Comparative Genomic Analysis of a Multidrug-Resistant Listeria monocytogenes ST477 Isolate

Abstract

Listeria monocytogenes is an opportunistic human foodborne pathogen that causes severe infections with high hospitalization and fatality rates. Clonal complex 9 (CC9) contains a large number of sequence types (STs) and is one of the predominant clones distributed worldwide. However, genetic characteristics of ST477 isolates, which also belong to CC9, have never been examined, and little is known about the detail genomic traits of this food-associated clone. In this study, we sequenced and constructed the whole-genome sequence of an ST477 isolate from a frozen food sample in China and compared it with 58 previously sequenced genomes of 25 human-associated, 5 animal, and 27 food isolates consisting of 6 CC9 and 52 other clones. Phylogenetic analysis revealed that the ST477 clustered with three Canadian ST9 isolates. All phylogeny revealed that CC9 isolates involved in this study consistently possessed the invasion-related gene vip. Mobile genetic elements (MGEs), resistance genes, and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system were elucidated among CC9 isolates. Our ST477 isolate contained a Tn554-like transposon, carrying five arsenical-resistance genes (arsA–arsD, arsR), which was exclusively identified in the CC9 background. Compared with the ST477 genome, three Canadian ST9 isolates shared nonsynonymous nucleotide substitutions in the condensin complex gene smc and cell surface protein genes ftsA and essC. Our findings preliminarily indicate that the extraordinary success of CC9 clone in colonization of different geographical regions is likely due to conserved features harboring MGEs, functional virulence and resistance genes. ST477 and three ST9 genomes are closely related and the distinct differences between them consist primarily of changes in genes involved in multiplication and invasion, which may contribute to the prevalence of ST9 isolates in food and food processing environment.

Introduction

L isteria monocytogenes is the etiological agent of listeriosis, which includes two deadly complications: central nervous system and maternal/fetal infections. This foodborne pathogen can survive under a wide range of temperatures and pH conditions (Walker et al., 1990) for a long period of time, and can easily contaminate a wide variety of foods (Kathariou, 2002; Gandhi and Chikindas, 2007). L. monocytogenes consists of four lineages (lineage I, II, III, and IV), among which lineage I strains are more likely to cause human cases than lineage II strains (Gray et al., 2004) and lineages III and IV strains are predominantly isolated from animal sources (Jeffers et al., 2001; Kuenne et al., 2013).

According to the Food and Drug Administration, over the past 2 years, extensive food products have been recalled due to the suspicions of L. monocytogenes contamination. Recent studies indicated multinational listeriosis outbreak due to L. monocytogenes transmission (Jackson et al., 2016; Angelo et al., 2017). Another study isolated L. monocytogenes in ∼20% of 1036 raw food samples from 24 Chinese cities from South to North China (Wu et al., 2015). In China, the predominant sequence types (STs) isolated from food are ST9, ST8, and ST87 (Wang et al., 2012; Chen et al., 2018). ST9 isolates, which belong to clonal complex 9 (CC9), were also found to infect immunodeficient humans and cause bacteremia (Ragon et al., 2008). However, potential pathogenicity of CC9 strains has not been performed previously.

L. monocytogenes chromosomal genes are highly conserved with respect to their structural organization (Kuenne et al., 2013). The diversity and wide variation are mainly reflected in their accessory genomes, although they only occupy a small fraction (12–23%) of the L. monocytogenes gene content compared with the core genomes (den Bakker et al., 2013). Accessory genetic materials, including mobile genetic elements (MGEs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, contribute to the phenotypic plasticity and adaptability of this pathogen (Kuenne et al., 2013). The Tn554-like transposon that introduces an arsenate resistance operon (arsCBADR) into the L. monocytogenes chromosome has been reported (Kuenne et al., 2013) but has not been characterized in detail. Studies on the structural organization of the Tn554-like transposon and genetic information are therefore necessary to assess the transmission and pathogenicity among L. monocytogenes isolates.

In the present study, we sequenced and analyzed the genetic sequence of an ST477 isolate and compared it with previously sequenced genomes of L. monocytogenes from human, animal, and food sources to (1) identify the genetic characteristics of the ST477 isolate; (2) investigate the genetic diversity of CC9 isolates from different sources and distant geographical regions; and (3) examine genome-level variation among CC9 isolates. Our results would serve as a framework for future analyses of CC9 strains.

