The occurrence, transmission, virulence and antibiotic resistance of Listeria monocytogenes in fish processing plant

International Journal of Food Microbiology 282 (2018) 71–83

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

The occurrence, transmission, virulence and antibiotic resistance of Listeria monocytogenes in fish processing plant

T

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Krzysztof Skowrona, , Joanna Kwiecińska-Piróga, Katarzyna Grudlewskaa, Agnieszka Świecab, Zbigniew Paluszakb, Justyna Bauza-Kaszewskab, Ewa Wałecka-Zacharskac, Eugenia Gospodarek-Komkowskaa a Department of Microbiology, Nicolaus Copernicus University in Toruń, L. Rydygier Collegium Medicum in Bydgoszcz, 9 M. Skłodowska-Curie St., 85-094 Bydgoszcz, Poland b Department of Microbiology and Food Technology, UTP University of Science and Technology, 6 Bernardyńska St., 85-029 Bydgoszcz, Poland c Department of Food Hygiene and Consumer Health, Wrocław University of Environmental and Life Sciences, 31 C.K. Norwida St., 50-375 Wrocław, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Listeria monocytogenes Salmon Fish processing plant Antibiotic resistance Genetic similarity Virulence genes Serogroups

The aim of this research was to investigate the occurrence of Listeria monocytogenes in fish and fish processing plant and to determine their transmission, virulence and antibiotic resistance. L. monocytogenes was isolated according to the ISO 11290–1. The identification of L. monocytogenes was confirmed by multiplex PCR method. Genetic similarity of L. monocytogenes strains was determined with the Pulsed-Filed Gene Electrophoresis (PFGE) method. The multiplex PCR was used for identification of L. monocytogenes serogroups and detection of selected virulence genes (actA, fbpA, hlyA, iap, inlA, inlB, mpl, plcA, plcB, prfA). The L. monocytogens isolates susceptibility to penicillin, ampicillin, meropenem, erythromycin, trimethoprim/sulfamethoxazole was evaluated with disc diffusion method according to EUCAST v. 7.1. The presence of 237 L. monocytogenes isolates (before genetic similarity assessment) in 614 examined samples was confirmed. After strain differentiation by PFGE techniques the presence of 161 genetically different strains were confirmed. The genetic similarity of the examined isolates suggested that the source of the L. monocytogenes strains were fishes originating from farms. All tested strains possessed all detected virulence genes. Among examined strains, the most (26, 38.6%) belonged to the group 1/2a-3a. The most of tested strains were resistant to erythromycin (47.1%) and trimethoprim/sulfamethoxazole (47.1%).

1. Introduction Since the beginning of the 1990s, the consumption of fish, and especially salmon, in the European Union member countries has increased dramatically (ReportLinker, 2016). With the increase in demand for fish products, the fish processing industry has also developed in Poland. A significant threat to product safety is microbial contamination in the farm environment, which can consequently lead to continuous fish infection (FAO Fisheries Report, 1999). Farms producing and exporting fish pose a direct contamination threat to the target processing plant environment and indirect threat to the health of the consumer (FAO Fisheries Report, 1999). Introducing microbiologically contaminated fish to the processing plant affects the whole production cycle. In subsequent stages of the technological process, the transfer of the microorganism to healthy fish may lead to the contamination of intermediate and ready-to-eat (RTE) products (FAO Fisheries Report,

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Corresponding author at: Skłodowska-Curie St., 85-094 Bydgoszcz, Poland. E-mail address: [email protected] (K. Skowron).

https://doi.org/10.1016/j.ijfoodmicro.2018.06.011 Received 12 February 2018; Received in revised form 7 May 2018; Accepted 11 June 2018 Available online 13 June 2018 0168-1605/ © 2018 Published by Elsevier B.V.

1999). Listeria spp. are Gram-positive, rod-shaped, facultatively anaerobes, and do not produce endospores. They have the ability to grow over a wide temperature range (0.5–45 °C), pH (4.7–9.2) and osmotic pressures. These characteristics allow Listeria spp. for survival in adverse environmental conditions (Dongyou, 2008; Vázquez-Boland et al., 2001). Among 17 known Listeria species, two - L. monocytogenes and L. ivanovii - are pathogenic for humans (Vos et al., 2009). L. monocytogenes, as well as non-pathogenic strains of L. innocua, are capable of colonizing the human gastrointestinal tract (Pappelbaum, 2007). The main source of human listeriosis is contaminated food. Direct infections from human to human are relatively rare (Kołakowska and Madajczak, 2011). L. monocytogenes may be present in RTE products with a long shelf life (EFSA, 2017). According to the report of the European Food Safety Authority (EFSA), in 2016 the presence of L.

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Fig. 1. Scheme of sampling.

monocytogenes has been detected in samples of fishes, poultry, beef and pork, fruits and vegetables, bread, delicatessen products and cheeses (EFSA, 2017). In 2016, EFSA reported 2536 confirmed human cases of listeriosis. (0.47 cases per 100,000 population), which was more than in 2015 (EFSA, 2017). It is estimated that in the USA L. monocytogenes causes almost 1600 cases each year, of which 1400 require hospitalization, and 250 of them result in death (Scallan et al., 2011). A study by EFSA (2017) found that in 2016 fish and fishery products, represented the highest rate of non-compliance with EU standards and stood at 5.6% for fishery products and 4.7% for fish.

Listeria spp. may be a component of water microbiota, especially when contaminated with sewage or animal excrements. L. monocytogenes is present on the outer surface of fish swimming in contaminated water (Miettinen and Wirtanen, 2005). The presence of these bacteria is also observed in the gastrointestinal mucosa and gills (Jami et al., 2014; Pizarro-Cerdá and Cossart, 2006). There are two pathways of potential fish contamination with L. monocytogenes in fish processing plants: (Autio et al., 1999) the spread of intestinal bile to other tissues (including muscles), especially when the time between death and removal of the bowel is longer than several hours;

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13 30.2 3 60.0

L. monocytogenes obtained directly from fish or production environment (after identification but before genetic similarity determination).

