The 392 L. monocytogenes strains used in this study were collected between 1999 and 2013 in Switzerland and Finland (Supplementary Table 1). The Swiss strains (n = 195) were collected at the Swiss National Reference Centre for Enteropathogenic Bacteria and Listeria (NENT). This strain collection differed from that recently described in the study by Ebner et al. (2015). Finnish strains (n = 197) were collected through the Department of Food Hygiene and Environmental Health of the Faculty of Veterinary Medicine at the University of Helsinki. Unlike the Swiss strain collection, the Finnish strain collection lacked human clinical isolates. The origin of the strains was summarized as: dairy (strains isolated from dairy products), meat (strains isolated from carcasses and raw meat products), fish (strains isolated from raw fish), ready to eat (RTE) food (strains isolated from RTE seafood, salad, sausage, ham, maize products), vegetables (strains isolated from raw vegetables), food animals (strains isolated from cows, goats, pigs, and sheep), birds (strains isolated from the feces of wild birds), food production environments (FPE; strains isolated from meat, RTE and dairy production environments), human (strains isolated from human listeriosis cases), others (strains isolated from quorn, rice, silage). Bacteria were stored at −80°C in brain heart infusion (BHI; Oxoid, Pratteln, Switzerland) broth plus 20% glycerol (Sigma-Aldrich, Buchs, Switzerland).

Strain serotypes were assigned by the slide agglutination test using the commercial set of Listeria O-factor and H-factor antisera from Denka Seiken (Pharma Consulting, Burgdorf, Switzerland) according to the manufacturer's instructions. Susceptibility to BC was tested using the previously described agar dilution method (Elhanafi et al., 2010). Strains were plated on blood agar plates (Difco, Columbia blood agar base, 5% sheep blood, Oxoid) and incubated for 18 h at 37°C. Single colonies were picked from each plate on the next day and suspended in 100 μl of Mueller Hinton broth (MHB; Oxoid, Pratteln, Switzerland). Five microliters of the suspensions were spotted in technical duplicates on Mueller Hinton Agar (MHA; Oxoid) plates supplemented with 2% defibrinated sheep blood (Oxoid) and various BC concentrations (0, 2.5, 5, 7.5, 10, 15, 20, 25, and 30 μg ml⁻¹; Sigma-Aldrich). Benzalkonium chloride minimal inhibitory concentrations (MICs) were read after incubating the plates at 37°C for 48 h. Spots could exhibit either no growth, growth of individual colonies, or confluent growth over the full area of the spot. Strains were considered resistant to a given concentration of BC if the spots showed confluent growth, and the MIC was defined as the lowest BC concentration preventing confluent growth of the spotted bacteria. Strains were considered BC^r if they exhibited confluent growth at or above 20 μg ml⁻¹. This cutoff was defined after the following considerations: we first determined the lowest BC concentration that killed >50% of all strains (10 μg ml⁻¹). Based on (Langsrud et al. (2003); Xu et al. (2014), we then defined resistance at a MIC that was double this concentration (20 μg ml⁻¹). Minimal inhibitory concentrations are indicated as >30 μg ml⁻¹ for strains that exhibited confluent growth at 25 and 30 μg ml⁻¹ BC.

To assess the contribution of efflux pump activity in BC^r strains, the BC MICs of such strains were also determined on MHB agar plates containing various BC concentrations (0, 2.5, 5, 7.5, 10, 15, 20, 25, and 30 μg ml⁻¹; Sigma-Aldrich) and supplemented with the efflux pump inhibitor reserpine (20 μg ml⁻¹; Sigma-Aldrich; Romanova et al., 2006). The BC resistance of the L. monocytogenes strains was classified as not efflux dependent (no effect of reserpine on the BC MIC), partially efflux pump dependent (addition of reserpine resulted in a decrease of the BC MIC of <10 μg ml⁻¹), or fully efflux pump dependent (addition of reserpine resulted in a decrease of the BC MIC of ≥10 μg ml⁻¹).

The DNA templates were extracted from L. monocytogenes strains that were grown overnight in BHI broth (37°C and 125 rpm), using the DNeasy blood and tissue kit (Qiagen). Genotyping by multilocus sequence typing (MLST) was performed as previously described (Ragon et al., 2008). Polymerase chain reactions (PCRs) to amplify seven housekeeping genes were performed using the HotStartTaq Master Mix (Qiagen) and 50 ng of genomic DNA template from each analyzed strain. The PCR products were sequenced at Microsynth (Balgach). The MLST types and genetic lineages were assigned using the L. monocytogenes MLST database website (http://bigsdb.web.pasteur.fr). The PCR analysis for the presence of bcrABC, Tn6188, and emrE genes was performed as previously described using primers shown in Table 1. The emrE primers were designed based on L. monocytogenes strain sequence 05–5578 described by Gilmoure et al. (2010). The R56 and R159 L. monocytogenes strains (Ebner et al., 2015) were used as bcrABC and Tn6188 positive controls, respectively whereas the LR39-1 strain (Kovacevic et al., 2012) was used as a positive control for emrE.

