Abstract
Listeria monocytogenes is a food-borne pathogen and a resilient environmental saprophyte. Dairy farms are a reservoir of L. monocytogenes and strains can persist on farms for years. Here, we sequenced the genomes of 250 L. monocytogenes isolates to investigate the persistence and mobile genetic elements of Listeria inhabiting dairy farms. We found that prophages and other mobile elements were significantly more numerous among persistent than sporadically occurring strains. We identified a remarkable diversity of mobile elements among farm isolates, including a novel group of plasmids infecting hypervirulent subtypes of L. monocytogenes and occasionally carrying biocide resistance determinants bcrABC or qacH. Resistance genes against bacitracin, arsenic and cadmium were significantly more prevalent among persistent than sporadic strains. Several of the mobile elements in Listeria were identical to the mobile elements of Enterococci, indicative of recent transfer between these genera. Finally, we demonstrated that the CRISPR-cas IIa system and a type II restriction-modification system were negatively associated with persistence on farms. Our findings suggest that mobile elements support the persistence of L. monocytogenes on dairy farms and that L. monocytogenes inhabiting the agroecosystem is a potential reservoir of mobile elements harbouring resistance genes against antimicrobials, biocides, and heavy metals.
Introduction
Listeria monocytogenes leads a double life: in one it is a potentially lethal, zoonotic foodborne pathogen and in the other, a ubiquitous environmental saprophyte (Gray et al., 2006). Agroecosystems provide a favourable habitat for L. monocytogenes and the pathogen is especially prevalent on dairy farms (Nightingale et al., 2004; Esteban et al., 2009). L. monocytogenes strains can inhabit dairy farms for years and be widely distributed in the farm environment, leading to the frequent contamination of milk (Ho et al., 2007; Castro et al., 2018). Raw milk and animals destined for slaughter are a major contamination source for the food industry (Samelis & Metaxopoulos 1999; Fox et al., 2009; Hellström et al., 2010). Knowledge of the pathogens ecology on farms is essential for controlling the spread of L. monocytogenes from farms to the food industry.
L. monocytogenes is extremely resilient and can tolerate various stresses used to control the pathogen in the food industry (Lundén et al., 2003; Aarnisalo et al., 2007). These phenotypic traits enable L. monocytogenes to survive in food processing environments for years, a phenomenon known as persistence (Lundén et al., 2000; Keto-Timonen et al., 2007; Stasiewicz et al., 2015; Pasquali et al., 2018; Hurley et al., 2019). Mobile genetic elements are common among L. monocytogenes isolates from food processing environments (Harvey & Gilmour, 2001; Pasquali et al., 2018; Hurley et al., 2019) and may harbour genes mediating tolerance to heat shock (Pöntinen et al., 2017), salt and acid stress (Naditz et al., 2019; Hingston et al., 2019) and biocides (Müller et al., 2013; Meier et al., 2017). These findings led us to the hypothesis that mobile genetic elements play a key role in the environmental adaptation and persistence of L. monocytogenes.
Although dairy farms are considered a reservoir of L. monocytogenes (Nightingale et al., 2004), and are known to harbour hypervirulent strains (Maury et al., 2019), the era of next generation sequencing has witnessed very few efforts to illuminate the pathogen’s ecology in the farm environment. How L. monocytogenes adapts to life in the farm ecosystem, and to what extent the farm environment acts as source of mobile genetic elements for L. monocytogenes persisting in food processing environments, are key issues to explore. Such insights would be instrumental in developing novel strategies to reduce contamination on farms and to prevent the emergence and spread of persistent strains.