2 40.0

2. Material and methods 2.1. Material The research material was 447 fish samples and 109 swabs collected from September 2013 to April 2014 in the fish processing plant located in Poland, at each stage of the production cycle (Fig. 1): raw material storage, decapitation hall, filleting hall, smokehouse, freezer, slicing hall, packing hall, freezer - finished product warehouse (Table 1). Raw fish came from 6 different packaging stations, from 5 farms located in Norway and Scotland. The fish were transported at −2 °C in polystyrene boxes filled with crushed ice. In this form, they were stored in a freezer at 0 °C. Each sample was assigned the origin of the raw material (farm), filleting date, smoking and packing. Samples were attributed to the date of manufacture, the type of salvage and the origin of the raw material. Samples were cut from the middle of the fillet. In addition, 109 cleaning control swabs were collected from the plant environment, including 56 samples in the filleting hall after night cleaning and sanitation (Table 2). Swabs were taken before starting work with any device that was in contact with fish meat. The places with which the employees contacted were also checked. Cleaning control swabs were also collected during the manufacturing process (Table 2). 2.2. Isolation of Listeria monocytogenes strains Analysis of the intermediate and finished product samples was carried out in accordance with the plant's internal procedures, based on ISO 11290–1 (ISO 11290-1, 2017). Samples of fresh or smoked fish meat (25 g) were homogenized with 225 ml of half-Fraser broth (Merck). In the case of swabs from raw material and purity tests swabs, the gauze was immersed in 100 ml of half-Fraser broth (Merck). Samples were incubated at 30 °C for 24 h. Then 0.1 ml of the culture was introduced into 10 ml of Fraser broth (Merck) and the secondary selective enrichment was performed at 37 °C for 48 h. After incubation, a reductive inoculation of material from the culture was performed on the agar plate according to Ottaviani and Agosti (ChromoCult® Listeria Selective Agar, ALOA®, Merck). Cultures were

a

139 31.1 17 32.7 2 20.0 0 0.0 Isolatesa

N %

32 26.9

24 44.4

45 40.9

5 8.8

14 40.0

Total N = 447 Storage warehouse n = 52 Packing hall n = 10 Fridge n = 10 Raw material storage n = 119

Decapitation hall n = 54

Filleting hall n = 110

Smokehouse n = 57

Slicing hall n = 35

10 17.9

Filleting hall n = 56 Slicing hall n = 5 Filleting hall n = 43 Decapitation hall n=5

28 25.7

Total n = 109 After cleaning During production

Swabs Fish samples

Table 1 Number of Listeria monocytogenes isolates in examined samples.

(Autio et al., 2003) cross contamination (improper transport, use of contaminated fish processing equipment) (Jami et al., 2014). In the production process there is a high risk of secondary contamination with L. monocytogenes of products processed with insufficient hygiene in the food processing plant. In the case of salmon fillet production, fodder and filleting are the most important stages of mechanical treatment, where pathogens are most commonly detected (FAO Fisheries Report, 1999). Brine preparation installations, filleting surfaces, scoops, injectors and slicers are the basic devices used in fish processing. These machines are difficult to effectively clean and disinfect, so they are most often contaminated by L. monocytogenes (Cheng-An Hwang, 2007; Djordjevic et al., 2002; Jami et al., 2014). Especially hard to keep clean are floors and niches where removal of L. monocytogenes is very difficult, e.g. contact surfaces or floor drains (Guðbjörnsdóttir et al., 2005). Further process steps such as salting, smoking or freezing do not affect the survival of microorganisms that have the capacity to reproduce under unfavourable environmental conditions (FAO Fisheries Report, 1999). The aim of the study was to determine the frequency and genetic similarity of L. monocytogenes isolates at each stage of the technological process in a selected fish processing plant and to determine the resistance of selected strains to selected antibiotics.

167 30.0

Total n = 556

K. Skowron et al.

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Table 2 The localizations of swabs collection. Technology line department Swabs after night cleaning Filleting hall

Specific place/device part

Number of swabs

Obtained isolates

Skin remover conveyor Fish pin bone remover machine Fish washer machine Sewage grate Squeegee handle Switch of the shoe washer

20 18 10 4 2 2 56

FH-SC1, FH-SC7, FH-SC8, FH-SC9, FH-SC10 FH-SC3, FH-SC5 FH-SC6 FH-SC4 FH-SC2 – 10

Weight display Trays for fish cutting Skin remover conveyor Fish pin bone remover machine Fish washer machine Saline injector Spine separator Main conveyor Fish slicer machine

3 2 8 8 7 8 8 4 5 53 109

– DH-S1, DH-S2, DH-S3 FH-S1, FH-S2, FH-S3, FH-S7 FH-S4, FH-S13 FH-S8 FH-S5, FH-S6, FH-S10, FH-S12 FH-S9 FH-S11 SH-S1, SH-S2 18 28

Total Swabs during production Decapitation hall Filleting hall

Slicing hall Total Total

selected strains of L. monocytogenes was determined with the PulsedField Gene Electrophoresis (PFGE). The procedure for genotyping was performed in accordance with the Standard Operating Procedure for PulseNet PFGE of Listeria monocytogenes (PNL04, April 2013) (PulsNet, 2013). To determine the degree of genetic similarity between isolates, a phylogenetic dendrogram was drawn in the program CLIQS 1D Pro (TotalLab). Clustering analysis was performed using hierarchical clustering with the UPGMA technique with Dice's coefficient.

