Prevalence and Antimicrobial Resistance of Bacterial Pathogens
During the study period, a total of 1000 bivalve molluscs samples were tested for the presence of Salmonella spp., L. monocytogenes, V. parahaemolyticus, and S. aureus, and the obtained isolates were evaluated for antimicrobial resistance. The results of these analyses are shown in Table 3, Table S1, and Figure 1, respectively. Thirty-one (3.1%) BMS samples were contaminated with Salmonella spp., mainly clams and mussels (13 and 11 samples, respectively). No statistically significant differences (p > 0.05) were observed in the presence of this bacterial pathogen in various types of bivalve molluscs. The Salmonella-contaminated BMS mostly originated from the Netherlands (15; 48.4% samples) and, to a lesser extent, from other countries ( Table 3). Serotyping of the isolates showed that 16 of them (51.6%) belonged to Salmonella Typhimurium, including two strains from oysters from the Netherlands, identified as monophasic S. Typhimurium Most of the tested Salmonella isolates (23 out of 31; 74.2%) were sensitive to all antimicrobial agents used, and only a few strains showed resistance to colistin (4; 12.9%), ampicillin, tetracycline, and sulfamethoxazole (3; 9.7% of each) (Figure 1a). Moreover, three Salmonella isolates (two from oysters and one from clams, all from the Netherlands), classified as S. Typhimurium, displayed multiresistant patterns: AMP-TET-SMX (two strains) and AMP-CHL-CIP-GEN-NAL-SMX (one isolate). Thirty-one (3.1%) BMS samples were contaminated with Salmonella spp., mainly clams and mussels (13 and 11 samples, respectively). No statistically significant differences (p > 0.05) were observed in the presence of this bacterial pathogen in various types of bivalve molluscs. The Salmonella-contaminated BMS mostly originated from the Netherlands (15; 48.4% samples) and, to a lesser extent, from other countries (Table 3). Serotyping of the isolates showed that 16 of them (51.6%) belonged to Salmonella Typhimurium, including two strains from oysters from the Netherlands, identified as monophasic S. Typhimurium Most of the tested Salmonella isolates (23 out of 31; 74.2%) were sensitive to all antimicrobial agents used, and only a few strains showed resistance to colistin (4; 12.9%), ampicillin, tetracycline, and sulfamethoxazole (3; 9.7% of each) (Figure 1a). Moreover, three Salmonella isolates (two from oysters and one from clams, all from the Netherlands), classified as S. Typhimurium, displayed multiresistant patterns: AMP-TET-SMX (two strains) and AMP-CHL-CIP-GEN-NAL-SMX (one isolate). L. monocytogenes was detected in 18 samples (1.8%), mainly in clams (12; 66.7%), but also in mussels (3 strains) and oysters (3 strains) (p > 0.05). Most of the contaminated molluscs originated from the Netherlands and France (six samples of each), and the remaining positive mussels were from Ireland (three samples), Italy (two samples), and Denmark (one sample). Molecular determination of the L. monocytogenes serogroups revealed that eight isolates (six from clams from the Netherlands and two from Irish oysters) belonged to serogroup IIa, six isolates (all from clams from France) to IIb, and four isolates (three from mussels and one from oysters from other countries) were classified to the IVb serogroup.
Most L. monocytogenes isolates were resistant to ceftriaxone (14 of 18; 77.8%) and oxacillin (10; 55.6%) (Figure 1b). However, only one isolate belonging to molecular serogroup IIa, obtained from clams from the Netherlands, was sensitive to all tested antimicrobials. Moreover, one L. monocytogenes strain isolated from French clams and classified in the IIb serogroup was multiresistant to clindamycin, erythromycin, linezolid, oxacillin, penicillin, rifampicin, and vancomycin.

V. parahaemolyticus
V. parahaemolyticus was identified by the ISO method in 261 (26.1%) samples, whereas PCR for the species-specific toxR gene confirmed the presence of this microorganism in 242 (24.2%) samples (Table 3). This number of isolates was used for the subsequent study. The majority of isolates were obtained from clams (151; 62.4%) and mussels (56; 23.1%), whereas the remaining 35 (14.5%) isolates were of oyster and scallop origin (Table 3). Statistically significant differences (p < 0.05) were observed when comparing the prevalence of V. parahaemolyticus in each type of bivalve molluscs with each other, except a pair of oysters being compared (29 positive samples) and scallops (6 samples) (p > 0.05). The greatest differences were observed when the pathogen was in the presence of clams and mussels (p = 0.0001), clams and oysters (p < 0.0001), and clams and scallops (p < 0.0001). Most of the V. parahaemolyticus-contaminated molluscs originated from the Netherlands (150; 62.0% samples).

