Veterinary Microbiology 185 (2016) 34–40
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Comparative genomics of toxigenic and non-toxigenic Staphylococcus hyicus Pimlapas Leekitcharoenphona,* , Sünje Johanna Pampa , Lars Ole Andresenb , Frank M. Aarestrupa a b
Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark Section for Diagnostics and Scientific Advice, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2015 Received in revised form 21 January 2016 Accepted 23 January 2016
The most common causative agent of exudative epidermitis (EE) in pigs is Staphylococcus hyicus. S. hyicus can be grouped into toxigenic and non-toxigenic strains based on their ability to cause EE in pigs and specific virulence genes have been identified. A genome wide comparison between non-toxigenic and toxigenic strains has never been performed. In this study, we sequenced eleven toxigenic and six nontoxigenic S. hyicus strains and performed comparative genomic and phylogenetic analysis. Our analyses revealed two genomic regions encoding genes that were predominantly found in toxigenic strains and are predicted to encode for virulence determinants for EE. All toxigenic strains encoded for one of the exfoliative toxins ExhA, ExhB, ExhC, or ExhD. In addition, one of these regions encoded for an ADPribosyltransferase (EDIN, epidermal cell differentiation inhibitor) and a novel putative RNase toxin (polymorphic toxin) and was associated with the gene encoding ExhA. A clear differentiation between toxigenic and non-toxigenic strains based on genomic and phylogenetic analyses was not apparent. The results of this study support the observation that exfoliative toxins of S. hyicus and S. aureus are located on genetic elements such as pathogenicity islands, phages, prophages and plasmids. ã 2016 Elsevier B.V. All rights reserved.
Keywords: S. hyicus Comparative genomics Genome Toxigenic S. hyicus
1. Introduction Staphylococcus hyicus is a gram-positive bacterium that can cause exudative epidermitis (EE) in pigs, and primarily piglets (Foster, 2012). EE is characterized by exfoliation of the skin and the formation of a thick, greasy, brown exudate (Jones, 1956) that may lead to dehydration and subsequent animal death (L’Ecuyer and Jericho, 1966). S. hyicus has been divided into pathogenic and nonpathogenic strains according to their ability to induce EE in pigs (Tanabe et al., 1996; Wegener et al., 1993) and their ability to produce exfoliative toxin which is the main virulence factor necessary to induce the disease (Andresen and Ahrens, 2004; Sato et al., 2000). Five exfoliative toxins from S. hyicus have been described so far, of which one has been characterized in Japan as SHETB (Sato et al., 2000) and four in Denmark as ExhA, ExhB, ExhC, ExhD (Andresen and Ahrens, 2004). The Exh toxins have been shown to cause loss of cell adhesion in the epidermis of porcine skin by cleaving desmoglein-1, while, human desmoglein-1 is
* Corresponding author at: National Food Institute, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark. Fax: +45 35 88 60 01. E-mail address: [email protected] (P. Leekitcharoenphon). http://dx.doi.org/10.1016/j.vetmic.2016.01.018 0378-1135/ ã 2016 Elsevier B.V. All rights reserved.
resistant to S. hyicus exfoliative toxins (Fudaba et al., 2005; Nishifuji et al., 2005). A genomic analysis and comparison between toxigenic and non-toxigenic strains potentially identifying other virulence markers has so far not been performed, and is the subject of the present study. A S. hyicus reference strain was recently completely sequenced (Calcutt et al., 2015). The present study was conducted to study the phylogeny of 17 different S. hyicus strains, and compare the genomes of toxigenic and non-toxigenic strains, and to identify potential additional virulence determinants. 2. Materials and methods 2.1. Bacterial isolates The eleven toxigenic and six non-toxigenic S. hyicus strains investigated in this study have been previously characterized by their ability to cause generalized EE after inoculation in healthy 14day-old piglets (Wegener et al., 1993) or their ability to produce exfoliative toxin (Andresen and Ahrens, 2004). The strains were from Denmark (n = 9), Germany (n = 5) and UK (n = 3).
