Assessment of the Prevalence and Drug Susceptibility of Listeria monocytogenes Strains Isolated from Various Types of Meat

Listeria monocytogenes are the etiological factor of listeriosis, and their main source for humans is food. The aim of the current study was to assess the contamination of various types of meat and the drug susceptibility of isolated L. monocytogenes. Between 2016–2018, 6000 swabs were taken (2000 annually) from the surface of pork, beef, and poultry. The analysis of intermediate and finished product samples was carried out in accordance with ISO 11290-1 (International Organization for Standardization). The genetic similarity assessment of the isolates obtained was based on the Pulsed Field Gel Electrophoresis (PFGE) method, and drug-sensitivity assessment using the disc-diffusion method. We found 2.1% of collected samples were L. monocytogenes positive. The level of meat contamination varied depending on its matrix. Most L. monocytogenes were isolated from poultry. It was shown that 39 (32.5%) strains were sensitive to all tested antibiotics and eight (6.7%) were resistant to all five tested antimicrobials. Most strains tested were resistant to cotrimoxazole (55; 45.8%) and meropenem (52; 43.3%), followed by erythromycin (48; 40.0%), penicillin (31; 25.8%), and ampicillin (21; 17.5%). High prevalence of this pathogen may be a serious problem, especially when linked with antibiotic resistance and high percentage of serotypes responsible for listeriosis outbreaks.


Isolation of L. monocytogenes Strains
Analysis of the meat samples was based on the procedures of the PN-EN ISO 11290-1:1999/A1:2005 [21]. The sponges taken from meat samples were immersed in 100 mL of half-Fraser broth (Merck, Darmstadt, Germany) and incubated at 30 • C for 24 h. The sampling sponge was then squeezed firmly several times into the bag. Secondary selective enrichment was performed for 48 h at 37 • C after transferring 0.1 mL of the culture into 9.9 mL of Fraser broth (Merck, Darmstadt, Germany). Next, a reductive inoculation of the bacterial cultures was performed on the selective agar medium according to Ottaviani and Agosti (ChromoCult ® Listeria Selective Agar (ALOA ® ; Merck, Darmstadt, Germany). Cultures were incubated for 24 h at 37 • C.
Bacterial colonies suspect to belong to Listeria spp. (one colony for each sample) were transferred to Columbia Agar with 5% sheep blood (bioMérieux, Marcy-l'Étoile, France). The hemolysis type was assessed and final identification using the PCR method, was performed. The identified L. monocytogenes isolates were frozen in brain-heart infusion broth (BHI; Merck, Darmstadt, Germany) with 15% glycerol (Avantor, Gliwice, Poland) and stored at −80 • C.

Isolation of Genomic DNA
In order to isolate L. monocytogenes genomic DNA, the column method with the Genomic Mini AX Bacteria Spin Kit (A&A Biotechnology, Gdynia, Poland) was applied, according to the protocol provided by the manufacturer.

Evaluation of Pulsotypes Similarity (PFGE)
The pulsotypes similarity analysis of the confirmed L. monocytogenes strains was performed with the pulsed-field gel electrophoresis (PFGE). The procedure was carried out according to the standard operating procedure for PulseNet PFGE of L. monocytogenes [23].
The degree of pulsotypes similarity between analyzed L. monocytogenes isolates was evaluated using a phylogenetic dendrogram drawn in the CLIQS 1D Pro program (TotalLab, Newcastle upon Tyne, UK). Clustering analysis was performed using hierarchical clustering with the Unweighted Pair-Group Method Using Arithmetic Averages (UPGMA) technique and Dice's coefficient.

Drug Susceptibility Analysis
The disk-diffusion method was applied to determine antibiotic susceptibility of L. monocytogenes strains tested. For this purpose, 24 h bacterial cultures diluted in 0.9% saline solution (Avantor, Gliwice, Poand) were plated on MHF medium (Mueller Hinton Agar with 5.0% horse blood and 20 mg/L β-NAD; bioMérieux, Marcy-l'Étoile, France). Next, following antibiotic discs were added: penicillin (1 IU), ampicillin (2 µg), meropenem (10 µg), erythromycin (15 µg), and cotrimoxazole (1.25-23.75 µg). The antibiotics for the studies were selected in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) v. 8.0 [26] recommendations. The antibiotics used in the study are the only ones that can be used in Europe to treat listeriosis and for which the interpretation of the results is possible. After 20 h of incubation at 35 • C, growth inhibition zones around the antibiotic discs were measured and analyzed in accordance with the EUCAST v. 8.0 [26].