incubated for 24 h at 37 °C. Selected colonies, identified according to the manufacturer's recommendations as a Listeria spp., were transferred to Columbia Agar with 5% sheep blood (Biocorp). L. monocytogenes exhibited β-hemolysis, which differentiated it from other Listeria species. Next, the selected isolates were frozen in brain-heart infusion broth (BHI, Merck) with 15% glycerol (Avantor) at −70 °C. 2.3. Genotypic and species identification

2.5. Molecular serotyping of Listeria monocytogenes strains

DNA was isolated using column methods with the Genomic Mini AX Bacteria Spin kit (A&A Biotechnology) according to the protocol provided by the manufacturer. The species identification of L. monocytogenes in the test samples was confirmed by PCR using two pairs of primers (L1 5′-CAG CAG CCG CGG TAA TAC-3′; L2 5′-CTC CAT AAA GGT GAC CCT-3′; LM1 5′-CCT AAG ACG CCA ATC GAA-3′; LM2 5′-AAG CAC TTG CAA CTG CTC′3) (Oligo.pl) (Pappelbaum, 2007). The standardized PCR protocol for 25 μl reaction mixture included 1 × PCR buffer (Promega), 2 mM MgCl2 (ABO), 1.25 mmol dNTPs (Promega), 0.5 μM of each primer (Oligo.pl), 1 unit of Taq DNA polymerase (Promega) and ultrapure water. DNA isolated from L. monocytogenes ATCC 19111 strain was the control. The PCR program was set as follows: initial denaturation 94 °C/2 min; 30 cycles of denaturation 94 °C/30 s, annealing 50 °C/30 s and duration 72 °C/1 min; extension 72 °C/1 min. The resultant PCR products were further analyzed by agarose gel electrophoresis (1.5% agarose); stained with Midori Green (NIPPON Genetics EUROPE GmbH) and visualized by a UV trans-illuminator. Two PCR products were found: 938 bp-length product specific for Listeria genus, and 700 bp-length product specific for L. monocytogenes species (Fig. 2).

Multiplex PCR for identification of the main L. monocytogenes serogroups (1/2a-3a, 1/2b-3b, 1/2c-3c, 4b-4d-4e) was performed as described previously Doumith et al. (2004). The L. monocytogenes strains tested by Wałecka-Zacharska et al. (2012) were used as reference strains for serogroups identification. 2.6. The frequency of selected virulence genes The multiplex PCR technique was used in order to determine the frequency of selected virulence genes among L. monocytogenes. The occurrence of 10 genes encoding selected virulence factors was evaluated. Three separate PCR reactions were prepared. The multiplex PCR reactions were carried out using previously isolated genomic DNA. As the reference strain, the L. monocytogenes IW 41 strain was used. The reactive mixture contained: 1 × PCR buffer (Promega), 25 mM MgCl2 (ABO), 10 mM dNTPs (Promega), 10 μM of each primer (Oligo.pl), 1 U Taq DNA polymerase (Promega), ultrapure water (Sigma Aldrich) and 2 μl DNA. The total volume of the reactive mixture was 25 μl. The amplification conditions and primers sequence are presented in Table 3. The electrophoretic separation of PCR products was performed in 1.5% agarose gel with an addition of the intercalating dye Midori Green (NIPPON Genetics EUROPE GmbH).

2.4. Genetic similarity After confirming the species identity, the genetic similarity of the

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meropenem, erythromycin and trimethoprim/sulfamethoxazole) were applied. Prepared antibiograms were incubated at 35 °C for 20 h. After the incubation period, growth inhibition zones around the antibiotic discs were measured. The results have been interpreted in accordance with the EUCAST v. 7.1. (2017) recommendations. 3. Results 3.1. Isolation of Listeria monocytogenes strains A total of 556 samples were taken: 447 from fish samples, and 109 from swabs collected from production hall. The presence of L. monocytogenes was found in 139 samples (31.1%) of fish samples, and in 28 samples (25.7%) of swabs. The highest percentage of L. monocytogenes isolates (24, 44.4%) was found in samples taken from fresh fillets in decapitation hall (Table 1). In 119 samples from the raw material, the presence of tested microorganisms was detected in 32 (26.9%) samples. Among the swabs collected from the production hall, L. monocytogens isolates were found in 18 (34.0%) samples taken during production and 10 (17.9%) samples taken after night time cleaning and sanitation. 3.2. Genetic similarity of Listeria monocytogenes strains For strains isolated from each type of sample, PFGE genetic similarity was assessed and isolates of the same genetic profile were identified (Fig. 3). These isolates were treated in further studies as one strain (Table 4). Among all examined isolated, 33 (19.8%) were classificated as strain RMS1, 16 (9.6%) – as RMS6, 13 (7.8%) – as RMS8, 24 (14.4%) – as RMS14, and 10 (6.0%) - as RMS28. All isolates cultured from raw fish samples belonged to one of above mentioned strains. In the biggest group of isolates (RMS1), isolates were mainly (81.8%) collected from fish samples delivered by farm A. Among 12 isolates cultured from raw fish samples, 10 were delivered by A farm since 17.09.2013 until 26.02.2014. Four isolates of RMS1 (RMS1, RMS2, RMS3, RMS4) were cultured in raw fish samples delivered in 17.09.2013. The same strain was isolated in samples collected during next stages of working fish samples the same day (DH1, DH2, DH3, FH1, FH2, FH3, SH1 SW1, S1, and S2) from the same farm. From raw fish samples were cultured also isolate delivered by B farm (RMS5), and by E farm (RMS15). The same strain was found in samples collected in storage warehouse, before transport products to consumers. None of the isolates of RMS1 strain was found in swabs collected during production. Isolates cultured from A farm's raw fish samples were found also in samples delivered in 16.04.2014, but they belong to another strain (RMS8: RMS31, RMS32). First isolate of RMS8 strain from raw fish sample was cultured in sample delivered by farm B in 08.10.2013 (RMS8, RMS9). It was also found in the fish samples collected half year later in storage warehouse (SW13). Among isolates of RMS14 strain, the same strain was found in fish sample delivered by E farm in 01.12.2013 (RMS14), in fish sample collected the same day in decapitation hall (DH15), and in environmental swabs collected during production (FH-S8, FH-S9). Another isolates of RMS14 strain were cultured also from swabs collected after night cleaning, from fish washer machine in 11.02.2013 (FH-SC6), and from skin remover conveyor in 16.0.2014

Fig. 2. Electrophoregram presenting the results of L. monocytogenes identification with PCR method (M – DNA Ladder 100–1000 bp (A&A Biotechnology), K + - standard strain L. monocytogenes ATCC 7644, L6–L22 – tested isolates).

