Atypical Listeria monocytogenes isolates, designated NTSN, XYSN, and 15LG, were identified as the causative agents in distinct listeriosis outbreaks across goat farms in Jiangsu Province, China, during 2011, 2012, and 2015. In one outbreak involving the lineage I serotype 4b strain NTSN, a significant mortality rate of 18 animals occurred within a flock of approximately 5000. Similarly, infections with XYSN and 15LG led to fatalities in 18% (36 out of 200) and 3% (5 out of 150) of goat populations, respectively. Notably, the 2015 outbreak saw a reduction in mortality due to prompt isolate identification and subsequent treatment with cephalexin and ampicillin.
Affected goats presented with meningitis and exhibited unusual gait and flock behavior, accompanied by severe neurological symptoms such as compulsive circling in one direction. Head tilting, uncoordinated movements, and an inability to feed or drink were also observed. In the terminal stages, goats were prostrate, with viscous saliva emanating from their mouths. Post-mortem examinations consistently revealed significant gross pathological changes in the goats’ brains, including meningeal congestion. Brain tissue cultures on CHROMagarTM plates yielded blue/green colonies with opaque halos, indicative of L. monocytogenes. Phenotypic characterization confirmed typical L. monocytogenes traits: Gram-positive rods, catalase-positive, oxidase-negative, unable to metabolize D-mannitol and D-xylose, but capable of fermenting α-methyl D-mannoside. However, atypically, these isolates failed to produce acid from L-rhamnose, a characteristic biochemical marker for L. monocytogenes. Colonies displayed hemolysis on blood agar plates and surprisingly, also tested positive in the Christie–Atkins–Munch-Petersen (CAMP) reaction using Rhodococcus equi. Standard PCR-based serogroup assays failed to type these isolates [7]. Agglutination studies using antisera to somatic antigens indicated membership in serogroup 4. Further analysis revealed reactivity with antisera for flagellar H-A and H-B antigens, but not H-C antigen, suggesting a novel serovar, designated 4h (Supplementary Table [5]). Antibiotic resistance profiling showed sensitivity to most antibiotics, with intermediate resistance to clindamycin (Supplementary Table [6]). A single rhamnose-negative environmental isolate (16E), sharing these characteristics, was recovered in 2016 from a goat-rearing farm. For comprehensive genomic analysis, three representative isolates—XYSN, 15LG, and 16E—were selected from the outbreaks and the environment. Experimental investigations were conducted primarily with XYSN, representing the earliest outbreak in 2011.
Hypervirulence Characterization of L. monocytogenes Isolates XYSN, 15LG, and 16E
To assess the virulence of isolates XYSN, 15LG, and 16E, their invasive capabilities were examined using the human intestinal epithelial Caco-2 BBe cell line. These were compared to known hypervirulent reference strains—LM11-00412 (Sv4b; Lineage I; CC1), LM13-00344 (Sv4b; Lineage I; CC4), LM6-01023 (Sv4b; Lineage I; CC6)—along with well-characterized strains EGD-e (Sv1/2a; Lineage II; CC9), NTSN (Sv4b; Lineage I; CC1), and L. ivanovii ZJU (Sv5). Invasion rates for XYSN, 15LG, and 16E were comparable and significantly higher than all other strains except L. ivanovii ZJU (Fig. 1a), indicating that enhanced invasiveness in human intestinal cells is a key feature of these isolates.
Fig. 1
Figure 1: Comparative analysis of virulence properties across Listeria monocytogenes lineage I and II strains alongside XYSN, 15LG, and 16E. (a) Demonstrates the invasion capacities of XYSN, 15LG, 16E, EGD-e, NTSN, LM11-00412, LM13-00344, LM6-01023, and L. ivanovii ZJU using the Caco-2 BBe cell line. (b) Shows survival curves of C57BL/6 mice after orogastric inoculation with various Listeria strains. (c) Illustrates body weight changes in mice post-infection. (d) Compares organ colonization propensities of different Listeria strains. (e) Presents survival curves for guinea pigs inoculated with XYSN and EGD-e.