Here, we sequenced the genomes of 250 L. monocytogenes isolates obtained from three Finnish dairy farms during 2013–2016 (Castro et al., 2018) to investigate the persistence and mobile genomic elements of L. monocytogenes in the farm environment. We found that prophages and other mobile genetic elements were significantly more abundant among persistent than sporadic strains. We identified a remarkable diversity of mobile elements among the dairy farm isolates and discovered several novel mobile elements that are relevant to food safety. We demonstrated that resistance genes against bacitracin, arsenic and cadmium were significantly more prevalent among persistent than sporadic strains. Finally, we found a negative association between persistence and three putative defence systems against invading prophages and plasmids (Garneau et al, 2010; Lee et al., 2012). Taken together, our findings suggest that prophages and mobile genetic elements confer an ecological advantage for persistence on farms, and that L. monocytogenes inhabiting the farm environment constitutes a reservoir of diverse mobile genetic elements having the potential to carry resistance genes against heavy metals, antimicrobials and biocides across the food chain.
Results
Persisting clades of L. monocytogenes were detected on all three farms
Whole genome sequencing and subsequent in silico subtyping of 250 Listeria monocytogenes isolates, collected from three dairy farms during 2013–2016 (Castro et al., 2018), yielded 25 unique STs (Fig. 1a, see Supplementary Data S1). The most frequently detected subtype was ST20, which represented 28% of all sequenced isolates. We identified in total 14 persistent clades (Table 1). Persistent clades represented 71% of all sequenced isolates and all persistent clades belonged to serogroup 1/2a. Clade C4 contained isolates from two different farms, suggesting that strains of L. monocytogenes can spread between farms faster than the rate of genomic diversification.
Mobiles genetic elements were more common among persistent clade isolates than singleton isolates
Prophages and other mobile genetic elements were significantly more numerous (Independent Samples Median Test; p<.01) among persistent clade isolates than singleton isolates (Fig. 1b-c). Overall, non-phage mobile genetic elements were detected in 72% of isolates in persistent clades and 66% of singleton isolates. All isolates contained at least one prophage, known as the monocin (Zink et al., 1995), and additional prophages were detected among 95% of isolates from persistent clades and 85% of singleton isolates.
Dairy farms harboured a novel plasmid that can infect hypervirulent STs and carry biocide resistance genes
Plasmids were detected among 10% of L. monocytogenes isolates in persistent clades and 11% singleton isolates. We detected three previously identified plasmids (pCFSAN010068, pLM58, pLMR479a), and three novel plasmids, labelled pHC143, pHC192 and pHC195-2 (Fig. 2a, see Supplementary Data S1). These plasmids were 55.5 – 86.7 Kb in size, except for pHC192, which was only 4.6 Kb. A Maximum-Likelihood phylogenetic analysis based on RepA grouped the large plasmids into three distinct groups (G1 – G3), which appear to be specific to the genus Listeria (Fig. 3a). In the small plasmid pHC192 the repA gene was truncated so it was excluded from the analysis.
Plasmid groups G1 and G2 composed of several well-characterized L. monocytogenes reference plasmids (Kuenne et al., 2010; Hingston et al., 2019; Nadiz et al., 2019). G3 represented a phylogenetically distinct, novel group of Listeria plasmids that all closely resemble pHC143, a plasmid we detected in five isolates of this study (see Supplementary Data S1). Visualisation of assembly graphs indicated that the plasmid was successfully assembled into a single 55.8 Kb contig in all five isolates. We identified three previously unreported variants of pHC143 among short-read sequence assembles deposited in GenBank, which all contained resistance genes against biocides (Fig. 3b, see Supplementary Fig. S1). Specifically, the plasmid of the L. monocytogenes ST1 environmental isolate FDA550584-30 (SAMN02923676) contained a benzalkonium chloride resistance cassette (bcrABC), and the plasmids of the ST6 human outbreak isolate YA00079283 (SAMN08970420) and the ST20 food isolate 967535 (SAMN15680309) contained a resistance gene against quaternary ammonium compounds (qacH). Additionally, pFDA550584-30 contained a mercuric resistance (mer) operon, and pYA00079283 contained a Tn554-family transposon carrying an arsenic resistance operon (arsABCD). This Tn554-family transposon was identified previously in the chromosome of L. monocytogenes ST9 (Kuenne et al., 2013), a subtype especially adapted to the food processing environment (Maury et al., 2019). All G3 plasmids contained a predicted fimbrial adhesin (WP_061691480.1), suggestive of a role associated with attachment and host colonisation (Ageorges et al., 2020). Unlike G1 and G2 plasmids, which primarily infect serogroups 1/2a and 1/2b (Hingston et al., 2019), G3 plasmids appear to primarily infect 4b, including L. monocytogenes STs 1 and 6, which are considered hypervirulent (Maury et al., 2019). In this study, pHC143 was detected among ST6 and ST149 isolates.