Materials and Methods

Isolate

The ST477 L. monocytogenes isolate (NH1) with the 1/2c serotype examined in this study was originally isolated from a quick-frozen food sample consisting of wheat flour, and meat was purchased in Hebei province, China, in 2010. The isolate displayed resistance to multiple antibiotics, including tetracycline, erythromycin, chloramphenicol, streptomycin, cefotaxime, and trimethoprim/sulfamethoxazole (Yan et al., 2010), as well as cotolerance to cadmium and benzalkonium chloride (Xu et al., 2014).

Genome sequencing and annotation

Genomic DNA was extracted from overnight culture grown in a brain/heart infusion broth at 37°C using the DNeasy DNA extraction kit (Axygen, Union City). Purified genomic DNA was sequenced using the PacBio RS II (Pacific Biosciences, Menlo Park, CA) and Illumina MiSeq (Illumina, San Diego, CA) platforms as previously described (Zheng et al., 2017). A single contiguous chromosome consisting of 3,002,491 bp and a 37.92% GC, DNA G+C content was obtained (Supplementary Fig. S1). The complete sequence of NH1 was deposited in GenBank under the accession number CP021325. Gene prediction and annotation were performed as previously described (Zheng et al., 2017).

Phylogenetic and clustering analyses

We constructed a phylogenetic tree of the ST477 isolate and 58 previously published genome sequences with different geographic areas and sources to investigate the genomic traits of the ST477 isolate (Supplementary Table S1). These 58 isolates include 25 human-associated, 27 food-associated, and 6 from other or unknown sources isolates, and comprised 6 CC9 and 52 other clones. This data set comprised two genomes from lineage III to root the phylogenetic tree. The tree was constructed under a maximum likelihood optimality criterion using RAxML v7.4.2 (Uhlemann et al., 2017).

In silico MLST analysis and determination of profiles of virulence and resistance genes

Clonal complexes were determined by the Institut Pasteur MLST database with default parameters (www.pasteur.fr/mls). To decipher the virulence heterogeneity between clones, 76 loci related to virulence or resistance were deduced in silico using the BIGSdb-Lm platform (http://bigsdb.pasteur.fr/listeria) (Jolley and Maiden, 2010) for each of the 59 genome sequences.

Analysis of the features of CC9 genomes harboring Tn554-like transposon

Based on the results of BLASTN search, eight isolates that harbored Tn554-like transposon and two isolates that did not were analyzed to compare the similarities of their genomic contents. Antimicrobial and heavy metal resistance genes, stress survival islet 1 (SSI-1), and IS3 were examined using a BLASTX search as described previously (Soge et al., 2016). PHASTER (www.phaster.ca) (Arndt et al., 2016) was used to identify prophage sequences in the 11 genomes. CRISPR arrays in each genome were identified using CRISPRFinder (Grissa et al., 2007), and the flanking sequences of these clusters were subsequently scanned for the presence/absence of cas gene.

Whole-genome alignment

Whole-genome alignment of seven CC9 genomes was performed. Mauve software was used to align the genomic sequences and identify locally collinear blocks shared by all genomes (Ortiz et al., 2016).

Single-nucleotide polymorphism identification and analysis

The WGS fastq data of six CC9 isolates were aligned against the chromosome of the ST477 to detect single-nucleotide polymorphisms (SNPs) using GATK and SAMtools as previously described (Cheng et al., 2014).

Results

Phylogenetic analysis of ST477 isolate

We determined a core gene sequence-based phylogeny based on 2037 single-copy orthologs present in the 59 genome sequences (Fig. 1). This tree resolved three lineages (lineage I–III). Unsurprisingly, the two lineage III isolates chosen as outgroup were clearly distant from all other isolates, whereas lineages I and II were strongly structured into major subgroups, each comprising multiple closely related isolates. Phylogenetic lineages exhibited strong association with serotype (Fig. 1). Lineage I contained two major clades: one consisted predominantly of serotype 4b plus one sample of serotype 4e and one of unknown serotype; in the other clade, the majority (6/11) of samples were of unknown serotype, the other samples belonged to serotypes 1/2b, 7, and one sample of serotype 1/2a. Lineage II consisted predominantly of serotype 1/2a, but contained two clades composed of serotypes 1/2c and 3c and a clade that included one serotype 3a isolate and an isolate of unknown serotype. The subgroups did not show any clear association with isolate source, although uncertainty of the infection source for the human clinical isolates could obscure such patterns. Also, isolates from one country did not cluster together phylogenetically.