Further virulence assessment was conducted using a mouse model of listeriosis. C57BL/6 mice, orogastrically inoculated with XYSN, 15LG, and 16E at 2 × 108 CFU, progressively succumbed to infection between days 2 and 5 (Fig. 1b). In contrast, no deaths were observed in mice inoculated with other Lm isolates or L. ivanovii ZJU at the same dose (Fig. 1b).
Organ colonization capabilities of XYSN, L. ivanovii ZJU, EGD-e, and reference CC1, CC4, and CC6 strains were then compared. All strains were administered via orogastric inoculation at 2 × 108 CFU. Two days post-infection, XYSN-infected mice exhibited significantly higher bacterial loads in the ileum, mesenteric lymph nodes (MLN), spleen, and liver compared to mice infected with EGD-e and the three hypervirulent CC1, CC4, and CC6 clone representatives [9]. L. ivanovii ZJU-infected mice showed significantly lower bacterial counts in individual MLN (1/7) and livers (2/7) than all other strains tested (Fig. 1d). Moreover, XYSN-infected mice experienced significantly greater body weight loss than mice in the other five groups (Fig. 1c). The colonization ability of XYSN across all four organs on day 5 post-infection also surpassed that of all other strains, even at a 100-fold lower infection dose (2 × 106 vs 2 × 108 CFU), as higher doses of XYSN proved lethal (Supplementary Fig. 1).
In a guinea pig orogastric infection model, the virulence of XYSN was further compared to strain EGD-e. All guinea pigs infected with 1 × 109 CFU of XYSN succumbed by day 7, whereas no fatalities occurred with the EGD-e strain (Fig. 1e). These collective findings underscore the hypervirulent nature of the L. monocytogenes isolates XYSN, 15LG, and 16E.
Discovery of the Hybrid Sub-Lineage HSL-II
Whole-genome sequencing (WGS) of XYSN, 15LG, and 16E was performed to elucidate the genetic basis of their hypervirulence and to understand the genetic underpinnings of their atypical phenotypes—rhamnose-negativity, sero-untypability, and CAMP reactivity.
The genome sizes of XYSN, 15LG, and 16E were approximately 2.99 Mb, with a high average nucleotide identity (ANI) of >99.5% among them. ANIb comparisons to the type strain NCTC 10357 yielded 97.01%, and in silico DNA–DNA hybridization (isDDH) values were around 76.20% [73.2–78.9%], confirming their classification as L. monocytogenes. Multilocus sequence typing (MLST) revealed that these isolates belong to a previously unidentified sequence type (ST) and clonal complex (CC). The Listeria MLST Pasteur database assigned them as ST626 and CC33, uniquely representing these categories within the database. This suggests the isolates are part of a novel Hybrid Subgroup.
The phylogenomic relationship of XYSN, 15LG, and 16E was compared to 144 closed genomes representing all four known Listeria monocytogenes lineages. Phylogenomic analysis, based on a concatenated set of 1977 core genes (1.69 Mb), grouped these 144 strains into the established four lineages, consistent with prior reports [8, 12, 13, 14]. XYSN, 15LG, and 16E formed a distinct, highly supported clade, separate from the four known lineages (Fig. 2). Based on these findings, these isolates are designated as members of a hybrid sub-lineage II (HSL-II). This discovery highlights the evolutionary diversity within Listeria monocytogenes and identifies a novel hybrid subgroup with unique characteristics.
Fig. 2
Figure 2: Phylogenomic tree illustrating the comparative analysis of L. monocytogenes strains. This tree, based on core gene analysis, positions XYSN, 15LG, and 16E within the broader L. monocytogenes phylogeny, highlighting their distinct hybrid sub-lineage II classification.
Genomic Features of Hybrid Sub-Lineage II Isolates
HSL-II genomes contain the Listeria Pathogenicity Island (LIPI)-1, which includes the hly gene essential for producing the hemolytic toxin listeriolysin LLO, a defining characteristic of Listeria monocytogenes. However, sub-lineage-specific pathogenicity islands LIPI-3 and LIPI-4, encoding bacteriocin LLS and a putative cellobiose-family phosphotransferase system, respectively, are absent in HSL-II genomes. LIPI-2, which harbors the sphingomyelinase smcL and internalins i-inlF and i-inlE and is typically exclusive to L. ivanovii, was found in a truncated form in HSL-II isolates (Supplementary Fig. 2). Other virulence-associated genomic traits common in Listeria monocytogenes, such as internalins (inlA, inlB, inlC, inlJ, and inlP), the invasion-associated protein (iap), and regulators (virRS), are also present in these hybrid sub-lineage isolates (Fig. 3; Supplementary Table 7).