Simultaneous Occurrence of Bacterial Pathogens
A total of 60 (6.0%) bivalve molluscs were contaminated with more than one pathogen, mainly with two bacterial species simultaneously (59 samples) ( Table 4). The majority of these bivalve molluscs were positive for V. parahaemolyticus and S. aureus (43; 71.6%). The remaining samples were contaminated with Salmonella spp. and S. aureus (7; 11.7%), Salmonella spp., and V. parahaemolyticus (5; 8.3%), as well as with L. monocytogenes and V. parahaemolyticus (4; 6.7%). Interestingly, one sample (a clam from the Netherlands) was positive for three pathogens: Salmonella spp., V. parahaemolyticus, and S. aureus (Table 4). On the other hand, a total of 618 (61.8%) of all samples used in the present study were free from all tested bacterial pathogens, whereas 322 (32.2%) samples were contaminated with only one bacteria, including Salmonella spp. (18 samples), L. monocytogenes (14 samples), V. parahaemolyticus (189 samples), and S. aureus (101 samples).

Discussion
Bivalve molluscs may be contaminated with bacterial pathogens, and there are serious safety concerns connected with the consumption of raw or undercooked food of marine origin [2,32]. For this reason, studies on the prevalence of pathogenic microorganisms in this kind of food have been performed in many countries. In the present study, the results of the long-term monitoring of the microbiological contamination of raw bivalve molluscs available on the Polish market are described.
One of the microbiological food safety criteria for live bivalve molluscs is the presence of Salmonella spp., which must not be present in any of the five analyzed samples [33]. In our study, this microorganism was found in 3.1% of the examined BMS, which makes such food potentially dangerous for consumers. In other investigations, these bacteria were found in 0-2.5% of raw BMS [3,34]. A high prevalence of Salmonella spp. was observed in Asian countries, particularly in tropical regions, where this pathogen was detected in 24.5% of the shrimp samples in Vietnam and 34.2% of the clams in India [35,36]. Environmental factors, including water quality, play a significant role in the contamination of foods of marine origin with Salmonella, which poses a risk for consumers [3,34,37]. Salmonella was isolated from a variety of seafood, such as fish, shrimp, clams, mussels, oysters, crabs, and others, and its prevalence was usually the highest in molluscs, clams, and shrimps [36]. In the current study, although a relatively few positive samples were detected, Salmonella was also identified mainly in clams and mussels. Among over 2500 Salmonella serovars, only some were connected with food of marine origin, i.e., S. Weltevreden, S. Rissen, S. Derby, and S. Typhimurium [36,38]. The latter one was also the most frequently identified during the present study (51.6% of Salmonella-positive samples).
L. monocytogenes is an important foodborne pathogen responsible for human listeriosis which is especially dangerous disease due to its high mortality rate [3,12]. Only 1.8% of the tested bivalve molluscs samples were contaminated with L. monocytogenes. Other surveys have shown that the presence of this pathogen in food of marine origin ranged from 0% (in Greece) to 68.0% (in Denmark) [39,40]. The Listeria species most frequently isolated from fish and BMS were L. innocua, L. monocytogenes, and L. welshimeri [40,41]. However, only L. monocytogenes is considered the most important cause of food-borne infections and deaths in developed countries [3,42,43]. Most were caused by strains of the IIa, IIb, and IVb serogroups, which were also identified in the present study. It has been described that four L. monocytogenes serotypes-1/2a, 1/2b, 1/2c, and 4b classified into the IIa, IIb, IIc, and IVb serogroups, respectively, are typically isolated from approximately 95% of human listeriosis cases [20]. Among them, L. monocytogenes serogroup IVb is considered to be the most pathogenic serogroup responsible for the majority of listeriosis outbreaks, whereas serogroup IIa is the most commonly isolated from food [12,20]. The L. monocytogenes serogroup IIa was predominant in the bacteria detected in fish in Ireland (>95.0%) and Finland (86.0%), whereas serogroup IVb was revealed only in 14.0% of the isolates [44,45]. Momtaz and Yadollahi [46] reported that L. monocytogenes serogroup IVb was most often (66.7%) detected in marine food samples, while serogroups IIa and IIb were identified in 5.6% and 27.8% of bacterial isolates, respectively.