Persistent clade C7 acquired a novel plasmid with a potentially broad host species range
The 4.6 Kb plasmid pHC192 represented a separate phylogenetic group (G4) than the previously reported 4.7 Kb Listeria plasmid pLMST6 (Kremer et al., 2017), which carries the benzalkonium chloride resistance gene ermC (Fig. 4a). Phylogenetically, pHC192 clustered closely with plasmids from Lactobacillus. Indeed, RepB of pHC192 (WP_035147907.1) was also detected in Lactobacilli and Brochothrix, suggestive of a broad host range for this plasmid. The closest relative of pHC192 in Listeria was the plasmid of the strain L. monocytogenes strain CFIAFB20130002, which possesses the lincosamide resistance gene lnuA (WP_001829870.1). Similarly, pLMST6 appears to also have a broad host range, as its RepB (WP_061092472.1) homologues were detected in Listeria, Salmonella and Enterococcus. Taken together, these findings suggest that unlike the large (>50 Kb) Listeria plasmids, the small (<10 Kb) plasmids of Listeria have a broad host range and several phylogenetically unrelated small plasmids have been acquired by Listeria through distinct transfer events across host species.
The plasmid pHC192 contains a putative tauE/safE -family sulphite exporter gene (WP_016896343.1) (Fig.4b) that is not typically present in Listeria plasmids (Hingston et al., 2019). The sequencing depth of coverage for pHC192 was approximately five times that of the chromosome, suggesting that pHC192 is a high copy number plasmid. This plasmid became increasingly prevalent among persistent clade C7 isolates during the sampling period and was detected in all isolates at the end of the study (Fig. 4c). An additional plasmid, pHC195-2, was detected in several isolates in the latter part of the study period. The pHC195-2 plasmid belonged to the phylogenetic group G2 (Fig. 3a) and closely resembled the reference plasmid pLMR479a (see Supplementary Fig. S2).
Transposons harbouring bacitracin and heavy metal resistance genes were more prevalent among persistent clades than singleton isolates
Among the 250 dairy farm isolates we identified six transposable elements: the L. monocytogenes IS3-like element (Kuenne et al., 2013); Listeria Genomic Island 2 (LGI-2) (Lee et al., 2017); Tn5801_B23 (León-Sampedro et al., 2016); and three novel transposons, which were submitted to the Transposon Registry (Tansirichaiya et al., 2019), and assigned the labels Tn7101, Tn7103 and Tn7104. The conjugative transposons LGI-2 and Tn5801_B23 were significantly more prevalent among persistent clade isolates than singleton isolates (Fisher’s Exact Test, p<.01) (Fig. 2b). LGI-2 is a conjugative transposon carrying cadmium and arsenic resistance cassettes and two multidrug transporters (see Supplementary Fig. S3). Identical (100% nucleotide identity) LGI-2 were present among all ST14 and ST145 isolates of this study. Moreover, BLASTn search identified identical LGI-2 in 11 L. monocytogenes and two Enterococcus faecalis complete genomes (see Supplementary Table S2), suggestive of recent transfer between these species.