Fig. 3
Figure 3: Circular genome map of L. monocytogenes HSL-II isolate XYSN. This genomic visualization details the locations of key features, including virulence genes and regions of difference, providing insights into the hybrid sub-lineage’s genetic architecture.
Compared to XYSN, isolates 15LG and 16E showed minor core genome variations of 4165 and 42 single nucleotide variants (SNVs), respectively, and contained 98 and 97 additional genes. Many of these extra genes are linked to metabolic functions, such as utilizing glucosides (arbutin- and salicin-phosphotransferase systems), amino acid metabolism (arginine and proline), and an ESAT-6-like secretion system (ESS) (Supplementary Table 8). A 39,213 bp prophage was absent in 15LG. These genomic similarities indicate a close relationship between 15LG, 16E, and XYSN, suggesting a recent common ancestry for this hybrid sub-lineage.
Evolutionary Path of XYSN Revealed by Comparative Genomics
Given the close phylogenomic relationship between XYSN and EGD-e, a detailed comparative genomic analysis was conducted. The core chromosomal content differed by 54,371 SNPs and included 78 regions of difference (RODs) (Fig. 3). The dN/dS ratio of coding-region-SNPs was 0.37, and the Ts/Tv (transition to transversion) ratio was 2.06, indicating long-term negative or purifying selection during evolution. Of the 78 RODs, 31 contained functional genes, while 47 were associated with transposases. Homology searches of the 31 ROD genes revealed that eight of the nine regions not previously seen in L. monocytogenes were L. ivanovii-specific (Supplementary Table 9). These included the truncated LIPI-2, a ugp operon for sn-glycerol-3-phosphate uptake, menaquinone biosynthesis genes, and a cobalt transporter operon. Two regions—one encoding a putative type seven secretion effector protein system (T7SS) followed by a polymorphic toxin with a LxG domain, and another encoding a toxin-immunity protein module tRNA-nuclease WapA—were exclusively present in XYSN. The remaining 22 RODs contained genes found in other Listeria monocytogenes lineages and other Listeria species, including a cell-wall teichoic acid modification gene cluster (XYSN-WTA), peptidoglycan-binding proteins, a putative lipase and oxidoreductase, ABC transporters, Type II CRISPR-Cas systems, leucine-rich cell surface proteins, a type I restriction modification system, a Fic-domain protein, and several hypothetical proteins (Fig. 3).
An exceptional feature of the XYSN genome was the high number of transposase genes (n=90), with 28 classified as IS5/IS1182-family, 16 as IS3-family, and 15 as IS256-family. Other transposases contained a helix-turn-helix (HTH) motif for DNA binding (Supplementary Table 10). Only two IS3-related transposases, ISLmo1 insertion elements, were previously reported in the L. monocytogenes chromosome [15]. The majority of these transposases were located near RODs, suggesting their role in genome plasticity within this hybrid sub-lineage.
Wall Teichoic Acid Modification and Bacterial Invasion
The enhanced invasive potential of XYSN in intestinal epithelial cells is linked to its hypervirulence. To identify genes contributing to invasion, genome-wide transposon mutagenesis was used to screen for mutants with reduced Caco-2 BBe cell invasion (Supplementary Fig. 3). Three mutants consistently showed a 20-fold decrease in invasion capacity. Transposon insertion sites in these mutants were mapped to loci LMxysn_0462, LMxysn_1095, and LMxysn_1098. LMxysn_0462 encodes InlA, a key factor for Listeria monocytogenes internalization into Caco-2 BBe cells [16].