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2.2. Whole genome sequencing Genomic DNA was extracted from the 17 S. hyicus strains using an Invitrogen Easy-DNATM Kit (Invitrogen, Carlsbad, CA, USA) and DNA concentrations were determined using the Qubit dsDNA BR
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assay kit (Invitrogen). The genomic DNA was prepared for Illumina pair-end sequencing using the Illumina (Illumina, Inc., San Diego, CA) NexteraXT1 Guide 150319425031942 following the protocol revision C (http://support.illumina.com/downloads/nextera_xt_sample_preparation_guide_15031942.html). A sample of the
Fig. 1. Pan-genome tree (A) and SNP tree (B) of non-toxigenic (green) and toxigenic (red) S. hyicus strains. The numbers shown in branches are bootstrap values.
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pooled NexteraXT Libraries was loaded onto Illumina MiSeq reagent cartridge using MiSeq Reagent Kit v2 and 500 cycles with a Standard Flow Cell. The libraries were sequenced using an Illumina platform and MiSeq Control Software 2.3.0.3. All the isolates were pair-end sequenced. Raw reads have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under study accession no.: ERP013882. The raw reads were de novo assembled using the assemble pipeline (version 1.0) available from the Center for Genomic Epidemiology (CGE) https://cge.cbs.dtu.dk/ services/Assembler/. The pipeline was based on the Velvet algorithms for de novo short reads assembly (Zerbino and Birney, 2008). Genomic information can be retrieved from Table S1. 2.3. SNP tree SNP tree was constructed from SNPs (Single Nucleotide Polymorphisms) that were identified using the pipeline CSI phylogeny (Kaas et al., 2014; Leekitcharoenphon et al., 2012a) available from the Center for Genomic Epidemiology (www. genomicepidemiology.org). Fundamentally, the paired-end reads were mapped to a reference genome using Burrows–Wheeler Aligner (BWA) version 0.7.2 (Li and Durbin, 2010). The reference genome was a recently sequenced S. hyicus strain ATCC 11249 (accession number NZ_CP008747, length 2,472,129 bp) (Calcutt et al., 2015). SNPs were determined using ‘mpileup’ module in SAMTools version 0.1.18 (Li et al., 2009). Qualified SNPs were chosen when meeting the following criteria: (1) a minimum distance of 15 bps between each SNP, (2) a minimum of 10% of the average depth, (3) the mapping quality was above 30, (4) the SNP
quality was higher than 20 and (5) all indels were excluded. The qualified SNPs from each S. hyicus genome were concatenated to a single alignment corresponding to the positions of the reference genome. The concatenated sequences were subjected to parsimony tree construction using PhyML (Guindon et al., 2010) with HKY85 substitution model and 100 bootstrap replicates. 2.4. Gene prediction and annotation Open reading frames (ORFs) were predicted on the genomes of S. hyicus using Prodigal software (Hyatt et al., 2010) and translated into proteins. Proteins were annotated by comparing them to the sequences of the complete microbial genomes deposited at NCBI using BLAST. In addition, functional protein domains were assigned by comparing the proteins against Pfam version 28.0. Secretory proteins were predicted by SignalP (Petersen et al., 2011), and transmembrane domains were predicted by TMHMM (Krogh et al., 2001). 2.5. Pan-genome tree A Pan-genome tree (Leekitcharoenphon et al., 2012b; Snipen and Ussery, 2010) was reconstructed based on a presence and absence matrix of all gene families of S. hyicus (protein sequences) across S. hyicus genomes. The absence and presence of proteins across genomes are represented by 0’s and 1’s respectively in a matrix. The genomes were clustered using hierarchical clustering of the relative Manhattan distance between genomes. The confidence of branches is shown as bootstrap values.
Fig. 2. Blast atlas of 11 toxigenic strains (red) and 6 non-toxigenic strains (green). The atlas showed the similarity in protein level of S. hyicus strains against the reference genome, S. hyicus ATCC 11249 using BLASTP. The atlas exhibited two regions (region 1 and 2) containing genes uniquely found in toxigenic strains.