The conjugative transposon Tn5801_B23 was detected in a subset of ST20 isolates, including the persistent clades C9 – C12 (see Supplementary Data S1). Tn5801_B23 detected in this study shared 97% identity with the Tn5801_B23 of the E. faecalis strain JH2-2 (see Supplementary Fig. S4). Tn5801_B23 is a conjugative transposon that contains an operon of resistance genes against the antimicrobial bacitracin (bcrABC) and a two-component system (baeSR) putatively involved in the regulation of the bcrABC operon (León-Sampedro et al., 2016). Unlike Tn5801_B23, other Tn5801-like transposons mediate tetracycline resistance in Enterococcus, Listeria and several other firmicute species (León-Sampedro et al., 2016). In L. monocytogenes ST20, Tn5801_B23 was inserted downstream of guaA (lmo1096), which is also the insertion site of the related conjugative transposon ICELm1 of the L. monocytogenes strain EGD-e, harbouring cadmium resistance genes (Kuenne et al., 2013).
Three novel transposons were identified among the farm isolates
The novel transposon Tn7101 was detected in the ST155 singleton isolate HC258, where it was inserted between lmo2596 and lmo2597 (see Supplementary Fig. S3). This transposon contains resistance genes against cadmium (cadA, cadC) and an arsenate reductase (arsC). Through a BLAST search we identified a variant of the Tn7101 containing a seven gene arsenic resistance cassette. This variant, labelled Tn7102, was detected in several L. monocytogenes and Enterococcus genomes deposited in GenBank (see Supplementary Fig. S2). The Tn7101 and Tn7102 of Listeria and Enterococcus were identical (100% nucleotide identity), suggestive of recent promiscuity between the two genera. Arsenic resistance genes in Tn7102 were distantly related (≥67% identity) to the arsenic resistance cassette of LGI-2 (see Supplementary Fig. S3).
The novel conjugative transposon Tn7103 was detected in the ST119 singleton isolate HC183, where it was inserted between lmo0810 and lmo0811. This transposon contained putative virulence genes encoding an InlJ-like internalin and a bacterial immunoglobulin (Big)-like protein (see Supplementary Fig. S5). A BLAST search confirmed the presence of Tn7103 in other L. monocytogenes strains, including N12-2532 (SAMN09947958), but we did not identify this element in other species.
The novel conjugative transposon Tn7104 was detected in the ST391 singleton isolate HC187 and was inserted between lmo1786 and lmo1787. This transposon contained a putative type I restriction-modification system (see Supplementary Fig. S6). Tn7104 was identified in several other L. monocytogenes strains deposited in GenBank, including the L. monocytogenes ST391 strain SHL013 (SAMN03265960), but we did not identify this element in other species.
Prophages were more prevalent among persistent clades than singleton isolates
All 250 dairy farm isolates from this study contained the L. monocytogenes monocin (Zink et al., 1995), and 0 – 3 additional prophages, which were detected at eight insertion sites (Fig. 2c). Prophages inserted into tRNA-Arg(tct) were significantly more prevalent among persistent clusters, and prophages inserted into tRNA-Lys(ctt) were significantly more prevalent among singleton isolates (Fisher’s Exact Test, p<.05).
Prophages harbouring cadmium resistance genes were detected in persistent clade C8
OPTSIL taxonomic clustering assigned prophages from this study into six genera. Prophages inserted into comK and tRNA genes were assigned to genera of Siphoviridae that are known to only infect Listeria. Surprisingly, in the isolate HC189, a 67 Kb Myovirus was inserted into comK, a site usually occupied by Siphoviridae (Pasechnek et al., 2020).
Prophages inserted between the rlmCD (lmo1703) and fosX (lmo1702) genes were not related to any of the Listeria specific phage genera, but instead represented a separate genus that infects a broad range of Firmicute species (Fig. 5). Many of the phages in this genus harbour antimicrobial and heavy metal resistance cassettes (see Supplementary Fig. S7). In this study, phages inserted between the rlmCD and fosX were detected among all isolates of persistent clade C8 and among three singleton isolates (see Supplementary Data S1). Among isolates of persistent clade C8, prophages inserted between rlmCD and fosX all harboured a cadmium resistance cassette (see Supplementary Fig. S7). In contrast, in the singleton isolates prophages inserted between rlmCD and fosX harboured no cadmium or antimicrobial resistance genes. Within Listeria genomes deposited in GenBank, we identified prophages inserted between rlmCD and fosX that carried resistance genes against cadmium (cadA), macrolides (mefA, msrD), tetracycline (tetM) and streptogramin (vatA).
