translate the following article into plain Chinese. Species name, scientific terms should not be translated
_____________________________________________________________________________________________________________________________________________________
Genomic analysis of the multi-host pathogen Erysipelothrix rhusiopathiae reveals extensive recombination as well as the existence of three generalist clades with wide geographic distribution

    Taya Forde, Roman Biek, Ruth Zadoks, Matthew L. Workentine, Jeroen De Buck, Susan Kutz, Tanja Opriessnig, Hannah Trewby, Frank van der Meer & Karin Orsel 

BMC Genomics volume 17, Article number: 461 (2016) Cite this article

    4484 Accesses

    36 Citations

    2 Altmetric

    Metrics details

Abstract
Background

Knowledge about how bacterial populations are structured is an important prerequisite for studying their ecology and evolutionary history and facilitates inquiry into host specificity, pathogenicity, geographic dispersal and molecular epidemiology. Erysipelothrix rhusiopathiae is an opportunistic pathogen that is currently reemerging in both the swine and poultry industries globally. This bacterium sporadically causes mortalities in captive marine mammals, and has recently been implicated in large-scale wildlife die-offs. However, despite its economic relevance and broad geographic and host distribution, including zoonotic potential, the global diversity, recombination rates, and population structure of this bacterium remain poorly characterized. In this study, we conducted a broad-scale genomic comparison of E. rhusiopathiae based on a diverse collection of isolates in order to address these knowledge gaps.
Results

Eighty-three E. rhusiopathiae isolates from a range of host species and geographic origins, isolated between 1958 and 2014, were sequenced and assembled using both reference-based mapping and de novo assembly. We found that a high proportion of the core genome (58 %) had undergone recombination. Therefore, we used three independent methods robust to the presence of recombination to define the population structure of this species: a phylogenetic tree based on a set of conserved protein sequences, in silico chromosome painting, and network analysis. All three methods were broadly concordant and supported the existence of three distinct clades within the species E. rhusiopathiae. Although we found some evidence of host and geographical clustering, each clade included isolates from diverse host species and from multiple continents.
Conclusions

Using whole genome sequence data, we confirm recent suggestions that E. rhusiopathiae is a weakly clonal species that has been shaped extensively by homologous recombination. Despite frequent recombination, we can reliably identify three distinct clades that do not clearly segregate by host species or geographic origin. Our results provide an essential baseline for future molecular epidemiological, ecological and evolutionary studies of E. rhusiopathiae and facilitate comparisons to other recombinogenic, multi-host bacteria.
Background

Uncovering population structure and its determinants is essential for understanding bacterial ecology and evolution [1]. Key questions concerning host specificity and generalism [2], global patterns of gene flow [3], and the genetic basis for clinical disease manifestations [1] can be addressed by examining the relationships among strains within a species. Additionally, molecular epidemiological studies rely on having an understanding of the population structure as a framework within which to interpret the genomic diversity of the target organism [4], and the identification of host- or geography-associated lineages can be helpful in source attribution [5]. Whole genome sequencing is providing new opportunities to address questions related to population structure within a phylogenetic framework; however, this undertaking is complicated by the need to consider the potential influence of recombination [4, 6].

Erysipelothrix rhusiopathiae, a Gram positive, facultative intracellular bacterium, is an important opportunistic pathogen for both humans and animals. Zoonotic infections with E. rhusiopathiae typically manifest as erythematous skin lesions known as erysipeloid, and tend to be occupationally associated (e.g. slaughterhouse workers, butchers, fishermen, etc.) [7]. E. rhusiopathiae has been documented in a wide range of wild and domestic species, including birds, mammals, reptiles, fish and arthropods [8]. Best known as the causative agent of swine erysipelas, E. rhusiopathiae can cause significant economic losses in swine production systems due to sporadic cases of acute septicemia, subacute cutaneous lesions, or chronic arthritis, which may be punctuated by larger outbreaks [9]; it is also among the most common causes of carcass condemnation for swine in the United States [10]. In recent years, the incidence of E. rhusiopathiae infection in swine has increased significantly in the mid-western United States, Japan and China [10–12]. Erysipelas is also reemerging in European poultry productions, likely in association with changes in housing systems [13, 14]. In captive marine mammals, E. rhusiopathiae is known to cause serious and often life-threatening infections [15, 16], while recent die-offs involving hundreds of muskoxen in the Canadian Arctic Archipelago have sparked interest in the potential conservation importance of this bacterium [17]. The broad ecological and geographic distribution of E. rhusiopathiae has been attributed to its ability to infect multiple host species which may act as healthy carriers, in combination with its long environmental persistence [18]. However, despite its ubiquity, importance for multiple host species including humans, and a highly variable clinical presentation, little is known about the genetic diversity, population structure, and host specificity of E. rhusiopathiae.

Recent whole genome sequencing projects have facilitated the taxonomic classification of the genus Erysipelothrix [11, 19]. A member of the phylum Firmicutes, the class Erysipelotrichia has the single order Erysipelotrichales and family Erysipelotrichaceae, the latter comprising 10 genera [20]. Within the genus Erysipelothrix, other recognized species are E. tonsillarum, E. inopinata, E. sp. strain 1, and E. sp. strain 2 [21, 22], as well as the recently identified E. larvae sp. nov. [23]. E. rhusiopathiae and E. tonsillarum have long been recognized as distinct species based on differences in pathogenicity, phenotypic characteristics, and serotype groups [21, 24], supported by DNA-DNA hybridization studies [25]. E. inopinata appears to have diverged prior to the split between E. rhusiopathiae and E. tonsillarum based on 16S rRNA gene sequence phylogeny [22]. The relationship of E. sp. strain 1 and E. sp. strain 2 to other Erysipelothrix species has not been explored.