FIG. 1.

Maximum-likelihood phylogeny of 59 Listeria monocytogenes isolates based on 2073 single-copy core genes. The annotation rings surrounding the tree, from inside to outside, depict (1) serotype, (2) sample isolation source, (3) geographic origin, (4) clonal cluster number, and (5) year of sample collection. Shading behind the branches represents lineage, and white indicates unknown information.

Although most of the 59 isolates were distributed among 35 known MLST types (Supplementary Table S1), we discovered that four displaying novel STs beared undescribed MLST alleles or previously unreported allelic combinations: three of these isolates, including two Canadian isolates from meat products and one clinical isolate from China, formed a clade within lineage I; the other isolate (an environmental sample from Canada) occupied a basal position in lineage II. Seven CC9 isolates derived from food and clinical sources formed a single clade within lineage II. In the CC9 subgroup, the ST477 isolate displayed the closest relationship to three ST9 food-associated isolates from Canada (10-5025, 10-5026, and 10-5027).

Biological features of different L. monocytogenes clonal complexes

Certain variations in virulence and stress resistance genes among L. monocytogenes lineages and subgroups were observed (Fig. 2). As expected, the major pathogenicity island LIPI-1 was highly conserved. Complete LIPI-3 and the LIPI-4 islands were almost exclusively detected within lineage I. The number of internalins ranged from two (CFSAN004330) to nine in the examined genomes. InlG was absent in lineage I except the CC6 subgroup. The ST477 isolate contained LIPI-1, nine inl genes, but not the LIPI-3 and LIPI-4 islands. Overall, there was rare difference between the ST477 and other CC9 isolates, except that the ST477 isolate lacked the bile-resistance gene mdrM. In addition, similar to clinical isolates, all CC9 isolates harbored inlH and inlK. Strikingly, a virulence gene (vip), encoding a surface protein, was universally present in CC9 isolates. Collectively, these results demonstrate the heterogeneity of biological features among L. monocytogenes subgroups and mark frequent virulence genes in CC9 clone.

FIG. 2.

Virulence and resistance profiles across the phylogeny of the 59 L. monocytogenes isolates. (a) Cluster analysis based on single-copy core orthologs. (b) Pattern of gene presence (colored line) or absence (white). The first and last columns correspond to the serogroup and sample source, respectively. The gene presence/absence matrix represents (from left to right) genes involved in teichoic acid biosynthesis (gltAB, tagB, gtcA), genes located in the LIPI-1 (prfA, plcA, hly, mpl, actA, plcB), LIPI-3 (llsAGHXBYDP), and LIPI-4 (LM9005581_70009 to LM9005581_70014), genes coding for internalins (inlABCEFGHJK), and other genes involved in adherence (ami, dltA, fbpA, lap, lapB), invasion (aut, aut_IVb, cwhA, lpeA, vip), intracellular survival (hpt, lplA1, oppA, prsA2, purQ, svpA), regulation of transcription and translation (agrAC, cheAY, fur, lisKR, rsbV, sigB, stp, virRS), surface protein anchoring (lgt, lspA, srtAB), peptidoglycan modification (oatA, pdgA), immune modulation (lntA), bile-resistance (bsh, mdrM, mdrT, brtA), resistance to detergents (qac, bcrABC, ermE), and biofilm formation and virulence (comK).