In the investigation, 24.2% of raw bivalve molluscs samples were positive for V. parahaemolyticus, and this result is similar to those reported by other authors, where the prevalence of vibrios varied from 0% in BMS from Greece to 35.0% in fish from Portugal [40,47]. The present results indicate that almost all V. parahaemolyticus isolates originated from clams, mussels, and oysters, mainly from the Netherlands. In a similar study conducted in Europe, V. parahaemolyticus predominantly contaminated oysters (42.2%), mussels (33.1%), and clams (24.7%) [48]. This bacterial pathogen was detected in the marine water of several European countries, including Great Britain, France, Spain, Italy, and Slovenia [2,49]. V. parahaemolyticus is often associated with molluscs; however, its high prevalence was also observed in fish, especially in Asian countries [50]. There is no microbiological criterion for raw bivalve molluscs in relation to V. parahaemolyticus in the European Union, but testing for the presence of this microorganism is recommended for molluscs harvested from water that was suspected to be contaminated with Vibrio sp. [32,40].
In the current study, coagulase-positive S. aureus was detected in 15.2% of samples of the examined bivalve molluscs. This is a lower prevalence than was identified in raw bivalve molluscs in Spain (37.0%) and mussels in Greece (56.6%) [40,51]. S. aureus is an important bacterial pathogen, causing food-borne infections and intoxications. The most common contamination of the food of marine origin occurs during its processing under poor hygienic conditions. However, for food-borne intoxication, a high number of S. aureus is needed. The current microbiological criteria do not provide any requirements for this microorganism [33]. Nevertheless, enterotoxigenic coagulase-positive S. aureus may be a serious risk for human health if present in the consumed raw bivalve molluscs [3,40].
The present study shows that some BMS samples (6.0%) were contaminated with more than one tested bacterial pathogens; mainly, they were simultaneously positive for V. parahaemolyticus and S. aureus. There is little information concerning the prevalence of more than one microorganism in bivalve molluscs. A correlation between the presence of Salmonella spp. and Vibrio spp. in various kinds of bivalve molluscs was observed by Hariharan and Amadi [15] and Atwill and Jeamsripong [52]. The latter authors noticed that the contamination of shellfish with Salmonella spp. was significantly associated with the type and source of molluscs, their sampling location, and the presence of E. coli and Vibrio spp. [52]. In the current study, it was also found that the presence of more than one pathogen was mainly identified in clams and mussels harvested in the Netherlands (Table 4).
However, a relatively low number of these samples did not allow for the determination of any correlation with bivalve molluscs species and their geographical origin.
It was previously shown that the prevalence of bacteria in bivalve molluscs depends on the seasons [8][9][10]. In the present study, V. parahaemolyticus was detected more often in the warmer months (32.1% positive samples), when the water temperature usually exceeds 15 • C, than in the colder period (17.1%). The results obtained by Ottaviani et al. [53] showed that this microorganism was identified at a slightly lower level (24.3%) in Adriatic mussels during the warmer months (May-September). Environmental conditions such as salinity and temperature have an important influence on the growth of V. parahaemolyticus [38,53,54]. Hatha and Lakshmanaperumalsamy [55] noticed that the lower temperatures during the monsoon months stimulated the growth of Salmonella spp. and the prevalence of these bacteria in seafood was significantly higher in the colder period (26.1%) than in the warmer pre-and post-monsoon seasons (6.4 and 7.1%, respectively). Moreover, L. monocytogenes can survive and multiply at low temperatures, including marine environments [38].
An antimicrobial resistance analysis of the current bacterial isolates from bivalve molluscs revealed that most Salmonella were sensitive to all antimicrobials dedicated to this pathogen and only a few strains showed resistance to colistin, ampicillin, tetracycline, and sulfamethoxazole. The recognized resistance, although referring to a single isolate, has a potential influence on public health because for the treatment of Salmonella infections. usually ampicillin, trimethoprim-sulfamethoxazole, and also chloramphenicol were used [56]. However, the crucial is resistance to colistin, which is the antimicrobial last chance in humans for the treatment of multidrug-resistant Gram-negative bacteria. The mcr colistin resistance gene may be disseminated between different bacteria because of its presence in the plasmid. It has been shown that many Salmonella isolates from various sources possess this gene [57]. In other studies, Gram-negative bacteria isolated from shellfish, including Salmonella, were resistant to ampicillin (60.0%), streptomycin (29.0%), amoxicillin-clavulanic acid (20.0%), tetracycline (17.0%), and chloramphenicol (8.6%), and none were resistant to enrofloxacin, ciprofloxacin, and trimethoprim-sulfamethoxazole [15]. In the study of Peruzy et al. [58], 54.0% of Salmonella isolates from molluscan shellfish were sensitive to all selected antibacterial agents, whereas 29.0% of the strains of this origin were resistant to one or two antimicrobial classes, and 17.0% were identified as multiresistant isolates.