Acquired heavy metal resistance genes were more prevalent among persistent clades than singleton isolates
The frequent occurrence of heavy metal resistance genes among L. monocytogenes persisting in food processing environments has led to the speculation that these determinants promote the environmental persistence of L. monocytogenes (Parsons et al., 2020). Indeed, heavy metal resistance genes were significantly more prevalent among persistent clade isolates than singleton isolates (Fig. 2d). Among the 250 sequenced isolates, we detected mobile genetic elements harbouring resistance genes against arsenic and cadmium (see Supplementary Fig. S8). Resistance genes against biocides and antimicrobials other than bacitracin were not detected in this study. However, variants of the mobile elements harbouring genes against biocides and antimicrobials were detected among farm and farm animal isolates of L. monocytogenes among genomes deposited in GenBank (see Supplementary Fig. S8 and Table S3). Taken together, the results of this study suggest that L. monocytogenes inhabiting the dairy farm environment constitutes a reservoir of diverse mobile genetic elements that have the potential to harbour resistance genes against antimicrobials, biocides, and heavy metals.
Three systems conveying immunity against invading DNA were negatively associated with persistence on dairy farms
The CRISPR-cas type IIA system, and the type II restriction-modification system LmoJ3 (Lee et al., 2012) were negatively associated with persistence in the dairy farm environment among L. monocytogenes Lineage II isolates (see Supplementary Table S4). CRISPR-cas systems and restriction-modification systems can act in synchrony to protect the host against invading prophages and other mobile elements (Price et al., 2016). Additionally, a putative recombination and DNA strand exchange inhibitor protein (WP_03166494.1) was negatively associated with persistence. These findings agree with the lower prevalence of mobile genetic elements and prophages among singleton isolates than persistent clade isolates and suggest that systems involved with inhibiting invading DNA are detrimental for the persistence of L. monocytogenes in the dairy farm environment.
Among the genes that were positively associated with persistence on dairy farms was a gene associated with biofilm formation (bapL) and a peptidoglycan hydrolase (murA). L. monocytogenes hypervariable hotspots 1 and 8 (Kuenne et al., 2013) contained genes both positively and negatively associated with persistence. Indeed, most genes associated with persistence belonged to L. monocytogenes hypervariable hotspots or prophages, suggesting that the role these components play in Listeria niche adaptation deems further study.
Discussion
Whole genome sequencing and subsequent analyses of 250 L. monocytogenes isolates from dairy farms illustrated that dairy farm isolates are hosts to a diversity of mobile genetic elements that carry, or have the potential to carry, resistance genes against antimicrobials, biocides and heavy metals. We found that prophages and other mobile genomic elements were significantly more numerous among isolates belonging to persistent clades than among singleton isolates. Moreover, systems that provide immunity against invading mobile genetic elements (Garneau et al, 2010; Lee et al., 2012; Price et al., 2016), namely the CRISPR-cas IIA system, the type II restriction-modification system LmoJ3, and a putative recombination and DNA strand exchange inhibitor protein, were negatively associated with persistence. Taken together, our findings suggest that for L. monocytogenes inhabiting the farm environment, the benefits of being receptive to potentially advantageous mobile elements outweigh the benefits protecting against elements that are potentially deleterious. Many of the mobile elements we identified carried genes encoding phenotypes that promote the survival of L. monocytogenes on farms, such as antimicrobial resistance genes or virulence factors. Moreover, genes responsible for the conjugation of mobile elements may have a dual role in promoting biofilm formation and invasion of the mammalian host (Barrios et al., 2005; Ageorges et al., 2020), thereby supporting the survival of L. monocytogenes on farms.