Genome comparisons of CC9 isolates

We identified a Tn554-like transposon sequence in the ST477 isolate. This transposon encodes three consecutive transposase genes (tnpABC), five arsenate resistance genes, and four genes encoding hypothetical proteins with unknown function (Fig. 3). Through BLASTN searches in GenBank using this nucleotide sequence as the query, highly conserved homologues (99% nucleotide sequence similarity) in eight other L. monocytogenes and two Listeria ivanovii genomes were found. The Tn554 transposon from Staphylococcus aureus and the Tn554-like transposon from the ST477 isolate shared a modular composition with a transposase module (consisting of the transposases TnpABC) and different resistance markers. Transposase genes of the Tn554-like transposon shared a low degree of similarity with the Tn554 transposon. Unlike the Tn554-like transposon, the Tn554 transposon is chromosomally integrated within the radC gene and displays a 5′-GATGTA-3′ and 5′-CAAGTT-3′ sequence at its left- and right-end junction, respectively. Thus, there are important differences between these two transposons. The GC content of Tn554 (32.6%) and Tn554-like (37.9%) transposons was similar to that of S. aureus and L. monocytogenes genomes (32.6–33% and 37.8–38%, respectively), suggesting that the ancestor of the Tn554-like transposon probably arose from L. monocytogenes rather than S. aureus. Not all CC9 isolates carry the Tn554-like transposon; however, eight isolates containing this transposon belong to CC9 clone, suggesting a single event of insertion into the CC9 background.

FIG. 3.

Organization of the L. monocytogenes Tn554-like transposon and comparison with the related transposon Tn554. ORFs are shown as arrows, indicating the transcription direction, and the colors of the arrows represent different fragments. Proteins with other or unknown functions are shown in yellow.

SSI-1 had been postulated to facilitate elevated resistance to several types of stress in certain L. monocytogenes strains. Our results indicated that no specific patterns existed among these strains (Table 1); however, both the ST477 isolate and two Canadian isolates (10-5026 and 10-5027) harbored this islet, further emphasizing their similarity.

Table 1.

Relevant Features of the Listeria monocytogenes Strain Genomes

Feature	Result for the following strains
Feature	NH1	10-5027	10-5026	10-5025	SLCC2372	SLCC2479	AT3E	AL4E	FSLR2-561	SLCC2482	10-092876-1559 LM1
Source	Frozen-food	Food processing	Food processing	Bacon flake	Human	N.A.	Food product	Equipment	Goat	Human	Chicken breast
Country	China	Canada	Canada	Canada	United Kingdom	N.A.	Finland	Finland	N.A.	N.A.	N.A.
Year	2007	2009	2009	2009	1935	1966	1995	1998	N.A.	1966	2006
Coding sequence	2983	2943	2941	2941	2988	2932	3119	2986	N.A.	2870	2954
GC content (%)	37.92	37.9	37.9	37.9	37.96	38	38	38	38	37.96	37.9
Total length	3,002,491	2,965,327	2,967,496	2,967,523	2,972,810	2.972,172	3,057,808	3,027,995	2,973,801	2.936,689	2,972,553
ST	477	9	9	9	122	9	9	9	9	3	37
Presence of:
Tn554-like	+	+	+	+	+	+	+	+	+	−	−
SSI-1	+	+	+	−	+	+	+	+	+	+	−
CRISPR	+	+	−	−	+	+	+	+	−	+	+
IS3	+	+	+	−	+	+	+	+	−	+	+
No. of prophages	4	3	3	3	3	3	5	3	3	2	3
Presence of the following prophages
Monocin	+	+	+	+	+	+	+	+	+	+	+
ϕLMC1	+	−	−	−	−	−	+	−	−	+	−
LP _101	+	+	+	+	−	−	+	+	+	−	+
A006	+	+	+	+	+	+	+	+	+	−	+
B025	−	−	−	−	+	+	−	−	−	−	−
PhiCT453A	−	−	−	−	−	−	+	−	−	−	−

The presence of mobile genetic elements is indicated by the “+” sign and the absence is indicated by the “−” sign.

GC, DNA G+C; N.A., unknown information; ST, sequence type.

PHASTER-based analysis of the fully closed ST477 genome predicted four putative complete prophages (Supplementary Table S2), including three common prophages (A006, B025, and LP_101) and a composite Φlmc1 prophage (A118, A006, and B025). Highly similar (99–100% identity) Φlmc1 sequences, including the site-specific integrase region, were detected in only two L. monocytogenes isolates (SLCC2482 and AT3E), isolated from humans and food from the United States and Finland, respectively (Table 1). Although the three Canadian isolates were devoid of this prophage, they still harbored the site-specific integrase region (Fig. 4), suggesting a possible recombination event that inserted the prophage into their chromosomes.