2.7. Susceptibility testing The antibiotics susceptibility of studied L. monocytogenes strains was determined using the disc-diffusion method. Based on 24-hour L. monocytogenes cultures, suspensions of the tested isolates were prepared in 0.9% saline solution (Avantor) with an optical density of 0.5 in Mac Farland standard. The suspensions prepared in this way were plated on MHF medium (Mueller Hinton Agar with 5.0% horse blood and β-NAD, bioMérieux), and then discs with antibiotics (penicillin, ampicillin,

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Table 3 Primers sequences and amplification conditions. Reactions

Virulence genes

Primers sequences (5′-3′)

Amplicons size [bp]

References

Amplification conditions

MIX I

fbpA

TTATTTCCTCGCATCCTAGC TATCAATTCGACCTGCTGAG ACACGAGCAATAAAATCCCT ATACTGACGAGGTGTGAATG TTTTCGATTGGCGTCTTAGGA ACTGAAGCAAAGGATGCATCTG GCAAGTGTTCTAGTCTTTCCGG ACCTGCCAAAGTTTGCTGTGA TCCGACTAAACAAGGCTATG TGTACCATAATTTTCCGCCA ACGAACAAAGCAGACCTAAT TGTACCATAATTTTCCGCCA ACAAGCTGCACCTGTTGCAG TGACAGCGTGTGTAGTAGCA CAGGCAGCTACAATTACACA ATATAGTCCGAAAACCACATCT TATGACGGTAAAAGCAGATT TTCCCAAGCTTCAGCAACTT CATGAACGCTCAAGCAGAAG AATTTTCCCAAGTAGCAGGA

435

Own sequence

278

Suo et al., 2010

Initial denaturation - 94 °C/2 min. 35 cycles: denaturation - 94 °C/15 s., annealing – 48.5 °C/30 s., elongation – 72 °C/50 s. Final elongation - 72 °C/1 min.

101

Own sequence

794 302

Franciosa et al., 2005 Own sequence

231

Own sequence

131

Rawool et al., 2007

2341

Franciosa et al., 2005

plcA hlyA MIX II

plcB inlB actA iap

MIX III

inlA mpl prfA

Initial denaturation - 94 °C/2 min. 16 cycles: denaturation - 94 °C/30 s., annealing – 57 °C/45 s., elongation – 72 °C/45 s. 19 cycles: denaturation - 94 °C/30 s., annealing – 49 °C/45 s., elongation – 72 °C/45 s. Final elongation - 72 °C/1 min.

1458 706

3.5. Evaluation of the Listeria monocytogenes susceptibility to selected antibiotics

(FH-SC6). It suggests that RMS14 strain might be more resistant to disinfections processes than strain RMS1. Similar situation was obtained for RMS6 isolates delivered by B farm. Isolates of RMS6 strain were found in two raw fish samples delivered in 08.10.2013 (RMS6, RMS7), in fish samples collected during next steps of production the same day, in decapitation hall (DH,7, DH8, DH9), in filleting hall (FH6, FH7), and in swabs collected two days later during production in decapitation hall from trays for fish cutting (DH-S1), and in filleting hall from skin remover (FH-S3), fish pin bone remover (FHS4), and saline injector (FH-S5). The genetic similarity of the examined isolates confirms that the source of the L. monocytogenes strains is probably fish originating from farms and that the procedures used in processing plants are often insufficient to eliminate the contamination of the final products (Fig. 3).

70 genetically different strains of L. monocytogenes were tested for drug susceptibility (Table 6). It was shown that 28 (40.0%) of them were susceptible to all tested antibiotics. Resistance to all tested antibiotics was found only in 5 (7.1%) strains of L. monocytogenes (Table 4). Most of the tested strains (82.9%) were ampicillin-susceptible. Among of all tested strains, most showed resistance to erythromycin (47.1%) and trimethoprim/sulfamethoxazole (47.1%). The strains obtained from the raw material were predominantly resistant to meropenem (80.0%), erythromycin (80.0%) and trimethoprim/sulfamethoxazole (80.0%). Most of these isolates (60.0%) remained susceptible to ampicillin. The results of the evaluation of the resistance of L. monocytogenes isolates to tested antibiotics are presented in Table 6. In each L. monocytogenes serogroup, the strains resistant to the tested antibiotics were found, however their percentage was different (Table 7). The highest percentage of penicillin resistant strains (44.4%) was found in the 1/2c-3a group, ampicillin resistant (30.0%) in the 1/ 2a-3c group, meropenem resistant (40.0%) and erythromycin resistant (60.0%) in the 1/2c-3c group, and trimethoprim/sulfamethoxazole resistant (52.2%) in the 1/2b-3b group. Strains that belong to the 4b-4d4e were most susceptible to examined antibiotics among all obtained serogroups (Table 7).