We identified a surprising diversity of mobile genetic elements encoding heavy metal resistance genes among the dairy farm isolates and acquired heavy metal resistance genes were encountered more frequently among persistent clade isolates that singleton isolates. Similarly, heavy metal resistance genes are more prevalent among persistent than sporadic L. monocytogenes subtypes from foods and food processing environments (Harvey & Gilmour, 2001; Pasquali et al., 2018). Whether heavy metal resistance genes contribute to persistence or merely co-occur with other fitness enhancing determinants remains unclear (Parsons et al., 2020). Nevertheless, heavy metal resistance genes may represent useful markers to aid the detection of L. monocytogenes strains with fitness-enhancing mobile genetic elements.
In the present study we found two novel plasmids, namely pHC143 (plasmid group G3) and pHC192 (plasmid group G4), that were unrelated to the previously described plasmids of Listeria. The comparison of these plasmids against the short-read sequence data deposited in GenBank led to the identification of three novel G3 plasmid variants carrying genes against biocides and heavy metals. Importantly, G3 plasmids can infect hypervirulent ST1 and ST6 strains, potentially enabling these STs to adapt to the food processing environment through the acquisition of biocide and heavy metal resistance. Indeed, we discovered a G3 plasmid harbouring the biocide resistance gene qacH and the arsenic resistance cassette arsABCD in the ST6 outbreak isolate YA00079283, associated with the largest listeriosis outbreak known to date (Smith et al., 2019).
We identified four transposons in Listeria, namely LGI-2, Tn5801_B23, Tn7101 and Tn7102, that closely resembled transposons in Enterococci, suggestive of recent transfer between the two genera. The co-occurrence of genomic elements in Enterococci to Listeria was unsurprising, as both genera are highly prevalent in animal faeces and farms (Franz et al., 1999; Hellström et al., 2010; Castro et al., 2018). Transfer of conjugative elements has been demonstrated both from Enterococci to Listeria and vice versa (Jahan & Holley, 2016; Haubert et al., 2018) indicating that both genera are potential donors. The extent to which Enterococci and other Firmicutes contribute to the horizontal spread of mobile elements and their associated antimicrobial, biocide and heavy metal resistance determinants in Listeria has implications for food safety and should be explored through further study.
Bacitracin resistance genes, mediated by Tn5801_B23, were significantly more prevalent among isolates in persistent clades than singleton isolates. Moreover, L. monocytogenes isolates harbouring Tn5801_B23 were detected frequently on all three farms investigated. It is plausible that the widespread use of bacitracin as a growth promoter in animal feeds during the mid-twentieth century facilitated the expansion of bacitracin resistance in Listeria. Although animal feeds are frequently contaminated by L. monocytogenes (Hellström et al., 2010; Castro et al., 2018), the frequent detection of Tn5801_B23 on the dairy farms investigated remains curious, as feed supplementation with bacitracin has been banned in the EU since the 1990’s.
We found that prophages were more prevalent among persistent clade isolates than singleton isolates. It is unclear whether the higher number of prophages among persistent clade isolates represents a beneficial role for these elements or is a side effect of being receptive to foreign DNA. There is increasing evidence that prophages can mediate beneficial phenotypes for their host. Phages mediate resistance or virulence properties in numerous bacterial species (Harrison & Brockhurst, 2017), and in Listeria, Siphoviruses inserted into comK were found to regulate the gene in a symbiotic manner (Pasechnek et al. 2020). Here, we discovered phage-mediated carriage of cadmium resistance and various antimicrobials in Listeria, suggesting that prophages contribute to the spread of phenotypes supporting persistence. Moreover, we noted that these phages belonged to a genus of Siphovirus with an apparently broad host species range that were introduced to Listeria through several distinct transfer evets. Host species jumps have the potential accelerate the transfer of novel resistance determinants between Listeria and other Firmicutes.