FIG. 4.

Linear comparison of the Φlmc1 prophage in four different L. monocytogenes isolates. The prophage is present in NH1, SLCC2482, and AT3E strains, and is absent from strain 10-5027. Homologous gene clusters in different isolates are shaded in gray (>97% nucleotide sequence similarity).

Our ST477 genome harbored resistance genes against several types of antibiotics, including sulfonamides (folP, thyA), aminoglycosides (adeC, strA), glycopeptides (cls), lincosamides (lmrB), and fosfomycin (fosX) (Table 2). In addition, this genome contained three efflux pump-related genes that confer resistance to tetracyclines (tetA) and quinolone (fepA and norB). Moreover, the small multidrug resistance (SMR) transporter qacE was identified in the ST477 and other CC9 strains, increasing their capacity to colonize food production plants. Notably, the antibiotic and heavy metal resistance genes were identical to those found in CC9 isolates, but greatly differed from those of non-CC9 isolates. The presence of multiple resistance genes in CC9 genomes further reiterates the health risk as these strains could be transmitted to humans via food contamination.

Table 2.

Comparison of Resistance Genes in Listeria monocytogenes from Different Countries

Resistance genes	CC9									Non-CC9
Resistance genes	NH1	10-5025	10-5026	10-5027	SLCC2372	SLCC2479	AT3E	AL4E	FSLR2-561	10-092876-1559 LM1	SLCC2482
Drug resistance genes
folP	+	+	+	+	+	+	+	+	+	+	−
fosX	+	+	+	+	+	+	+	+	+	+	−
adeC	+	+	+	+	+	+	+	+	+	−	−
thyA	+	+	+	+	+	+	+	+	+	+	−
Cls	+	+	+	+	+	+	+	+	+	+	−
strA	+	+	+	+	+	+	+	+	+	+	−
lmrB	+	+	+	+	+	+	+	+	+	+	−
Heavy metal resistance genes
Cad	+	+	+	+	+	+	+	+	+	+	+
arsA	+	+	+	+	+	+	+	+	+	−	−
arsB	+	+	+	+	+	+	+	+	+	−	−
arsC	+	+	+	+	+	+	+	+	+	−	−
arsD	+	+	+	+	+	+	+	+	+	−	−
arsR	+	+	+	+	+	+	+	+	+	−	−
Efflux pump
SMR transporter (qacE)	+	−	+	+	−	+	+	+	−	+	−
MFS efflux pump (tetA)	+	−	+	+	−	+	+	+	−	+	−
MATE transporter (fepA)	+	−	+	+	−	+	+	+	−	+	−
MFS transporter (norB)	+	−	+	+	−	+	+	+	−	+	−

The presence of resistance genes is indicated by the “+” sign and the absence is indicated by the “−” sign.

MFS, major facilitator superfamily; MATE, mulitidrug and toxic compound extrusion; SMR, small multidrug resistance.

The ST477 and three closely related isolates contained an identical CRISPR cluster, consisting of four spacer sequences interspersed between five highly conserved 29 bp direct repeat regions, and were not associated with any cas genes (Supplementary Table S3). All CC9 isolates shared identical spacer arrays, which were directed versus known listeriaphages (B054, P70). However, the number of spacers in CC9 was much less than other clones.

SNP analysis among CC9 isolates

The initial comparative analysis of the ST477 genome with three closely related ST9 sequences was performed following the genome alignment of seven CC9 isolates using Mauve (Supplementary Fig. S2). The NH1 genome is ∼37 kbp larger than the 10-5027 genome and 35 kbp larger than the other two genomes (Supplementary Table S4). Annotation of the predicted coding sequence regions for the four genomes using the NCBI database revealed that the ST477 genome contained the highest number of genes (n = 3084) and pseudogenes (n = 24). The ST477 genome was highly similar to the three ST9 isolates, except for two major regions, ranging from ∼669–705 and 2754–2761 kbp in ST477 (Fig. 5). These regions contain genes that encode hypothetical proteins, metabolism enzymes, and phage-related proteins. Thus, the ST477 isolate and these Canadian ST9 isolates may share a common gene pool.

FIG. 5.