3.3. Serogroups of isolated Listeria monocytogenes strains PCR products allowing the serological classification of L. monocytogenes strains are shown in an example electrophoregram (Fig. 4). Among the tested L. monocytogenes strains, the strains belonging to all 4 main serogroups were found. Of the total number of strains, the most (27, 38.6%) belonged to the group 1/2a-3a, and the least to 4b-4d4e and to 1/2c-3c (Table 5). The same trends were found for strains isolated from fish. In turn, the most strains isolated from the production environment belonged to the group 1/2b-3b, and the least to 1/2a-3a (Table 5).

4. Discussion The presence of L. monocytogenes on the food production line poses a serious threat to the trader, who may lose potential customers, and to the consumers exposed to infectious agent associated with consumption of contaminated fish. The incidence of L. monocytogenes in the samples tested during the

3.4. The frequency of selected virulence genes It was shown, that all examined L. monocytogenes strains possessed all detected virulence genes (Fig. 5).

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eight months of testing was estimated at 22.8%. Hertemink and Georgsson (1991) found that in Iceland 56.0% of fresh fish for sale were contaminated by L. monocytogenes and other microorganisms belonging to this genus. In 1991, Noah et al. (1991) noted that in 28.0% of the samples were Listeria spp., among which fresh products were contaminated at 49.0% and processed product at 20.0%. ŁaniewskaTrokenheim et al. (2006) found relatively few cases of fish contamination. Of the 112 samples tested, only two cases of L. monocytogenes were detected. In a study by Wieczorek and Osek (2017) in Poland it was shown that 18.9% of samples of fresh and smoked fish were contaminated with L. monocytogenes. Smoked salmon (33.8%), fresh salmon (32.0%) and fresh cod (31.8%) were the most common sources of these bacteria. For three samples, the level of contamination was higher than 100 CFU/g (exceeding safety level for RTF products established by EFSA) (EFSA, 2017; Wieczorek and Osek, 2017). On the other hand, Mędrala et al. (2003) obtained that contamination of raw materials (mainly raw-smoked fish and its products, including cold smoked slices and vacuum packed) is maintained at 4.3–15.4%. The final products were markedly higher (61.3%) contaminated. It was suggested that these products were contaminated by L. monocytogenes during their contact with the production environment. Vogel et al. (2001) have shown that product contamination is predominantly along the technological line, recently confirmed by Fallah et al. (2013). Hites et al. (2004) stated that microbial contamination of the farmed environment is a major threat, which in turn leads to continuous infection of fish. Also from present research it can be concluded that cyclically exported fishes from farm pose a risk for the environmental contamination of the target processing plant. In subsequent stages of the technological process, the transfer of the microorganism to the processed batches of fish can lead to contamination of the intermediate and the ready-to-eat product. However, Autio et al. (2003) did not find the same L. monocytogenes strains in raw fish and final product. Similarly, Lundén et al. (2003) showed that strains derived from raw poultry products were genetically different from those isolated from the finished one. In our studies it was shown that both isolates obtained from fresh fillets and from further products were related to the isolates from the raw material. The results of own research also indicate the inaccurate washing of surfaces and devices, since genetically identical isolates were isolated from different surfaces. The isolate FH-SC6, obtained 11.02.2014 from a swab taken after overnight washing from a fish washer machine, was identical to the isolates RMS24, RMS25, RMS26 obtained from raw fish samples. Ryser and Marth (2007), as potential sources of contamination with L. monocytogenes, indicated hand tools, gloves and gowns of people in contact with meat. Dirty gloves could have caused the strain transmission from the fish to the squeegee and to the display. The worker who weighed the trolley also transported it to the smokehouse. It is possible that on a worker's gloves the operator, after touching the scale display, moved the strain on the trolley and onto the handles opening the smokehouse door. Subsequent workers were able to transfer the strain to further stages of production. Other studies also show that the products were contaminated in the production processes, and that deep tissues were most often infected with head shearing, skin removal and filleting (Eklund et al., 1995). The presence of L. monocytogenes in final product is more often a results of secondary contamination (ŁaniewskaTrokenheim et al., 2006). Also Lundén et al. (2003) confirm that contamination of meat products can occur inside the plant at the production stage. Another device that posed a high risk of spreading L. monocytogenes was the injector. The number of positive trials was significantly higher when salted by injection than by dry rubbing. Also

Fig. 3. Dendrogram presenting the genetic similarity of tested L. monocytogenes isolates and their drug susceptibility profile (RMS – raw material storage, DH – decapitation hall, FH – filleting hall, S - smokehouse, SH – slicing hall, …-S – swab during production, …-S.C. – swab after cleaning).

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Table 4 Genetic similarity of isolates, serogroups and antibiotic resistance of strains. Processing stage

Raw material storage (RMS)

Strain

RMS 1

RMS 6

RMS 8

RMS 14

RMS 28

Decapitation hall (DH)