In conclusion, our study indicates that L. monocytogenes inhabiting the dairy farm environment are receptive to a diversity of prophages and mobile genetic elements. We suggest that mobile elements enable L. monocytogenes to adapt to the stresses encountered in the farm ecosystem and in general improve the fitness of the pathogen on farms, thereby supporting persistence. Given the abundance of L. monocytogenes on farms farms (Nightingale et al., 2004; Esteban et al., 2009; Castro et al., 2018) and the apparent exchange of mobile genetic elements between Listeria and other Firmicute species, L. monocytogenes occurring in agroecosystems should be viewed as a reservoir of mobile genetic elements. Importantly, many of these elements have the potential to carry and spread antimicrobial, biocide, and heavy metal resistance genes. The spread of mobile genetic elements and resistance determinants from primary production to Listeria in the food processing environments has important food safety implications and should be explored further. The present study represents a leap forward in this effort and in our understanding of listerial ecology in the agroecosystem.
Methods
Whole genome sequencing
Altogether 250 L. monocytogenes isolates obtained from three Finnish dairy cattle farms during 2013–2016 (Castro et al., 2018) were selected for whole genome sequencing in the present study (see Supplementary Data S1). DNA was extracted from overnight cultures using the guanidium thiocyanate extraction method (Pitcher et al. 1989). DNA samples were standardized to a concentration of 10 ng/μl using the dsDNA BR Assay Kit (Thermo Fisher Scientific; Waltham, MA, USA) using the Qubit Fluorometer (Thermo Fisher Scientific). Genomic libraries were constructed form the DNA samples using the Nextera XT DNA Sample Preparation Kit (Illumina; San Diego, CA, USA), and paired-end sequencing (2×250 bp) was performed using the Illumina HiSeq platform.
Genome assembly, pangenome construction, subtyping
Following the removal of adapter sequences and low-quality reads using Trimmomatic 0.36 (Bolger et al., 2014), draft genomes were assembled using SPAdes 3.9 with K-mer values 55, 77, 99, 113 and 127 (Bankevich et al., 2012). Assembly quality was assessed using QUAST 4.0 (Gurevich et al., 2013) and taxonomic assignment was performed using Kraken (Wood & Salzberg, 2014). The assemblies were annotated using Prokka 1.12 (Seemann, 2014). The pangenome of the sequenced isolates was constructed using Roary 3.8.0 (Page et al., 2015) with the protein identity cut-off value set at 90%. Multi Locus Sequence Types (ST) were determined in silico from the assembled genomes using the BIGSdb-Lm database (Moura et al., 2017). Genome assemblies were deposited in GenBank under the BioProject number PRJNA704814 (see Supplementary Data S1).
Maximum-Likelihood Phylogenomic Analysis
Phylogenomic reconstruction of the L. monocytogenes isolates was performed using the Lyve-SET 1.1.4f pipeline (Katz et al., 2017). For each ST, a draft assembly from the present study with the best quality statistics, i.e. the highest N50 value and lowest number of contigs (see Supplementary Data S1), was used as a reference genome. The Lyve-SET pipeline was run using Listeria monocytogenes pre-sets (Katz et al., 2017), with additional options --mask-phages, --mask-cliffs, and --read_cleaner CGP. In brief, the pipeline generated genome alignments by mapping quality-filtered reads to a reference genome. To improve the accuracy of phylogenomic inference, putative prophage genes were removed from the reference genome prior to mapping. Mapping was followed by the detection of high-quality SNPs, having ≥ 10x depth of coverage and ≥ 75% consensus among reads. Recombinant sites within the genome alignments generated by Lyve-SET were identified and removed using (Croucher et al., 2015). PhyML 3.3 (Guindon et al., 2010) was used to infer Maximum-Likelihood - phylogeny of each ST using a general time reversible model (GTR) with 100 bootstrap replicates. Persistent clades of L. monocytogenes were defined monophyletic clades of isolates with PWDs < 20 SNPs (Pightling et al., 2018) that were isolated from the same farm from ≥3 samples during ≥6 months.