The other two mutants had insertions in closely located CDS, LMxysn_1095 and LMxysn_1098. Bioinformatic analysis indicated these genes are part of an operon involved in wall teichoic acid (WTA) glycosylation, a major somatic antigen in Gram-positive bacteria. As both mutants showed similar invasion defects, further study focused on LMxysn_1095, predicted to encode an enzyme transferring an unidentified sugar to WTA.
An isogenic deletion mutant, LMxysn_∆l095, was created and tested for Caco-2 BBe cell adhesion and invasion. While adhesion levels were similar to the parental strain (Fig. 4a; Supplementary Fig. 4, Supplementary Videos 1-3), internalization of LMxysn_∆l095 was significantly reduced (Fig. 4a, b; Supplementary Videos 4-6). Complementation restored the invasive phenotype. In vivo virulence contribution of LMxysn_1095 was assessed using a mouse oral infection model. The mutant’s ability to colonize deeper tissues and organs was severely impaired at 24 hours post-infection (h p.i.), and by 72 h p.i., no mutant bacteria were recovered from the spleen or liver (Fig. 4c, d). In contrast, parental XYSN colonized these organs at high levels even at 24 h p.i.. Complementation restored both entry and virulence to parental XYSN levels. These results confirm LMxysn_1095’s critical role in intestinal barrier translocation and organ colonization, highlighting the importance of WTA modification in the hypervirulence of this hybrid sub-lineage.
Fig. 4
Figure 4: Analysis of invasive capacities of XYSN and its isogenic WTA mutants. (a) Quantifies adhesion and invasion rates. (b) Presents confocal microscopy images of GFP-expressing strains XYSN, Δ1095, and Δ1095::1095 in Caco-2 BBe cells. (c, d) Compare organ colonization properties of XYSN and its isogenic WTA mutants at 24 and 72 hours post infection.
Galactose Decoration on WTA of Hybrid Sub-Lineage II Isolate XYSN
Since WTAs define Listeria monocytogenes serogroup specificity, the role of LMxysn_1095 in serogroup 4 antiserum agglutination was examined. The ∆l095 mutant did not agglutinate with this antiserum, while the complemented strain did, indicating LMxysn_1095’s role in serogroup specificity (Supplementary Fig. 6). Despite HSL-II isolates being phylogenetically closer to lineage II strains, they agglutinate with lineage I serogroup 4 antiserum. To understand this, WTA polymers were purified from XYSN, LMxysn_∆l095, and serotype 4b strain WSLC 1042 and their structures analyzed by UPLC-MS/MS. Chromatograms showed XYSN produces a type II WTA similar to serogroup 4 strains. While serovar 4b WTA glycosylation involves both glucose and galactose on GlcNAc, XYSN WTA only had galactose (Fig. 5a). Deletion of LMxysn_1095 abolished galactose decoration on WTA, confirming it encodes a galactosyltransferase. This unique galactose modification on the WTA is a defining characteristic of this hybrid sub-lineage.
Fig. 5
Figure 5: Structural analysis of wall teichoic acid in L. monocytogenes HSL-II strain XYSN. (a) Compares the structure and UPLC-MS/MS analysis of type II WTA from L. monocytogenes WSLC 1042 serotype 4b strain with XYSN and its Δ1095 mutant. (b) Confocal images show immunofluorescence staining of XYSN and mutants with Listeria O-antiserum 4. (c) Transmission electron microscopy images reveal cell wall structures. (d) Quantifies bacterial viability after treatment with antimicrobial peptides.
Confocal microscopy of L. monocytogenes stained with fluorescein-labeled Listeria O-antiserum 4 showed that galactose decoration on WTA is associated with O-antigen integrity (Fig. 5b). TEM revealed cell wall ultrastructural changes in the ∆l095 mutant (Fig. 5c). The mutant was also more sensitive to antimicrobial peptides (AMPs) LL-37 and CRAMP, while complementation restored AMP resistance (Fig. 5d). Thus, the unique WTA structure, particularly the galactose glycosylation, is crucial for the hybrid sub-lineage XYSN’s AMP resistance.