Whole-genome alignment of L. monocytogenes using the Mauve, version 2.3.1, and the figure was generated using the Mauve viewer. Larger white portions identify areas of low similarity. Areas that are completely white within a block are not aligned and most likely contain sequence elements specific to a particular genome.

Variant detection, based on read mapping of each of the CC9 isolates to our ST477 isolate, showed that 291 nonsynonymous SNPs occurred in ST477 genome (Supplementary Table S5). A further comparison of SNPs among three closely related Canadian ST9 isolates indicated that 60 nonsynonymous substitutions were shared by these isolates (Table 3). Of note, nonsynonymous substitutions occurred in important functional genes smc, ftsA, and essC and other genes encoding cell surface proteins. We also identified nine different indels in each of the Canadian isolates. However, only one indel encoding a hypothetical protein was identified in all of these isolates. This result showed that these genes play key role in the evolution pattern of ST477 and ST9.

Table 3.

Single-Nucleotide Polymorphisms and Indels Shared in Three Canadian ST9 Isolates

No.	Pos ^a	Consensus	SNP in isolate			Affected gene/gene reference	Amino acid change	Gene product ^a
No.	Pos ^a	Consensus	10-5025	10-5026	10-5027	Affected gene/gene reference	Amino acid change	Gene product ^a
1	59615	A	G	G	G	CA173_RS00275	Thr→Ala	Adenylosuccinate synthase
2	68354	A	C	C	C	essC	Ile→Leu	Type VII secretion protein EssC
3	214617	A	G	G	G	CA173_RS01050	Ile→Thr	L-lactate dehydrogenase
4	244106	G	A	A	A	CA173_RS01180	Ala→Thr	Protein arginine kinase
5	311439	G	A	A	A	CA173_RS01495	Ala→Thr	Serine protease
6	324944	C	A	A	A	CA173_RS01565	Gly→Cys	Hypothetical protein
7	325857	G	A	A	A	CA173_RS01570	Asp→Asn	Hypothetical protein
8	331276	A	T	T	T	CA173_RS01595	Glu→Asp	Hypothetical protein
9	418322	C	T	T	T	CA173_RS02055	Ala→Val	Transcriptional antiterminator
10	459841	G	A	A	A	CA173_RS02230	Glu→Lys	NAD(P)-dependent oxidoreductase
11	486437	T	C	C	C	CA173_RS02335	Cys→Arg	Hypothetical protein
12	508397	T	A	A	A	CA173_RS02460	Leu→His	Hypothetical protein
13	547406	T	C	C	C	CA173_RS02670	Leu→Ser	Hypothetical protein
14	568326	G	A	A	A	CA173_RS02755	Gly→Glu	Zn-dependent hydrolase
15	714418	C	T	T	T	CA173_RS03540	Arg→Trp	Amino acid permease
16	721744	A	C	C	C	CA173_RS03570	Lys→Gln	GntR family transcriptional regulator
17	733634	A	G	G	G	CA173_RS03660	Thr→Ala	NAD(P)-dependent dehydrogenase
18	736865	A	G	G	G	mogR	Phe→Leu	Motility repressor MogR
19	788677	T	A	A	A	CA173_RS03980	Val→Asp	Cell surface protein
20	923189	T	C	C	C	CA173_RS04615	Thr→Ala	LacI family transcriptional regulator
21	967301	A	G	G	G	CA173_RS04875	Ile→Met	Osmotically inducible protein C
22	1000236	A	T	T	T	CA173_RS05040	Gln→His	NADPH-dependent oxidoreductase
23	1011774	T	G	G	G	CA173_RS05110	Phe→Leu	AI-2E family transporter
24	1036973	G	C	C	C	CA173_RS05260	Asp→His	Branched chain amino acid aminotransferase
25	1041627	A	G	G	G	CA173_RS05285	Tyr→Cys	Glutathione peroxidase
26	1089332	C	T	T	T	CA173_RS05540	Leu→Phe	Transketolase
27	1161438	T	G	G	G	CA173_RS05870	Tyr→Ser	Hypothetical protein
28	1215806	T	C	C	C	CA173_RS06195	Phe→Leu	Propanediol utilization protein