Detection and analysis of plasmids
Putative plasmid sequences were assembled from read files using SPAdes with option --plasmid and the assembled contigs were annotated using Prokka 1.12. Additionally, whole-genome assembly graphs generated by SPAdes were visualized using Bandage 0.8.1 (Wick et al., 2015) and extrachromosomal elements were inspected manually. Similarity of the plasmid sequences we identified to plasmids deposited in GenBank was assed using the BLAST suite (http://www.ncbi.nlm.nih.gov/blast). Maximum-Likelihood phylogeny of the plasmids, based on the translated sequence alignments of the repA gene, were generated with MEGA7 (Kumar et al., 2016), using the Jones-Taylor-Thornton substitution model with 100 bootstraps. Alignments of the translated sequences of the repB gene were used to compare plasmids in which repA was truncated or absent. Plasmid alignments were generated and visualized using BRIG 0.95 (Alikhan et al., 2018) and EasyFig 1.2 (Sullivan et al., 2011).
Detection and analysis of transposons
The occurrence transposons ICELm1 (Kuenne et al., 2013), LGI-2 (Lee et al., 2017), Tn5422 (Lebrun et al., 1994), Tn6188 (Müller et al., 2013), and the IS3-like and TN554-like transposons of L. monocytogenes (Kuenne et al., 2013), among isolates from this study was assed using BLAST. Additionally, the pangenome was searched for annotations including “recombinase”, “integrase”, “transposase”, “transposon”, “cadmium”, “arsenic”, “mercuric”, “ardA”, “ftsK”, “P60” and “iap” and hits were inspected manually. EasyFig 1.2 was used to align and visualize the identified transposons and their occurrence among genomes deposited in GenBank was assessed using BLAST.
Detection and analysis of prophages
Prophages inserted into the L. monocytogenes genomes were identified using PHASTER (Arndt et al., 2016) and the insertion sites were inspected manually. Phylogeny and taxonomic clustering of prophages classified by the PHASTER algorithm as “intact” were inferred using VICTOR (Meier-Kolthoff & Göker, 2017). Nineteen additional Listeria phage genomes and one Streptococcal phage genome obtained from GenBank were included in the analyses for reference (see Supplementary Table S1). In brief, VICTOR applies the Genome-BLAST Distance Phylogeny (GBDP) method (Meier-Kolthoff et al., 2013) to obtain pairwise distances, from which balanced minimum evolution trees are inferred. VICTOR utilizes OPTSIL (Göker et al., 2009) to obtain taxonomic clustering. Duplicate phage genomes are removed from the analysis. Trees generated by VICTOR were visualized using FigTree 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). BLAST was used to identify phages inserted between rlmCD and fosX in the genomes of Listeria and other bacterial species deposited in GenBank, and hits were inspected manually. Phylogeny and taxonomic clustering of prophages inserted between rlmCD and fosX were inferred using VICTOR.
Identification of genes associated with predominance
Scoary 1.6.16 (Brynildsrud et al., 2016) was used to identify genes significantly associated with occurrence in persistent clade isolates versus singleton isolates. Scoary was executed using default options, using the gene_presence_abence.csv -file generated by Roary as input. Associations with a Bonferroni corrected p<.05 were considered significant. As all predominant clades belonged to Lineage II, the analysis was limited to the 233 Lineage II isolates of this study to reduce noise arising from population structure bias.
Author Contributions
H.C. conducted the analyses and wrote the manuscript. H.C. and M.L. provided the study materials and designed the study. F.D., H.K. and M.L. supervised the study. All authors participated in data interpretation and reviewed the manuscript.
Competing Interests
The authors have no competing interests to declare.
Acknowledgements
This research was supported by the Finnish Ministry of Agriculture and Forestry (grant number 618/03.01.02/2017) and by the Walter Ehrström Foundation. The authors want to acknowledge Esa Penttinen for his assistance with DNA extraction.