DH DH DH DH DH DH

10 11 12 13 22 23

Genetically identical isolates/fish farm/isolation date

RMS 1 (A, 17.09.2013), RMS 2 (A, 17.09.2013), RMS 3 (A, 17.09.2013), RMS4 (A, 17.09.2013), RMS5 (B, 08.10.2013), RMS10 (A,27.10.2013), RMS11 (A, 27.10.2013), RMS15 (E, 01.12.2013), RMS16 (A, 14.10.2013), RMS17 (A, 14.10.2013), RMS18 (A, 14.10.2013), RMS27 (A, 26.02.2014), DH1 (A, 17.09.2013), DH2 (A, 17.09.2013), DH3, (A, 17.09.2013), DH4 (B, 08.10.2013), DH18 (C, 31.03.2014), DH19 (C, 31.03.2014), FH1 (A, 17.09.2013), FH2 (A, 17.09.2013), FH3 (A, 17.09.2013), FH12 (A, 27.10.2013), FH13 (A, 27.10.2013), FH14 (A, 27.10.2013), FH40 (A, 16.04.2014), FH41 (A, 16.04.2014), S1 (A, 17.09.2013), S2 (A, 17.09.2013), SH1 (A, 17.09.2013), SH7 (C, 19.11.2013), SW1 (A, 17.09.2013), SW2 (A, 19.09.2013), SW12 (A, 16.04.2014) RMS 6 (B, 08.10.2013), RMS 7 (B, 08.10.2013), RMS22 (D, 21.01.2014), DH7 (B, 08.10.2013), DH8 (B, 08.10.2013), DH9 (B, 08.10.2013), DH20 (D, 19.04.2014), DH21 (D, 19.04.2014), DH24 (C, 30.04.2014), FH6 (B, 08.10.2013), FH7 (B, 08.10.2013), FH20 (D, 08.01.2014), FH21 (D, 08.01.2014), FH22 (D, 08.01.2014), SW15 (B, 26.04.2014), SW16 (B, 26.04.2014), DH-S1 (B, 10.10.2013), DH-S2 (A, 27.10.2013), DH-S3 (A, 26.02.2014), FH-S3 (B, 10.10.2013), FH-S4 (B, 10.10.2013), FH-S5 (B, 10.10.2013) RMS 8 (B, 08.10.2013), RMS9 (B, 08.10.2013), RMS12 (C, 19.11.2013), RMS13 (C, 19.11.2013), RMS31 (A, 16.04.2014), RMS32 (A, 16.04.2014), DH14 (E, 01.12.2013), FH10 (B, 08.10.2013), S4 (B, 08.10.2013), SW3 (A,17.09.2013), SW4 (A, 17.09.2013), SW13 (B, 26.04.2014), FH-S6 (A, 27.10.2013) RMS 14 (E, 01.12.2013), RMS19 (D, 08.01.2014), RMS20 (D, 08.01.2014), RMS21 (D, 08.01.2014), RMS23 (d, 08.01.2014), RMS24 (B, 11.02.2014), RMS25 (B, 11.02.2014), RMS26 (B, 11.02.2014), DH5 (B, 08.10.2013), DH6 (B, 08.10.2013), DH15 (E, 01.12.2013), DH16 (D, 08.01.2014), DH17 (D, 08.01.2014), FH4 (A, 17.09.2013), FH5 (A, 17.09.2013), FH30 (B, 03.03.2014), FH31 (B, 03.03.2014), SH3 (B, 10.10.2013), SH4 (B, 08.10.2013), FH-S7 (C, 19.11.2013), FH-S8 (E, 01.12.2013), FH-S9 (E, 01.12.2013), FH-SC6 (B, 11.02.2014), FH-SC7 (A, 16.04.2014) RMS 28 (C, 31.03.2014), RMS29 (C, 31.03.2014), RMS30 (C, 31.03.2014), FH43 (C, 30.04.2014), SH12 (D, 22.04.2014), SW11 (A, 16.04.2014), SW14 (B, 26.04.2014), SW17 (B, 26.04.2014), FH-S12 (B, 03.03.2014), SH-S1 (C, 19.11.2013) C (19.11.2013) C (19.11.2013) C (19.11.2013) E (01.12.2013) D (19.04.2014) C (30.04.2014)

Serogroup

Antibiotic susceptibility P

AM

MEM

E

SXT

1/2a-3a

S

S

R

R

R

1/2b-3b

R

R

R

R

R

1/2c-3c

R

R

R

R

R

1/2a-3a

R

S

R

R

R

1/2b-3b

S

S

S

S

S

1/2b-3b 1/2b-3b 1/2b-3b 1/2b-3b 1/2b-3b 1/2a-3a

R R R R S S

S S R S S S

R R R R S S

R R R R S S

R R R R S S

(continued on next page)

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Table 4 (continued) Processing stage

Filleting hall (FH)

Filleting hall (FH)

Smokehouse (S) Slicing hall (SH)

Packing hall (PH) Storage warehouse (SW)

Strain

FH 8 FH 9 FH 11 FH 15 FH 16 FH 17 FH 18 FH 19 FH 23 FH 24 FH 25 FH 26 FH 27 FH 28 FH 29 FH 32 FH 33 FH 34 FH 35 FH 36 FH 37 FH 38 FH 39 FH 42 FH 44 FH 45 FH-S1 FH-S2 FH-S10 FH-S11 FH-S13 FH-SC1 FH-SC2 FH-SC3 FH-SC4 FH-SC5 FH-SC8 FH-SC9 FH-SC10 S3 S5 SH 2 SH 5 SH 6 SH 8 SH 9 SH 10 SH 11 SH 13 SH 14 SH-S2 PH 1 PH 2 SW 5 SW 6 SW 7 SW 8 SW 9 SW 10

Genetically identical isolates/fish farm/isolation date

B (08.10.2013) B (08.10.2013) B (08.10.2013) C (19.11.2013) C (19.11.2013) C (19.11.2013) C (19.11.2013) D (08.01.2014) D (21.01.2014) B (11.02.2014) B (11.02.2014) B (11.02.2014) A (26.02.2014) A (26.02.2014) A (26.02.2014) B (03.03.2014) B (03.03.2014) C (31.03.2014) C (31.03.2014) C (31.03.2014) A (16.04.2014) A (16.04.2014) A (16.04.2014) D (22.04.2014) C (30.04.2014) C (30.04.2014) B (10.10.2013) B (10.10.2013) D (21.01.2014) D (21.01.2014) A (16.04.2014) C (19.11.2013) C (19.11.2013) E (01.12.2013) D (08.01.2014) B (11.02.2014) A (16.04.2014) D (17.04.2014) D (17.04.2014) B (08.10.2013) B (11.02.2014) A (17.09.2013) B (08.10.2013) C (19.11.2013) D (21.01.2014) D (21.01.2014) B (03.03.2014) D (22.04.2014) C (30.04.2014) C (30.04.2014) D (08.01.2014) E (01.12.2013) B (11.02.2014) B (08.10.2013) B (08.10.2013) C (19.11.2013) A (26.02.2014) A (26.02.2014) A (16.04.2014)