Statistical Analysis
The obtained results were subjected to statistical analysis in the Statistica 13 PL program (StatSoft, Round Rock, TX, USA). The normality of the distribution of the obtained results was assessed with the Shapiro-Wilko test. ANOVA with the Tukey post-hoc test was used to determine significant differences between strains number in particular groups.

Results
The research showed that L. monocytogenes was isolated from 127 (2.1%) of 6000 swabs taken from all kind of meat. The level of contamination of all meat samples, regardless of their type, in particular years was similar and ranged from 1.8% (35 positive samples) in 2018 to 2.5% (50 positive samples) in 2017. These differences were not statistically significant. In each year, the largest number of positive samples was obtained from poultry (1.9-4.2%), and in 2016-2017 the differences were statistically significant ( Figure 1). In turn, the lowest L. monocytogenes contamination was noted in pork samples (0.3-1.6%; Figure 1). In 2016-2017, the percentage of contaminated pork samples was statistically significantly lower compared to other types of meat ( Figure 1). Foods 2020, 9, x FOR PEER REVIEW 4 of 18

Drug Susceptibility Analysis
The disk-diffusion method was applied to determine antibiotic susceptibility of L. monocytogenes strains tested. For this purpose, 24 h bacterial cultures diluted in 0.9% saline solution (Avantor, Gliwice, Poand) were plated on MHF medium (Mueller Hinton Agar with 5.0% horse blood and 20 mg/L β-NAD; bioMérieux, Marcy-l'Étoile, France). Next, following antibiotic discs were added: penicillin (1 IU), ampicillin (2 μg), meropenem (10 μg), erythromycin (15 μg), and cotrimoxazole (1.25-23.75 μg). The antibiotics for the studies were selected in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) v. 8.0 [26] recommendations. The antibiotics used in the study are the only ones that can be used in Europe to treat listeriosis and for which the interpretation of the results is possible. After 20 h of incubation at 35 °C, growth inhibition zones around the antibiotic discs were measured and analyzed in accordance with the EUCAST v. 8.0 [26].

Statistical Analysis
The obtained results were subjected to statistical analysis in the Statistica 13 PL program (StatSoft, Round Rock, TX, USA). The normality of the distribution of the obtained results was assessed with the Shapiro-Wilko test. ANOVA with the Tukey post-hoc test was used to determine significant differences between strains number in particular groups.

Results
The research showed that L. monocytogenes was isolated from 127 (2.1%) of 6000 swabs taken from all kind of meat. The level of contamination of all meat samples, regardless of their type, in particular years was similar and ranged from 1.8% (35 positive samples) in 2018 to 2.5% (50 positive samples) in 2017. These differences were not statistically significant. In each year, the largest number of positive samples was obtained from poultry (1.9-4.2%), and in 2016-2017 the differences were statistically significant ( Figure 1). In turn, the lowest L. monocytogenes contamination was noted in pork samples (0.3-1.6%; Figure 1). In 2016-2017, the percentage of contaminated pork samples was statistically significantly lower compared to other types of meat ( Figure 1).

Evaluation of Pulsotypes Similarity (PFGE)
The analysis of pulsotypes similarity (PFGE) distinguished the L. monocytogenes isolates into 127 patterns, among which seven pairs of isolates represent the identical pulsotypes ( Figure 2). In most cases, strains with identical pulsotypes were obtained from the same material. The exception was a pair of strains 07/16 and 17/16, which were isolated from pork and beef, respectively. Moreover, all strains with identical pulsotypes were always isolated in the same year. The cut-off value was set at 80% similarity. The comparison of pulsotypes allowed us to assign 62 (48.8%) of the tested strains to 20 clusters. Apart from genetically identical strains, the highest degree of similarity (95.0%) was shown by three pairs of isolates: no. 08/16 and 21/16 (isolated in the same year from pork); 23/17 and 27/17 (isolated in the same year from beef and pork, respectively); 06/18 and 23/16 (isolated in 2018 and 2016, respectively from pork). However, the analysis of pulsotypes showed that strains from pairs: 08/16, 21/16 and 06/18, 23/16 were the least similar with strains 09/16 (isolated from beef in 2016) and 05/18 (isolated from pork in 2018), respectively. The remaining 65 (51.2%) strains created unique PFGE patterns and were not classified into any of the clusters at the adopted cut-off value.