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Fig. 3. heatmaps showing similarity of genes from region 1 (A) and region 2 (B) against all S. hyicus genomes. The degree of similarity has been divided into different colors. Any similarities less than 65% were displayed in white. Toxigenic strains of S. hyicus were colored in red and non-toxigenic strains were colored in green. Genes colored in orange (A) were genes found in at least four toxigenic strains, and one non-toxigenic strain (A3793/76) with high similarity (more than 95% similarity) and found in other nontoxigenic strains with low similarity (less than 85% similarity). Genes colored in orange (B) were genes found in toxigenic strains with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) to non-toxigenic strains. Genes colored in purple (B) were genes present only in some toxigenic strains. Genes without function described are hypothetical proteins.
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2.6. Genomic deletions Genomic deletions were determined and visualized by genomic comparisons from BLAST using a reference genome in a BLAST atlas. All predicted proteins from the reference genome were aligned against other S. hyicus genomes using BLASTP. The presence and absence of genes were displayed in a circle, with increasing intensity of color representing higher similarity (Hallin et al., 2008). The homology of genes from genomic deletions against genomes was displayed in a heatmap based on a BLASTN analysis with a cut off at 65% similarity. 2.7. Homology of exfoliative toxins and bacteriophage FETA Exfoliative toxin protein sequences of S. hyicus were retrieved from NCBI by the following GenBank IDs; ExhA (AF515453), ExhB (AF515454), ExhC (AF515455), ExhD (AF515456) (Ahrens and Andresen, 2004), SHETB (AB036767) (Watanabe et al., 2000). Toxins from S. aureus were PSMa1 (Wang et al., 2007), PSMb1 (Wang et al., 2007), Selx1 (Wilson et al., 2011), LukD (Baba et al., 2002; Barrio et al., 2006; Lindsay and Holden, 2006), Sed_Sej (Bayles and Iandolo, 1989; Omoe et al., 2003; Zhang et al., 1998), Ser (Bayles and Iandolo, 1989; Omoe et al., 2003), ETA (M17347), ETB (M17348), ETD (AHC54578), and ExhB from S. chromogenes (AY691553). Eta-encoding bacteriophage FETA genome from S. aureus strain E-1 was retrieved by NCBI with GenBank accession number, AP001553 (Yamaguchi et al., 2000). The homology of the toxin proteins and proteins from FETA phage against S. hyicus genomes was identified by BLASTP. 3. Results 3.1. Phylogeny of toxigenic and non-toxigenic S. hyicus The pan-genome tree based on presence and absence of genes across genomes is shown in Fig. 1A. The tree consisted of toxigenic (red) and non-toxigenic strains (green). The tree showed a cluster of five toxigenic strains, whereas other toxigenic strains were clustered with non-toxigenic strains. The tree composed three subclusters containing non-toxigenic strains but they were close to the toxigenic strains 1289E-88, 842G-88 and 842A-88. In addition, the phylogenetic tree based on SNPs (Fig. 1B) showed two sub-clusters of toxigenic strains. The larger cluster of toxigenic strains was
clustered with other non-toxigenic strains. The toxigenic S. hyicus strains tended to cluster according to geography of isolation in both the pan-genome tree and the SNP tree as the toxigenic strains from the UK clustered together, as well as certain strains from Germany and Denmark, respectively. 3.2. Comparative genomics The results from protein comparisons of the reference strain S. hyicus ATCC 11249 (Calcutt et al., 2015) with the toxigenic and nontoxigenic strains were visualized in a BLAST atlas (Fig. 2). The atlas contains eleven toxigenic strains in red color intensity and six nontoxigenic strains in green color intensity. The atlas revealed two interesting regions, region 1 (41806-72839) and region 2 (10721061087194), where some genes from toxigenic strains were present but genes from non-toxigenic strains were largely absent, or vice versa. The genes from those regions were extracted and aligned against all 17 S. hyicus genomes. The similarity of genes from region 1 and region 2 against all genomes are shown in heatmaps in Fig. 3A and B respectively. The heatmap constructed from region 1 showed 12 genes (Fig. 3A, genes colored in orange) being present in at least four out of eleven toxigenic