The intraspecific classification of E. rhusiopathiae strains is less clearly defined, with a variety of different tools used for categorizing isolates. Serotyping, which involves testing for agglutination with specific antisera recognizing different peptidoglycan antigens of the cell wall [26], is one of the approaches that has been most frequently implemented. Although it has diagnostic value, as the different Erysipelothrix species have distinct sets of serotypes [27], serotyping is an inappropriate tool for inferring evolutionary relatedness among isolates due to the frequent horizontal exchange of capsule-specific genes in many bacterial species [28]. More recently, differences in immunogenic proteins known as surface protective antigens (Spa) have also been used to distinguish between strains of E. rhusiopathiae [13, 29]. The genetic relationship among E. rhusiopathiae isolates has also been examined using various comparative genotyping methods such as pulsed-field gel electrophoresis (PFGE) [30]. However, sequence-based typing methods, which allow for easier inter-laboratory comparison and investigation of functional differences, have only very recently been applied to E. rhusiopathiae: a comparison was made among the three E. rhusiopathiae whole genome sequences (two complete and one draft) available on GenBank at the time of writing [11], and the population structure of this bacterium was examined using multi-locus sequence typing (MLST), focusing primarily on European poultry isolates [13]. Little is known about the importance of recombination in E. rhusiopathiae, although the latter study found the species to be weakly clonal [13], suggesting recombination may contribute significantly to genetic variability in this species. Until now there have been no large-scale genomic comparison studies to fully investigate the genetic diversity of E. rhusiopathiae.

The objectives of this study were to describe the global genomic diversity of E. rhusiopathiae and to examine its population structure while accounting for the presence of recombination, in order to provide an essential baseline for future studies into the epidemiology and ecology of this multi-host pathogen. A secondary objective was to examine the phylogenetic relationship among different Erysipelothrix species based on whole genome sequence data.
Results
Sequencing and pan-genome statistics

Eighty-three newly sequenced E. rhusiopathiae isolates were included in this analysis, with representation from North America (Canada and US), South America (Argentina), Europe (Belgium, Hungary and the UK), Asia (Japan), South Africa, and Australia, with the majority originating from Canada (n = 37), US (n = 20) and Belgium (n = 17) (see Additional file 1: Table S1 for a table of the isolates included in this study). Dates of isolation ranged from 1958 to 2014. Host species of origin encompassed swine (n = 18), poultry (n = 14), captive marine mammals (dolphins and beluga whales; n = 8), fishes (n = 5), wild birds (primarily water fowl; n = 7), wild ungulates (muskoxen, moose and caribou; n = 28), and a wolf. Background information regarding the clinical manifestations associated with the isolates was only available for 12 isolates (Additional file 1: Table S1), but cases of acute and subacute septicemia, as well as E. rhusiopathiae isolated from skin lesions were included. Serotype was known for 16 isolates previously described in the literature (Additional file 1: Table S1); the newly serotyped isolate VI11-2_lu was found to belong to serotype 5.

A total of 1137 core genes were present among all the E. rhusiopathiae isolates included in this analysis, representing 67 % of the coding sequences present in the Fujisawa reference genome (Fig. 1a and Additional file 2: Figure S1). A total of 512 singleton genes were identified (i.e. present in only one isolate), with an average of 6.6 new unique genes discovered per additional genome sequenced (Fig. 1b). Among the core genes were the ComEC (membrane pore) and dprA (recombination mediator) genes, as well as putative Type II/IV secretion system proteins (Additional file 2: Figure S1), the presence of which is highly suggestive of natural competency [31]. Based on the pairwise comparisons made in the program LS-BSR, no major differences in overall gene content were detected among host species or geographic origin, or among clades that were identified during analyses described below.
Fig. 1
figure 1

Erysipelothrix rhusiopathiae pan-genome. Plots were generated in LS-BSR and visualized using PanGP. a Convergence of the core genome with concurrent accumulation of coding sequences in the pan-genome in relation to the number of genomes analyzed. b Number of unique coding sequences for each additional genome analyzed. Publicly available E. rhusiopathiae isolates and de novo assembled isolates whose average depth of coverage was greater than 15X were included in this analysis
Full size image
A high proportion of the E. rhusiopathiae core genome has experienced recombination

The core genome alignment generated using Parsnp was 1,049,431 base pairs, representing 58 % of the reference genome. Overall there was a high degree of sequence similarity, with 99.3 % pairwise identity. The BratNextGen analysis, which included all isolates, inferred that 58 % of the overall core genome alignment had experienced recombination (Additional file 2: Figure S2). There were major differences among the clades in terms of the proportion of the alignment with inferred recombination (Table 1, Additional file 2: Figures S2 and S3). Both recombination detection methods estimated that a greater proportion of the genome of Clade 2 isolates has experienced recombination. BratNextGen did not detect any recombinogenic segments in Clade 1, while this was also the clade with the least recombination across the core genome as detected by Gubbins. Although a large proportion of the alignment was influenced by recombination, average recombination to mutation (r/m) rates were moderate, at 0.96, 2.18 and 0.55 in Clades 1, 2 and 3 respectively.
Table 1 Recombination detected in the core genome of Erysipelothrix rhusiopathiae
Full size table
Phage and plasmid-related sequences were detected in several isolates