29	1232411	A	C	C	C	cobA	Gln→Pro	Uroporphyrinogen-III C-methyltransferase
30	1281867	G	T	T	T	CA173_RS06590	Lys→Asn	Hypothetical protein
31	1290664	T	G	G	G	CA173_RS06645	Ile→Arg	Hypothetical protein
32	1293391	A	G	G	G	CA173_RS06670	Lys→Glu	Terminase
33	1294091	A	C	C	C	CA173_RS06670	Asp→Ala	Terminase
34	1309498	A	G	G	G	CA173_RS06735	Met→Val	Hypothetical protein
35	1373205	T	G	G	G	hflX	Phe→Val	GTPase HflX
36	1386004	T	C	C	C	CA173_RS07145	Asp→Gly	Phosphoadenosine phosphosulfate sulfurtransferase
37	1462311	G	T	T	T	CA173_RS07545	Arg→Leu	CD4+ T cell-stimulating antigen
38	1552496	G	A	A	A	CA173_RS07965	Arg→Cys	50S ribosomal protein L11 methyltransferase
39	1663648	T	C	C	C	CA173_RS08495	Asn→Asp	DNA polymerase III subunit alpha
40	1708552	T	C	C	C	CA173_RS08720	Arg→Gly	MFS transporter
41	1757081	C	T	T	T	CA173_RS08935	Arg→Cys	Hypothetical protein
42	1912429	A	G	G	G	CA173_RS09750	Asn→Ser	MBL fold metallo hydrolase
43	1926635	C	T	T	T	smc	Gly→Ser	Chromosome segregation protein SMC
44	1938744	G	C	C	C	CA173_RS09870	Pro→Ala	Hypothetical protein
45	2075247	T	C	C	C	lysA	Met→Val	Diaminopimelate decarboxylase
46	2093204	T	C	C	C	CA173_RS10660	Lys→Arg	PTS ascorbate transporter subunit IIC
47	2163002	G	A	A	A	ftsA	Ala→Val	Cell division protein FtsA
48	2174875	T	C	C	C	CA173_RS11025	Arg→Gly	MFS transporter
49	2248967	C	G	G	G	CA173_RS11420	Gly→Ala	Hypothetical protein
50	2271402	A	C	C	C	CA173_RS11540	Leu→Arg	XRE family transcriptional regulator
51	2274442	A	G	G	G	CA173_RS11555	Ser→Gly	N-acetyltransferase
52	2352014	G	A	A	A	CA173_RS11920	Thr→Ile	ABC transporter permease
53	2412573	T	G	G	G	CA173_RS12245	Asn→Thr	Hypothetical protein
54	2518160	G	A	A	A	CA173_RS12925	Glu→Lys	Cell surface protein
55	2542413	G	A	A	A	CA173_RS13075	Pro→Leu	Hypothetical protein
56	2797024	G	A	A	A	CA173_RS14410	Val→Ile	Transposase
57	2871761	C	T	T	T	CA173_RS14780	Gly→Gl	HlyC/CorC family transporter
58	2951629	T	C	C	C	CA173_RS15170	His→Arg	tRNA uridine-5-carboxymethylaminomethyl(34) synthesis enzyme MnmG
59	2977146	G	A	A	A	CA173_RS15285	Ser→Phe	Glycosyl hydrolase family 65
60	2981290	A	C	C	C	CA173_RS15300	Phe→Leu	Alcohol dehydrogenase
Indels
1	1080196	T	/	/	/	CA173_RS05495	Frameshift deletion	Hypothetical protein

Refers to genomic nucleotide positions and gene products from the mapping reference sequence of the novel ST477 isolate.

MBL, metallo β-lactamase; MFS, major facilitator superfamily; SMC, structural maintenance of chromosomes; SNP, single-nucleotide polymorphism.

Discussion

CC9 isolates are frequently isolated in various kinds of foods (Yan et al., 2010; Wu et al., 2016). This is one of the clones with the highest geographical transition rates (Chenal-Francisque et al., 2011). However, its genetic characteristics and pathogenesis are poorly understood. Whole-genome sequence of ST477 isolate has never been reported as of now. The phylogenetic analysis shows that strains from one country did not cluster together phylogenetically, which is consistent with the previous observation that L. monocytogenes clones could have broad geographic distributions (Chenal-Francisque et al., 2011). Furthermore, ST477 isolate has a close evolutionary relationship with three other ST9 isolates from Canada.