strains, and one non-toxigenic strain (A3793/ 76) with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) in non-toxigenic strains. The predicted function of the proteins encoded by those genes are N-6 DNA methylase, type I restriction-modification system, thioesterase, transferase, AMP ligase, equibactin nonribosomal peptide synthase, polyketide synthase, salicylate synthase, DEAD/DEAH box helicase and hypothetical protein (see Fig. S1 and Table S2). There were two polyketide synthase genes in this region. They have different Pfam domains and have different sequence length. The co-location of multiple polyketide synthase have been described (Heather et al., 2008). The heatmap from region 2 showed 5 genes (Fig. 3B, genes colored in orange) found in four to ten toxigenic strains with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) in non-toxigenic strains. Several of these proteins are hypothetical proteins and one of them is phage tail protein. Furthermore, there were 10 genes (Fig. 3B, genes colored in purple) present only in three to eleven toxigenic strains. One of the 10 genes (NZ_CP008747_CDS_1079564-1078743) encodes for exfoliative toxin ExhA that is an important virulence factor in
Table 1 The presence/absence of exfoliative toxins in S. hyicus genomes. Exfoliative toxin proteins (% sequence similarity) S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus
genomes ATCC 11249 toxigenic (Denmark) NCTC10350 toxigenic (Denmark) 1289D-88 toxigenic (Denmark) 1403E-88 toxigenic (Denmark) S3588 toxigenic (Germany) 842A-88 toxigenic (Denmark) 9390-88 toxigenic (Denmark) A2869C toxigenic (Germany) P119 toxigenic (UK) P411 toxigenic (UK) 842G-88 toxigenic (Denmark) 1289E-88 toxigenic (Denmark) 842J-88 non-toxigenic (Denmark) A3793/76 non-toxigenic (Germany) A4596/76 non-toxigenic (Germany) A72/75 non-toxigenic (Germany) SK170 non-toxigenic (UK) 1403B-88 non-toxigenic (Denmark)
ExhA + [100] + [100] – – + [100] – + [100] – + [100] + [100] – – – – – – – –
ExhB – – + [100] + [100] – – – – – – – – – – – – – –
ExhC – – – – – + [100] – – – – – – – – – – – –
ExhD – – – – – – – + [100] – – + [100] + [100] – – – – – –
SHETB – – – – – – – – – – – – – – – – – –
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S. hyicus. The other genes of the 10 genes are hypothetical proteins, glutamyl endopeptidases, a predicted peptidase S8, an epidermal cell differentiation inhibitor protein (ADP-ribosyltransferase) and a putative RNase toxin (polymorphic toxin). Other genes from region 2 (Fig. 3B, genes colored in black) that were found in many toxigenic and some non-toxigenic strains include genes encoding for DNA polymerase, DNA repair protein and bacteriophagerelated proteins (see Fig. S2 and Table S2). Sequences of genes from region 1 and region 2 can be retrieved from File S1 and File S2. The genome sequence of Staphylococcus aureus phage FETA (Yamaguchi et al., 2000) was compared with the whole genome of of S. hyicus strain ATCC 11249. There were 11 genes out of 66 genes from FETA phage found in ATCC 11249. Four genes from the eleven genes were found in region 2 (Table S2). The genes from region 2 that were similar to FETA phage were hypothetical proteins, phage PVL and ExhA. The other seven genes from the phage were found in other regions away from the regions 1 and 2. Using ISfinder (Siguier et al., 2006) to search for insertion sequences, we found ISSep3 in the reference genome, S. hyicus strain ATCC 11249. The insertion site was found from position 1,570,484 to 1,571,023, which is not in the range of region 1 and region 2 or even close to the regions. In addition, we found proteins encoded by the different S. hyicus strains, both toxigenic and non-toxigenic ones, that were not detected in the reference genome S. hyicus ATCC 11249. The number of additional proteins in toxigenic strains (excluding NCTC10350) ranged from 81–203. The numbers in non-toxigenic strains ranged from 39–164. The majority of the additional proteins were phage- and plasmid-related (Table S3). The five exfoliative toxin-encoding genes, ExhA, ExhB, ExhC, ExhD, SHETB, were compared against all S. hyicus genomes. Four of the exfoliative toxins were found in S. hyicus with 100% similarity (Table 1). SHETB was absent from all S. hyicus under study with the cut off 65% similarity. Toxigenic strains encoded for either ExhA, ExhB, ExhC or ExhD. Non-toxigenic strains appear not encode for any of the exfoliative toxins. In addition, toxigenic strains were clustered according to the type of exfoliative toxin genes in heatmaps showing similarity of the genes from region 1 and 2 (Fig. 3A and B). Nine toxin genes from S. aureus and one toxin gene from S. chromogenes were also aligned against S. hyicus genomes. With the cut-off at 65 percent similarity, the exhB gene from S. chromogenes was found in two S. hyicus toxigenic strains, 1403E-88 and 1289D88. The other genes were not detected in any S. hyicus. 