Drug Susceptibility Evaluation
Among 120 L. monocytogenes strains isolated from all types of meat 22 antimicrobial resistance patterns were defined (Table 1). Profile I included 39 (32.5%) strains susceptible to all antibiotics

Drug Susceptibility Evaluation
Among 120 L. monocytogenes strains isolated from all types of meat 22 antimicrobial resistance patterns were defined (Table 1). Profile I included 39 (32.5%) strains susceptible to all antibiotics tested. Statistically the highest number (11,29.7%) of all antibiotic-susceptible strains was reported in poultry meat in 2016 (Table 1 and Figure 3). On the other hand, eight L. monocytogenes strains, classified to profile III, showed resistance to all antibiotics used in the research-six were isolated from poultry and beef meat (four and two, respectively) in 2017 and two from poultry meat in 2018. L. monocytogenes strains resistant to all antibiotics has not been isolated from pork (Table 1 and Figure 4). Resistance to cotrimoxazole only (profile II) was confirmed in 14 strains (11.67%). Seven strains (5.8%) were ampicillin-susceptible only (profile IV). Profiles XVII-XXII were represented by single L. monocytogenes strains. In 2017 and 2018, the statistically significantly largest number of strains resistant to all tested antibiotics were isolated from poultry ( Figure 4). tested. Statistically the highest number (11,29.7%) of all antibiotic-susceptible strains was reported in poultry meat in 2016 (Table 1 and Figure 3). On the other hand, eight L. monocytogenes strains, classified to profile III, showed resistance to all antibiotics used in the research-six were isolated from poultry and beef meat (four and two, respectively) in 2017 and two from poultry meat in 2018. L. monocytogenes strains resistant to all antibiotics has not been isolated from pork (Table 1 and Figure  4). Resistance to cotrimoxazole only (profile II) was confirmed in 14 strains (11.67%). Seven strains (5.8%) were ampicillin-susceptible only (profile IV). Profiles XVII-XXII were represented by single L. monocytogenes strains. In 2017 and 2018, the statistically significantly largest number of strains resistant to all tested antibiotics were isolated from poultry ( Figure 4).    tested. Statistically the highest number (11,29.7%) of all antibiotic-susceptible strains was reported in poultry meat in 2016 (Table 1 and Figure 3). On the other hand, eight L. monocytogenes strains, classified to profile III, showed resistance to all antibiotics used in the research-six were isolated from poultry and beef meat (four and two, respectively) in 2017 and two from poultry meat in 2018. L. monocytogenes strains resistant to all antibiotics has not been isolated from pork (Table 1 and Figure  4). Resistance to cotrimoxazole only (profile II) was confirmed in 14 strains (11.67%). Seven strains (5.8%) were ampicillin-susceptible only (profile IV). Profiles XVII-XXII were represented by single L. monocytogenes strains. In 2017 and 2018, the statistically significantly largest number of strains resistant to all tested antibiotics were isolated from poultry ( Figure 4).     Overall analysis of L. monocytogenes drug susceptibility showed that the most numerous group included strains resistant to cotrimoxazole (55; 45.8%) and meropenem (52; 43.3%). The remaining antimicrobials-erythromycin, penicillin and ampicillin-showed no activity against 48 (40.0%), 31 (25.8%), and 21 (17.5%) strains, respectively ( Table 1). The results of the first-year analyses (2016) confirmed the presence of L. monocytogenes antibiotic resistant strains in poultry and beef only and 12 of these strains were resistant to cotrimoxazole (32.4%). On the contrary, for the next two years, all the types of meat tested were contaminated with antibiotic resistant strains. Statistically, the highest number of antibiotic-resistant strains was isolated from poultry meat in 2017-19 strains were resistant to cotrimoxazole and the same number to meropenem. It was reported, that the lowest number of L. monocytogenes antibiotic resistant strains was isolated from pork meat during each year of the experiment ( Figure 5). Overall analysis of L. monocytogenes drug susceptibility showed that the most numerous group included strains resistant to cotrimoxazole (55; 45.8%) and meropenem (52; 43.3%). The remaining antimicrobials-erythromycin, penicillin and ampicillin-showed no activity against 48 (40.0%), 31 (25.8%), and 21 (17.5%) strains, respectively ( Table 1). The results of the first-year analyses (2016) confirmed the presence of L. monocytogenes antibiotic resistant strains in poultry and beef only and 12 of these strains were resistant to cotrimoxazole (32.4%). On the contrary, for the next two years, all the types of meat tested were contaminated with antibiotic resistant strains. Statistically, the highest number of antibiotic-resistant strains was isolated from poultry meat in 2017-19 strains were resistant to cotrimoxazole and the same number to meropenem. It was reported, that the lowest number of L. monocytogenes antibiotic resistant strains was isolated from pork meat during each year of the experiment ( Figure 5).