The virulence potential of clones is determined, at least in part, by vertical transmission (Maury et al., 2016). Thus, the distribution and variation of known virulence determinants can not only suggest important differences among strains regarding virulence potential but also indicate the evolutionary patterns among L. monocytogenes lineages and subgroups. Notably, all CC9 strains consistently carried vip and nine inl genes, whereas these genes were scattered among non-CC9 strains. The Vip protein is required for entry into some mammalian cells (Martins et al., 2012). Internalins play essential roles in host cell invasion and virulent phenotypes in animals (Balandyte et al., 2011). Of which, internalin G and H are more important for the survival of L. monocytogenes. Gene inlK was previously described to play an important role in escape of autophagic recognition (Dortet et al., 2012). Together, these results provide preliminary evidence that CC9 isolates have at least a theoretical pathogenic potential and could pose a significant threat to the public health.

Notably, we identified the exclusive presence of Tn554-like transposon in the CC9 isolates, which has been reported in a previous study (Kuenne et al., 2013) but has not been characterized in detail. To our knowledge, only a few Tn554-related transposons have been identified to date: Tn6188 is the only transposon identified in L. monocytogenes that confers tolerance to disinfectants. Tn558, Tn559, and Tn5406, found in various Firmicutes isolates, confer resistance to antibiotics (Müller et al., 2013). Tn554 and related transposons are neither flanked by direct or inverted repeats, nor generate a duplication of the target sequence at their integration site (Murphy and Löfdahl, 1984). Whole-genome sequencing demonstrated that the Tn554-like transposon contains five arsenic-resistance genes, which are present in the resistant phenotype. However, subsequent functional studies of this transposon are necessary. In addition, the implication from this study, in which the emergence of this transposon may be linked to the CC9 background, deserves further investigation.

SSI-1 is a five-gene cluster known to enhance the adaptation of L. monocytogenes to food environments based on deletion mutant studies (Ryan et al., 2010). However, no significant differences in the stress tolerances of naturally occurring L. monocytogenes isolates with or without SSI-1 were found (Hingston et al., 2017). The former mechanism appears less probable given that CC9 isolates are prevalent in foods but lack the SSI-1 cluster.

The distribution of different spacers indicates that the seven CC9 strains compared in this work were infected by the listeriaphages B054 and P70. Self-defense relies on the Cas enzymatic machinery (Barrangou et al., 2007), the absence of cas genes would likely render the entire CRISPR system nonfunctional. Thus, CC9 isolates appear more likely to acquire foreign genetic elements, including antibiotic resistance genes, and pose a greater risk for public health compared with other isolates containing the cas genes.

Whole-genome comparison indicated that the ST477 isolates and the three Canadian isolates show high genomic similarity and may share a common gene pool. Further inspection of SNPs showed that most of nonsynonymous mutations presented in metabolism genes. A previous study showed that smc played a central role in chromosome condensation and segregation to affect the bacterial growth (Wang et al., 2014). Another study indicated that ftsA encoded cell division protein essential for the survival of the bacteria (Furusato et al., 2018). Moreover, essC and other genes encoding cell surface proteins played important roles in the capacity of invasion to the host cell (Martins et al., 2012; Pinheiro et al., 2017). The common ancestor of ST477 and ST9, during the course of its evolution, has continuously evolved their metabolism and physical characteristics to adapt to different environments. This may also explain why ST9 isolates are more prevalent in food products.

Conclusions

The present study preliminarily suggests that the wide distribution of CC9 clone in various kinds of foods is likely due to the presence of MGEs and functional virulence and resistance genes. Our ST477 isolate has close evolutionary relationship with ST9 isolates. Genomic differences between ST477 and ST9 include changes in the genes for origin segregation and cell surface protein, suggesting these genes may contribute to the adaption/colonization of L. monocytogenes in food processing environments. Further research should be carried out to confirm the evolutionary relationships between ST477 isolates and pathogenicity of CC9 strains.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 31571934), the Science and Technology Planning Project of Guangdong Province, China (2014A020214001, 2016A020219001), the Fundamental Research Funds for the Central Universities, SCUT (D2170320), and the Central Universities construct the world first-class university (discipline) and Characteristic Development Guidance Special Fund (K5174960).

Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

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