Serogroup

1/2a-3a 1/2b-3b 1/2c-3c 1/2a-3a 1/2b-3b 1/2c-3c 1/2a-3a 1/2a-3a 1/2a-3a 1/2b-3b 1/2b-3b 1/2a-3a 4b-4d-4e 4b-4d-4e 4b-4d-4e 1/2b-3b 4b-4d-4e 1/2a-3a 1/2a-3a 1/2a-3a 4b-4d-4e 1/2a-3a 4b-4d-4e 1/2a-3a 1/2a-3a 4b-4d-4e 1/2c-3c 1/2c-3c 1/2b-3b 1/2b-3b 1/2b-3b 1/2a-3a 1/2b-3b 1/2b-3b 4b-4d-4e 1/2c-3c 1/2b-3b 4b-4d-4e 1/2b-3b 1/2b-3b 1/2b-3b 1/2a-3a 1/2c-3c 1/2a-3a 1/2a-3a 1/2a-3a 1/2a-3a 1/2a-3a 1/2a-3a 1/2a-3a 1/2b-3b 1/2a-3a 1/2b-3b 1/2c-3c 1/2c-3c 1/2c-3c 4b-4d-4e 1/2a-3a 1/2a-3a

Total strains number Total isolates number

Antibiotic susceptibility P

AM

MEM

E

SXT

R S R S S S R S R R S R S S R S S S S S S S S S S S S S R R S R R S S R S S S S R S S S R R R S S R S S S S S S S R R

S S R S S S S S S S R R S R S S S S S S S S S S S S S S S S S R S S S S S S S S R S S S S R S S S S S S S R S R S S S

R S R R R R R S S S S R S R R S R S S S S S S S S S S S R R S S R S S R S S S S S S S S R S R S S S S S S S S S S R R

R S R S R R R R S R S R R S R S R S S S S S S S S S S S R R S R R S S R S S S R S S S S S S R S S R S S S S R R S R R

R S R R R S R S S R R R R S R S R S S S S S S S S S S S R R S S R S S R S S R S S S S R S R R S S R S R S S S S S R R

70 167

RMS – raw material storage, DH – decapitation hall, FH – filleting hall, S - smokehouse, SH – slicing hall, …-S – swab during production, …-S.C. – swab after cleaning.

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Fig. 4. Electrophoregram presenting the PCR products allowing the serological classification of L. monocytogenes.

Table 5 Phylogenetic groups of tested L. monocytogenes strains (n = 70). Group

I.1. (1/2a-3a) I.2. (1/2c-3c) II.1. (4b-4d-4e) II.2. (1/2b-3b) Total

Number of strains depending the source of origin [n (%)] Fish

Production environment

Total

26 (46.4%) 7 (12.5%) 8 (14.3%) 15 (26.8%) 56 (100.0%)

1 (7.2%) 3 (21.4%) 2 (14.3%) 8 (57.1%) 14 (100.0%)

27 10 10 23 70

Fig. 5. Electrophoregram presenting the results of PCR technique used for virulence genes detection.

80

(38.6%) (14.3%) (14.3%) (32.8%) (100.0%)

International Journal of Food Microbiology 282 (2018) 71–83

29 41 12 58 28 42 33 37 33 37 (37.6%) (62.3%) (12.6%) (87.6%) (25.0%) (75.0%) (37.6%) (62.3%) (37.6%) (62.3%) 3 5 1 7 2 6 3 5 3 5 (0.0%) (100.0%) (0.0%) (100.0%) (0.0%) (100.0%) (0.0%) (100.0%) (0.0%) (100.0%) 0 1 0 1 0 1 0 1 0 1 (40.0%) (60.0%) (0.0%) (100.0%) (40.0%) (60.0%) (40.0%) (60.0%) (40.0%) (60.0%) SXT

E

MEM

AM

P – penicillin, AM – ampicillin, MEM – meropenem, E – erythromycin, SXT – trimetophrim/sulfamethoxazole; R – resistance, S – susceptible.

2 3 0 5 2 3 2 3 2 3 (42.9%) (57.1%) (19.6%) (80.4%) (42.9%) (57.1%) (50.0%) (50.0%) (50.0%) (50.0%) 24 32 11 45 24 32 28 28 28 28 (50.0%) (50.0%) (33.3%) (66.7%) (33.3%) (66.7%) (66.7%) (50.0%) (33.3%) (66.7%) 3 3 2 4 2 4 4 3 2 4 (0.0%) (100.0%) (0.0%) (0.0%) (0.0%) (100.0%) (0.0%) (100.0%) (50.0%) (50.0%) 0 2 0 0 0 2 0 2 1 1 (44.4%) (55.6%) (11.1%) (88.9%) (22.2%) (77.8%) (22.2%) (77.8%) (44.4%) (55.6%) 4 5 1 8 2 7 2 7 4 5 (50.0%) (50.0%) (50.0%) (50.0%) (0.0%) (100.0%) (50.0%) (50.0%) (0.0%) (100.0%) 1 1 1 1 0 2 1 1 0 2 9 (34.6%) 17 (65.4%) 4 (15.4%) 22 (84.6%) 12 (46.2%) 14 (53.8%) 13 (50.0%) 13 (50.0%) 13 (50.0%) 13 (50.0%) (66.7%) (33.3%) (16.7%) (83.3%) (66.7%) (33.3%) (66.7%) (33.3%) (66.7%) (33.3%) 4 2 1 5 4 2 4 2 4 2 (60.0%) (40.0%) (40.0%) (60.0%) (80.0%) (20.0%) (80.0%) (20.0%) (80.0%) (20.0%) 3 2 2 3 4 1 4 1 4 1 R S R S R S R S R S P

Raw material storage n = 5

Decapitation hall n=6

Filleting hall n = 26

Smokehouse n=2

Slicing hall n=9

Packing hall n=2

Storage warehouse n=6

Total N = 56

Filleting hall n=5

Slicing hall n=1

Filleting hall n=8

5 (35.7%) 9 (64.3%) 1 (7.1%) 13 (92.9%) 4 (28.6%) 10 (71.4%) 5 (35.7%) 9 (64.3%) 5 (35.7%) 9 (64.3%)

Total n = 14 After cleaning During production

Swabs Fish samples

Table 6 Antibiotic susceptibility of L. monocytogenes strains (n = 70).