4. Discussion In pigs, the common cause of EE is infection with S. hyicus (Foster, 2012). Differentiation between toxigenic and non-toxigenic S. hyicus strains can readily be obtained by the use of PCR (Andresen and Ahrens, 2004; Futagawa-Saito et al., 2007) but other significant virulence factors with regard to EE have not been examined. In this study, we have sequenced and compared toxigenic and non-toxigenic strains of S. hyicus. The S. hyicus strains exhibited no relationship between phylogeny and virulence according to phylogenetic tree based on single-nucleotide polymorphisms (SNPs). The phylogenetic trees seemed, however, to indicate clustering of toxigenic S. hyicus strains according to geographic location. The protein comparison of S. hyicus genomes against the reference genome revealed two genomic regions containing genes that were predominantly present in toxigenic strains but absent or present with low similarity in non-toxigenic strains. These genes included the exfoliative toxin, exhA. Region 1 appears to encode for non-ribosomal peptide synthetases. It has similarity with equibactin (yersiniabactin) potentially involved in iron-aquisition. The
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genes in the region 2 encode for several toxins/proteins with enzymatic function for example exfoliative toxin (Trypsin-like), two additional Trypsin-like enzymes, EDIN/3C3stau-homolog (ADP-ribosyltransferase), Toxin 46 (polymorphic toxin) (Zhang et al., 2012) and Peptidase S8. The proteins all have predicted signal peptide domains (except Peptidase S8), and hence are likely to be secreted. Peptidase S8 has one TM domain and may be located in the membrane. In addition, region 2 contained four genes from S. aureus phage FETA and we did not identify any insertion sequences in the regions including sequences around the regions. The genes found in the two genomic regions might encode for potential additional virulence determinants for EE. Fig. 3B shows that the strains that carry exhA also harbour the majority of the genes encoded in region 2. Strains carrying exhB, exhD and exhC have 17, 8 and 1 gene(s), respectively, that are highly homologous to the 28 genes from region 2 shown in Fig. 3B. This would indicate that only the gene encoding ExhA of the exfoliative toxins from S. hyicus is associated with region 2. Region 2 is similar to the genomic island previously identified in the genome of S. hyicus strain ATCC 11249T (Calcutt et al., 2015) which is the same strain as NCTC 10350 in this study. These results indicate that strains that carry exhA are very similar with regard to the genes that surround exhA, and gives further support to the indication that exhA may be located on a pathogenicity island or a prophagerelated element as suggested by Calcutt et al. (Calcutt et al., 2015). The strains carrying the other types of exfoliative toxin, ExhB, ExhC and ExhD, have fewer genes that are homologous to the genes from region 2 compared to the ExhA-positive strains, suggesting that these toxin genes have a different genetic background than ExhA. The results of this study adds to the notion that different types of exfoliative toxin from S. hyicus and from S. aureus, although predicted to be part of the same protein family (Ahrens and Andresen, 2004), do have quite different genetic background for the genes of the these toxins, such as bacteriophage (Yamaguchi et al., 2000), plasmid (Yamaguchi et al., 2001) and pathogenicity island (Calcutt et al., 2015; Yamaguchi et al., 2002). In summary, we found that there are other genetic differences between toxigenic and non-toxigenic strains of S. hyicus besides well-known exfoliative toxins. Some of the genes might potentially be virulence determinants. There is however, no apparent relationship between