L. monocytogenes is usually sensitive to most antimicrobials used against Gram-positive bacteria, except a natural resistance to the first-generation of quinolones, fosfomycin, and the third-generation of cephalosporins [59,60]. Listeriosis requires effective antimicrobial therapy, which usually includes β-lactam antibiotic (ampicillin or penicillin), sometimes together with gentamicin. The second choice of treatment is a combination of sulfonamide (e.g., sulfamethoxazole) and trimethoprim. In the present investigation, the majority of L. monocytogenes isolates were resistant to ceftriaxone and oxacillin, i.e., the antimicrobials that are not clinically important in listeriosis treatment. In a similar study, high resistance rates of these bacteria (isolated from ready-to-eat food, including seafood) to ceftriaxone (49.3%) and oxacillin (90.4%) were also shown [61]. Moreover, in the present investigation, one L. monocytogenes isolate was multiantimicrobial-resistant to clindamycin, erythromycin, linezolid, oxacillin, penicillin, rifampicin, and vancomycin, whereas only one isolate of IIa serogroup was sensitive to all the tested antimicrobials.
Vibrio spp., including V. parahaemolyticus, are usually susceptible to most antibiotics used in veterinary and human medicine. The infection caused by these bacteria usually do not require antimicrobial treatment; however, the antibiotics such as tetracycline or ciprofloxacin are sometimes used in severe or prolonged illnesses [62]. The antimicrobial resistance among the tested V. parahaemolyticus revealed that most isolates were resistant to ampicillin and streptomycin. These data are similar to results originating from other countries, where V. parahaemolyticus of shellfish origin was mainly resistant to β-lactams (ampicillin) [63,64]. It was also noted that all V. parahaemolyticus analyzed in the present study were sensitive to chloramphenicol, whereas other investigations demonstrated that 35-100% of such isolates were resistant to this antimicrobial [63,64].
S. aureus is widespread in the environment and in the human body. The resistance of these bacteria to antimicrobials increased worldwide, especially methicillin-resistant S. aureus (MRSA), which is clinically important problem, because this group of S. aureus is not only resistant to β-lactams but also to many other classes of antimicrobial agents, such as aminoglycosides, quinolones, and macrolides [65]. Nowadays, vancomycin is the drug of choice for treating MRSA infection. Most of the current S. aureus isolates showed resistance to penicillin, whereas the levels of resistance to sulfamethoxazole, tetracycline, and erythromycin were much lower. Moreover, none of the S. aureus was resistant to gentamicin. In the study conducted by Marijani, [66], among 16 S. aureus isolates, 31% and 5% were resistant to tetracycline and gentamicin, respectively. However, a resistance to ciprofloxacin, clindamycin, and penicillin was observed in 19%, 13%, and 13% of isolates, respectively.
Bacteria resistant to antimicrobials occur naturally the environment, including water reservoirs, aquatic plants, and animals. Some substances such as tetracyclines and fluoroquinolones may persist in the environment for months or even years [67]. The resistance-encoding genes can be transferred between the same species but also among not closely related bacteria by horizontal gene transfer of mobile genetic elements. The reservoir of these genes in the environment may be naturally resistant bacteria that also often possess genetic elements carrying multiple traits of resistance.

Conclusions
In conclusion, the results of the present long-term study investigation indicate that raw bivalve molluscs may be contaminated with various bacterial pathogens, including simultaneous contamination with more than one of the bacterial species. The relatively high prevalence of V. parahaemolyticus highlights the importance of raw bivalve molluscs as potential vehicles for transmission of these bacteria. Moreover, this pathogen was detected more often in the warmer period than in the colder months. On the other hand, few molluscan shellfish were contaminated with L. monocytogenes, and for 3.1% of the samples, the food safety criterion for Salmonella spp. was exceeded. Considering the simultaneous occurrence of bacterial pathogens, it was found that V. parahaemolyticus and S. aureus were most frequently isolated from bivalve molluscs, mainly clams. Several of these microorganisms were resistant to the commonly used antimicrobials. High levels of resistance to ceftriaxone and oxacillin were observed among L. monocytogenes isolates, to ampicillin and streptomycin in V. parahaemolyticus, and to penicillin in S. aureus isolates, whereas most Salmonella spp. were sensitive to all tested antimicrobials. Although, only a few of the bacterial isolates showed multiresistant patterns, continued investigation of the antimicrobial resistance of these pathogens is important to establish effective treatment in humans. To prevent food-borne diseases due to the consumption of raw bivalve molluscs, it would be reasonable to test such food, looking for the bacteria that constitute a potential threat to public health and not only those included in the official microbiological criteria.