Statistical analysis was performed using the JMP program (Version 11.0.0, SAS Institute Inc., NC, USA). Fisher's exact test was used in a series of individual pairwise comparisons using 2 × 2 tables to compare proportions of BC^r and BC^s within the serotypes and the sources per country. P < 0.05 were considered to be statistically significant.

A panel of 195 Swiss (CH) and 197 Finnish (FIN) L. monocytogenes strains that were isolated from diverse sources including foods, food production environments, food animals, wild birds, and human listeriosis cases was serotyped. Table 2 presents an overview of the distribution of these strains based on serotypes and isolation sources. Strains examined from these two countries belonged to serotypes 1/2a (CH: 105/195, 53.8%, and FIN: 134/197, 68.0%), 4b (CH: 47/195, 24.1%; FIN: 23/197, 11.7%), 1/2b (CH: 26/195, 13.3%; FIN: 18/197, 9.1%), 1/2c (CH: 17/195, 8.7%; FIN: 18/197, 9.1%), and 3a (CH: 1/195, 0.5%; FIN: 4/197, 2.0%). In terms of isolation sources, the Swiss strains came from human listeriosis (80/195, 41.0%), meat (59/195, 30.3%), RTE food (17/195; 8.7%), dairy (14/195, 7.2%), FPE (17/195, 8.7%), fish (2/195, 1.0%), food animals (1/195, 0.5%), vegetables (1/195, 0.5%), and other sources (4/195, 2.1%). Finnish strains originated from meat (69/197, 35%), FPE (31/197, 15.7%), fish (27/197, 13.7%), birds (25/197, 12.7%), food animals (19/197, 9.6%), raw vegetables (11/197, 5.6%), dairy (8/197, 4.0%), RTE food (3/197, 1.5%), and other sources (4/197, 2.0%).

The BC susceptibility profiles of the Swiss and Finnish L. monocytogenes strains were assessed. Benzalkonium chloride MICs ranging from 7.5 to >30 μg ml⁻¹ were found (Figure 1; Table 2). Strains with BC MICs ≥20 μg ml⁻¹ were classified as BC^r; strains with BC MICs <20 μg ml⁻¹ were classified as BC^s.

By this definition, the majority (348 out of 392) of strains from both countries were BC^s. There were however 24 (12.3%) Swiss and 21 (10.6%) Finnish strains classified as BC^r, with BC MICs ranging from 20 to >30 μg ml⁻¹. The largest group within the Swiss strains (104/195; 53.3%) had BC MICs of 10 μg ml⁻¹ whereas the largest group within the Finnish strains (96/197; 48.7%) had BC MICs of 7.5 μg ml⁻¹.

None of the dairy, food animal and other category strains from either Switzerland or Finland from this strain collection exhibited a BC^r phenotype. In both countries strains exhibiting BC^r phenotypes were recovered from the FPE, raw meat and RTE food categories. Swiss BC^r strains also included isolates from human listeriosis cases, whereas the Finnish BC^r strains included isolates from raw fish, vegetables, and wild birds. The prevalence of BC^r strains in both countries also varied with regard to the isolation sources (Figure 2A).

The overall frequency of a BC^r phenotype in the Swiss strains was 12.3% and sources included, in descending order of relative frequency: FPE (5/17, 29.4%), meat (12/59, 20.3%), human isolates (6/80, 7.5%), and RTE food (1/17, 5.9%). The overall frequency of a BC^r phenotype in the Finnish strains was 10.2% and the sources included RTE (2/3, 66.7%), vegetables (3/11, 27.3%), the FPE (5/31, 16.1%), fish (3/27, 11.1%), and meat (4/69, 5.8%). Statistical analysis detected significant differences between the prevalence of a BC^r phenotype in Swiss vs. Finnish strains that were isolated from meat (CH > FIN) and RTE (CH < FIN) food products (p > 0.05). It is possible that such differences might have been biased due to discrepancies in the number of tested strains since there were only three Finnish RTE food strains compared to 17 Swiss strains examined in this category. No significant (p > 0.05) differences were detected in BC^r prevalence between the FPE, vegetables and raw fish strain categories in the two countries. BC^r strain prevalence in human listeriosis (6/80; 7.5%) and bird (3/24; 12.5%) categories in the two countries could however not be compared as they were not represented in both locations.