phylogeny and virulence. Other factors are likely involved in S. hyicus colonization and virulence in pigs, such as interactions with other members of the skin microbiota as hypothesized for S. aureus (Yan et al., 2013), as well as host determinants (Daugaard et al., 2007) including immunological factors. Previous investigations in pigs indicated that colonization by a certain strain evokes macroscopic and microscopic signs of disease (EE) to different degrees in different animals (Wegener et al., 1993). However, such differences may also be due to differences in the structure and amino acid composition of the exfoliative toxins (Ahrens and Andresen, 2004). Acknowledgements This work was supported by the Center for Genomic Epidemiology at the Technical University of Denmark funded by grant 09-067103/DSF from the Danish Council for Strategic Research. Sünje J. Pamp was supported by a grant from Carlsbergfondet. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2016.01.018.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Comparative genomics of toxigenic and non-toxigenic Staphylococcus hyicus Pimlapas Leekitcharoenphona,* , Sünje Johanna Pampa , Lars Ole Andresenb , Frank M. Aarestrupa a b
Research Group for Genomic Epidemiology, National Food Institute, Technical University of Denmark, Kgs. Lyngby, Denmark Section for Diagnostics and Scientific Advice, National Veterinary Institute, Technical University of Denmark, Bülowsvej 27, 1870 Frederiksberg C, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2015 Received in revised form 21 January 2016 Accepted 23 January 2016
The most common causative agent of exudative epidermitis (EE) in pigs is Staphylococcus hyicus. S. hyicus can be grouped into toxigenic and non-toxigenic strains based on their ability to cause EE in pigs and specific virulence genes have been identified. A genome wide comparison between non-toxigenic and toxigenic strains has never been performed. In this study, we sequenced eleven toxigenic and six nontoxigenic S. hyicus strains and performed comparative genomic and phylogenetic analysis. Our analyses revealed two genomic regions encoding genes that were predominantly found in toxigenic strains and are predicted to encode for virulence determinants for EE. All toxigenic strains encoded for one of the exfoliative toxins ExhA, ExhB, ExhC, or ExhD. In addition, one of these regions encoded for an ADPribosyltransferase (EDIN, epidermal cell differentiation inhibitor) and a novel putative RNase toxin (polymorphic toxin) and was associated with the gene encoding ExhA. A clear differentiation between toxigenic and non-toxigenic strains based on genomic and phylogenetic analyses was not apparent. The results of this study support the observation that exfoliative toxins of S. hyicus and S. aureus are located on genetic elements such as pathogenicity islands, phages, prophages and plasmids. ã 2016 Elsevier B.V. All rights reserved.
Keywords: S. hyicus Comparative genomics Genome Toxigenic S. hyicus
1. Introduction Staphylococcus hyicus is a gram-positive bacterium that can cause exudative epidermitis (EE) in pigs, and primarily piglets (Foster, 2012). EE is characterized by exfoliation of the skin and the formation of a thick, greasy, brown exudate (Jones, 1956) that may lead to dehydration and subsequent animal death (L’Ecuyer and Jericho, 1966). S. hyicus has been divided into pathogenic and nonpathogenic strains according to their ability to induce EE in pigs (Tanabe et al., 1996; Wegener et al., 1993) and their ability to produce exfoliative toxin which is the main virulence factor necessary to induce the disease (Andresen and Ahrens, 2004; Sato et al., 2000). Five exfoliative toxins from S. hyicus have been described so far, of which one has been characterized in Japan as SHETB (Sato et al., 2000) and four in Denmark as ExhA, ExhB, ExhC, ExhD (Andresen and Ahrens, 2004). The Exh toxins have been shown to cause loss of cell adhesion in the epidermis of porcine skin by cleaving desmoglein-1, while, human desmoglein-1 is
* Corresponding author at: National Food Institute, Technical University of Denmark, Søltofts Plads, Building 221, DK-2800 Kgs. Lyngby, Denmark. Fax: +45 35 88 60 01. E-mail address: [email protected] (P. Leekitcharoenphon). http://dx.doi.org/10.1016/j.vetmic.2016.01.018 0378-1135/ ã 2016 Elsevier B.V. All rights reserved.