Autio et al. (1999) and Rørvik (2000) note that this device can play an important role in the propagation of L. monocytogenes in the plant environment. Autio et al. (2003) found related strains in smoked trout samples and isolated from the production plant environment, especially in brine tanks and slicers. Wałecka (2011) states that even short time of sodium chloride action and its low concentration was sufficient to induce maximum invasiveness of L. monocytogenes. Wesche et al. (2009) add that stress cause a specific bacterial response that changes its survival and resistance to subsequent stress. Our results confirmed the claims of other authors, that cold smoking does not eliminate L. monocytogenes. Isolates of the same strain were found in raw fish product and in the fish samples collected in smokehouse the same day (RMS8, RMS9, S4). Moreover, in the case of the presence of strains in deeper tissues, keeping and intensive multiplication of the microorganism may occur (Eklund et al., 1995). Furthermore, the increase in NaCl concentration and the addition of sodium nitrite in smoked salmon do not inhibit the ability of Listeria species to multiply during cooling (Pelroy et al., 1994; Peterson et al., 1993). The serotypes 1/2a, 1/2b and 4b are responsible for over 90% of cases of listeriosis in humans, of which 1/2a and 1/2b are mostly isolated from food, and 4b from clinical cases (Swaminathan and Gerner-Smidt, 2007). The obtained results showed that the most of the tested strains (38.6%) belonged to serogroup 1/2a-3a, and slightly less (32.8%) to 1/2b-3b. Our results are in agreement with the previous investigations of fish in several countries, where 1/2a and 4b serotypes were predominant (Fallah et al., 2013; Jami et al., 2014; Kramarenko et al., 2013; Lambertz et al., 2013; Lambertz et al., 2012; Miya et al., 2010; Terentjeva et al., 2015; Wieczorek and Osek, 2017). In turn, Su et al. (2016) showed that most strains isolated from food belonged to serogroups 1/2c-3c (39.1%) and 1/2a-3a (36.7%). Our studies showed that all tested L. monocytogenes strains had all detected virulence genes. Su et al. (2016) found the presence of the hly, plcA, plcB, prfA, actA, inlA and inlB genes in all examined strains. Also, Wieczorek and Osek (2017) detected all 10 tested virulence genes among all strains from fish. This confirms that L. monocytogenes strains isolated from food are potentially pathogenic and play an important role in epidemics. In our study, 5 (7.1%) of the 70 strains showed resistance to all tested antibiotics. In Jamali et al. (2015), 14.0% of L. monocytogenes strains resistant to more than two antibiotics were recorded. According to Komora et al. (2017), there has been an increase in the incidence of resistant L. monocytogenes strains to at least one antibiotic. In current study, strains showed the highest resistance to erythromycin (47.1%), and co-trimoxasole (47.1%). In the Jamali et al. (2015) study, 20.9%, 16.3%, 14.0%, 11.6% of L. monocytogenes strains isolated from fresh fish and the environment showed resistance to ampicillin, penicillin, erythromycin, and co-trimoxasole, respectively. Analysing the results obtained from the different stages of the production process, it was found that the highest percentage of resistant strains to the tested antibiotics were found among isolates isolated from the raw material. The experiment confirmed that L. monocytogenes is a serious problem in the fish industry. It was shown that the main source of contamination of the production environment was the raw material delivered to the processing plant from individual fish farms. However, failure to employ Good Manufacturing Practice in fish processing plants is also a serious cause of the spread of this pathogen. Moreover, antibiotic resistance in isolated strains is also an important problem. Therefore, it is necessary to monitor the transmission pathways of L. monocytogenes in fish processing plants and to implement appropriate hygiene plans.

(41.4%) (58.6%) (17.1%) (82.9%) (40.0%) (60.0%) (47.1%) (52.9%) (47.1%) (52.9%)

Total n = 70

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Table 7 The percentage of strains (n = 70) resistant to the tested antibiotics, including serogroup appurtenance. Antibiotics

P AM MEM E SXT

Serogroup 1/2a-3a (n = 27)

1/2c-3c (n = 10)

4b-4d-4e (n = 10)

1/2b-3b (n = 23)

44.4% 11.1% 37.0% 40.7% 48.1%

30.0% 30.0% 40.0% 60.0% 30.0%

10.0% 10.0% 30.0% 30.0% 30.0%

43.5% 17.4% 39.1% 47.8% 52.2%

(Franciosa et al., 2005) (Buchanan et al., 1997) (Fallah et al., 2013) (FAO, 1999) (Guðbjörnsdóttir et al., 2005)

(Buchanan et al., 1997) (Buchanan et al., 1997) (Hwang, 2007) (Dongyou, 2008) (Buchanan et al., 1997)

(Autio et al., 1999) (Autio et al., 1999) (Buchanan et al., 1997) (Buchanan et al., 1997) (Buchanan et al., 1997)

(Fallah et al., 2013) (Hwang, 2007) (Eklund et al., 1995) (FAO, 1999) (Franciosa et al., 2005)

P – penicillin, AM – ampicillin, MEM – meropenem, E – erythromycin, SXT – trimetophrim/sulfamethoxazole.

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