Prevalence of the BC^r strains also varied in each country with regard to the different L. monocytogenes serotypes. As expected given the composition of our strain collection, the majority of BC^r strains detected in both countries belonged to serotypes 1/2a (28/45, 62.2%) and 1/2c (12/45, 26.7%), although BC^r serotype 4b (3/45, 6.7%), and 1/2b (2/45, 4.4%) strains were also found. Interestingly, the highest prevalence of BC resistance was detected in serotype 1/2c strains from both countries (CH 4/16, 25%; FIN 7/18, 38.9%; Figure 2B). The second highest BC^r prevalence in Swiss strains was found in serotype 1/2a strains (19/105, 18.1%), and in Finnish strains among serotype 4b strains (3/23, 13%; Figure 2B). Low prevalence of BC resistance was found in serotype 1/2b isolates from both countries (CH: 1/26, 3.8%; FIN: 1/18, 5.5%), and no BC^r phenotypes were detected in Swiss serotype 4b (n = 47), as well as serotype 3a (n = 5) strains from both countries. Statistical comparison revealed significantly higher BC^r prevalence among the Swiss (19/105, 18.1% vs. 10/134, 7.5%; p < 0.05) serotype 1/2a strains compared to their Finnish counterparts. On the other hand the Swiss serotype 4b strains displayed significantly lower BC^r prevalence (0 vs. 13%; p < 0.05) compared to those from Finland. No significant differences were observed in BC^r prevalence associated with serotype 1/2c and 1/2b strains from the two countries. We are however aware that our observations could be biased due to overall differences in the examined sample sizes between some of the serotype categories in the two countries.

Molecular genotypes associated with the Swiss and Finnish BC^r strains were assessed based on MLST genotyping. The 45 BC^r strains from the two countries were assigned to 14 sequence types (ST), which included two newly described sequence types (ST25, ST28; Table 3). BC^r strains in both countries belonged predominantly to sequence types ST121 (14/45; 31.1%) and ST9 (11/45; 24.4%) although there were some country specific differences. Sequence type 121 (50 vs. 19%) predominated among the Swiss BC^r strains while ST9 (28.6 vs. 20.8%) was predominant in Finnish BC^r strains. Sequence types ST403 (n = 4), ST204 (n = 1), ST25 (n = 1), and ST 28 (n = 1) were exclusive to Swiss BC^r strains whereas ST155 (n = 3), ST1 (n = 1), ST101 (n = 1), ST120 (n = 1), ST194 (n = 1), and ST515 (n = 1) were exclusive to the Finnish BC^r strains. Strains were grouped into 10 MLST clonal complexes based on their sequence types showing that BC^r in this strain collection is mainly associated with serotype 1/2a, CC121 (16/45; 35.6%) and serotype 1/2c, CC9 (11/45, 24.4%). Overall, most BC^r strains belonged to evolutionary genetic lineage II (39/45; 86.7%). There were only five (11.1%) genetic lineage I BC^r strains observed, one of which was isolated in Switzerland and four were from Finland. One serotype 1/2c strain that originated from a FPE in Finland was untypable using the current MLST scheme. In this strain, primers for one (bglA) out of the seven MLST genes amplified a PCR product bearing a sequence that is unrelated to the L. monocytogenes bglA gene.

Benzalkonium chloride resistant strains were also examined for the distribution of genes encoding the three efflux pump systems (brcABC, qacH, and emrE) currently known to confer BC resistance in L. monocytogenes (Table 3). The PCR-based analysis detected genes associated with such efflux pump systems in 79% (19/24) Swiss and 57% (12/21) Finnish BC^r strains, respectively. Swiss strains harbored both qacH (11/24; 45.8%) and brcABC (8/24, 33.3%) associated genes, and no strains harboring emrE were found. A majority of the BC^r strains from Finland harbored qacH genes (11/21; 52.4%), no bcrABC genes were found, and in one BC^r strain, an emrE gene was detected. With respect to associated serotypes and MLST genotypes, the qacH genes were detected in serotype 1/2a, CC121 (ST121 and ST28), serotype 1/2a, CC204 (ST204), and serotype 1/2c, CC9 (ST9) strains. The brcABC genes were associated with serotype 1/2a, CC121 (ST121 and ST25), serotype 1/2b, CC5, and serotype 1/2c, CC9 strains. The emrE gene was associated with a serotype 1/2a, CC8 (ST120) strain. In terms of origins, the qacH-encoding strains came from FPE, raw meat, fish RTE food, and human listeriosis cases. The brcABC harboring strains were from FPE and raw meat, and the emrE strain originated from an RTE food product. None of the three known BC resistance determinants were detected in 21% (5/24) and 43% (9/21) of the Swiss and Finnish BC^r strains, respectively (Table 3). This group included serotype 4b, CC1 (ST1 and ST515), serotype 1/2a, CC8 (ST8), CC101 (ST101), CC155 (ST155), and serotype 1/2b, CC5 (ST5) strains, which were isolated from diverse sources. At this stage, sequence alterations affecting PCR primer binding sites across different strains cannot be completely ruled out as a possible reason for false negative results in some of the BC^r strains found to lack the known BC^r genes.

A screen with reserpine showed that the BC resistance in 4 out of 45 strains depended on reserpine sensitive efflux pump systems. The BC resistance in an additional 14 strains was classified as partially efflux pump dependent while the addition of reserpine had no effect on the BC MIC in 27 strains. As mentioned above, a subset of 14 BC^r strains lacked known BC resistance genes. Reserpine addition had no impact on BC MICs in four of those strains. The BC resistance in an additional three strains was classified as efflux pump dependent, and in seven strains as partially efflux dependent. (Table 3).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.