L. monocytogenes Serotypes Determination and Distribution in Meat
The serotype distribution of analyzed L. monocytogenes strains in meat samples was shown in Figure 6. It was found that all four main serogroups were identified within tested L. monocytogenes strains.

L. monocytogenes Serotypes Determination and Distribution in Meat
The serotype distribution of analyzed L. monocytogenes strains in meat samples was shown in Figure 6. It was found that all four main serogroups were identified within tested L. monocytogenes strains. Although the antibiotic resistance of the strains belonging to individual serogroups varied depending on the year of their isolation, some common trends were also observed. Each year of the research the highest number of cotrimoxazole-resistant strains was noted in 4b-4d-4e serotype. Over 50% of strains resistant to cotrimoxazole and meropenem in 2017 (13, 27.1% and 12, 25.0%, respectively) belonged to this serogroup, it was a statistically significant value. A high resistance to all antibiotics tested was confirmed in strains of 1/2a-3a serotype in 2017-2018. On the contrary, isolates of group 1/2c-3c presented a low resistance to penicillin and ampicillin, it was a statistically significant value (Figure 7). The most common serogroup isolated from all types of meat samples was 4b-4d-4e (51 strains, 42.5%), followed by 1/2a-3a (29 strains, 24.2%), 1/2b-3b (21 strains, 17.5%), and 1/2c-3c (19 strains, 15.8%). Almost 50% of all 4b-4d-4e strains originated from poultry ( Figure 6), it was a statistically significant value. In turn, the least numerous serotype in this type of meat was 1/2b-3b, represented by eight strains only. The 1/2c-3c and 1/2a-3a serogroups were the least frequent within the strains isolated from beef and pork, respectively; it was a statistically significant value ( Figure 6).
Although the antibiotic resistance of the strains belonging to individual serogroups varied depending on the year of their isolation, some common trends were also observed. Each year of the research the highest number of cotrimoxazole-resistant strains was noted in 4b-4d-4e serotype. Over 50% of strains resistant to cotrimoxazole and meropenem in 2017 (13, 27.1% and 12, 25.0%, respectively) belonged to this serogroup, it was a statistically significant value. A high resistance to all antibiotics tested was confirmed in strains of 1/2a-3a serotype in 2017-2018. On the contrary, isolates of group 1/2c-3c presented a low resistance to penicillin and ampicillin, it was a statistically significant value (Figure 7).  The results of the research confirmed L. monocytogenes presence in raw poultry, beef, and pork meat. High prevalence of this pathogen may be a serious problem, especially when linked with antibiotic resistance and high percentage of serotypes responsible for listeriosis outbreaks. Although the sensitivity of the L. monocytogenes strains tested in our study to penicillin and ampicillin is not so small, the presence of ampicillin-resistant strains of serotype 4b-4d-4e in the analyzed samples is a reason for concern, considering that this serotype is a cause for majority of human listeriosis and ampicillin is a first choice antibiotic in Listeria-linked infections treatment. In order to the increase in antibiotic resistance in L. monocytogenes emphasized by many studies and researches, the need for constant surveillance of this trend is undisputed.