Galactose Glycosylation of WTAs Promotes Surface Association of Virulence Factors
WTA modification can affect surface protein localization [17]. The influence of galactose glycosylation on the location of virulence factors was examined. Deletion of LMxysn_1095 caused redistribution of GW-motif proteins Ami and ActA from the cell surface to the secreted fraction (Fig. 6a), leading to loss of actin tail formation and defects in intracellular motility during Caco-2 BBe cell infection (Fig. 6b). Complementation restored ActA surface association to parental levels. The amount of InlA, an LPXTG-anchored cell wall protein, was also affected by LMxysn_1095 deletion (Fig. 6a). These data indicate that galactose decoration of WTA is essential for stable surface localization of key virulence proteins in this hybrid sub-lineage.
Fig. 6
Figure 6: Impact of WTA galactosylation on the surface association of Ami, ActA, and InlA in Listeria monocytogenes. (a) Shows protein extracts from surface-associated and secreted fractions of XYSN and mutants. (b) Confocal images demonstrate ActA localization and actin tail formation in Caco-2 BBe cells infected with XYSN and mutants.
smcL Contribution to Bi-zonal Hemolysis in the Hybrid Sub-Lineage
The genes for listeriolysin (hly) and sphingomyelinase (smcL), both cytolytic proteins, are located on LIPI-1 and LIPI-2 in XYSN. Since smcL‘s role in Listeria monocytogenes is understudied, the contributions of hly and smcL to hemolysis and CAMP phenotype were examined. XYSN and its ∆hly mutant produced the characteristic shovel-shaped cooperative lytic reaction with R. equi (Fig. 7a). However, ∆smcL deletion abolished this CAMP reaction, and a double mutant, XYSNΔsmcLΔhly, was nonhemolytic and CAMP-negative. Hemolytic titers of XYSN, EGD-e, and NTSN were similar (Supplementary Fig. 6). Recombinant strains EGD-e∷smcL and NTSN∷smcL, expressing smcL, exhibited shovel-shaped CAMP-like reactions with R. equi. Thus, smcL in Listeria monocytogenes XYSN contributes to the strong bi-zonal hemolysis, previously seen only in L. ivanovii. This highlights a unique characteristic of this hybrid sub-lineage, borrowing a trait typically associated with a different Listeria species.
Fig. 7
Figure 7: Role of smcL in CAMP assay, invasion, and virulence. (a) Shows the CAMP reaction on blood agar for XYSN, EGD-e, NTSN, and their smcL and hly mutants. (b) Quantifies invasion rates of XYSN and its mutants in Caco-2 BBe cells. (c) Compares invasion rates of NTSN and EGD-e with smcL-expressing variants. (d, e) Present mice infection assays with XYSN and EGD-e mutants. (f) Compares translocation of NTSN and EGD-e recombinant strains harboring smcL.
Virulence Roles of Listeriolysin and Sphingomyelinase in the Hybrid Sub-Lineage
Given XYSN’s strong invasive phenotype, the roles of hly and smcL in invasion were assessed using Caco-2 BBe cells. Loss of hly reduced XYSN invasion sevenfold, indicating listeriolysin’s role in bacterial entry. The ∆smcL mutant showed a ~1.6-fold invasion reduction (Fig. 7b). Introducing smcL into EGD-e and NTSN increased epithelial cell invasion at least twofold (Fig. 7c). Thus, both hly and smcL contribute to XYSN entry into human intestinal epithelial cells.
In a mouse infection model, the roles of hly and smcL were further examined. The ∆smcL mutant showed no significant reduction in spleen or liver bacterial load. In contrast, the ∆hly mutant was significantly impaired in colonizing these organs (Fig. 7d). The double mutant (∆hly∆smcL) showed even greater restriction in spleen and liver colonization (Fig. 7d).
The contribution of smcL to early bacterial translocation post-oral infection was also assessed using recombinant strains. Introducing smcL into serotype 1/2a EGD-e promoted colonization, though less than in NTSN (Fig. 7e). Introducing smcL into serotype 4b NTSN significantly increased invasion, colonization, and migration to mesenteric lymph nodes at 24 h p.i. (Fig. 7f). These findings demonstrate that smcL plays a significant role in both invasion and overall virulence of XYSN and the hybrid sub-lineage it represents.