resistant to S. hyicus exfoliative toxins (Fudaba et al., 2005; Nishifuji et al., 2005). A genomic analysis and comparison between toxigenic and non-toxigenic strains potentially identifying other virulence markers has so far not been performed, and is the subject of the present study. A S. hyicus reference strain was recently completely sequenced (Calcutt et al., 2015). The present study was conducted to study the phylogeny of 17 different S. hyicus strains, and compare the genomes of toxigenic and non-toxigenic strains, and to identify potential additional virulence determinants. 2. Materials and methods 2.1. Bacterial isolates The eleven toxigenic and six non-toxigenic S. hyicus strains investigated in this study have been previously characterized by their ability to cause generalized EE after inoculation in healthy 14day-old piglets (Wegener et al., 1993) or their ability to produce exfoliative toxin (Andresen and Ahrens, 2004). The strains were from Denmark (n = 9), Germany (n = 5) and UK (n = 3).
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2.2. Whole genome sequencing Genomic DNA was extracted from the 17 S. hyicus strains using an Invitrogen Easy-DNATM Kit (Invitrogen, Carlsbad, CA, USA) and DNA concentrations were determined using the Qubit dsDNA BR
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assay kit (Invitrogen). The genomic DNA was prepared for Illumina pair-end sequencing using the Illumina (Illumina, Inc., San Diego, CA) NexteraXT1 Guide 150319425031942 following the protocol revision C (http://support.illumina.com/downloads/nextera_xt_sample_preparation_guide_15031942.html). A sample of the
Fig. 1. Pan-genome tree (A) and SNP tree (B) of non-toxigenic (green) and toxigenic (red) S. hyicus strains. The numbers shown in branches are bootstrap values.
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pooled NexteraXT Libraries was loaded onto Illumina MiSeq reagent cartridge using MiSeq Reagent Kit v2 and 500 cycles with a Standard Flow Cell. The libraries were sequenced using an Illumina platform and MiSeq Control Software 2.3.0.3. All the isolates were pair-end sequenced. Raw reads have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena) under study accession no.: ERP013882. The raw reads were de novo assembled using the assemble pipeline (version 1.0) available from the Center for Genomic Epidemiology (CGE) https://cge.cbs.dtu.dk/ services/Assembler/. The pipeline was based on the Velvet algorithms for de novo short reads assembly (Zerbino and Birney, 2008). Genomic information can be retrieved from Table S1. 2.3. SNP tree SNP tree was constructed from SNPs (Single Nucleotide Polymorphisms) that were identified using the pipeline CSI phylogeny (Kaas et al., 2014; Leekitcharoenphon et al., 2012a) available from the Center for Genomic Epidemiology (www. genomicepidemiology.org). Fundamentally, the paired-end reads were mapped to a reference genome using Burrows–Wheeler Aligner (BWA) version 0.7.2 (Li and Durbin, 2010). The reference genome was a recently sequenced S. hyicus strain ATCC 11249 (accession number NZ_CP008747, length 2,472,129 bp) (Calcutt et al., 2015). SNPs were determined using ‘mpileup’ module in SAMTools version 0.1.18 (Li et al., 2009). Qualified SNPs were chosen when meeting the following criteria: (1) a minimum distance of 15 bps between each SNP, (2) a minimum of 10% of the average depth, (3) the mapping quality was above 30, (4) the SNP
quality was higher than 20 and (5) all indels were excluded. The qualified SNPs from each S. hyicus genome were concatenated to a single alignment corresponding to the positions of the reference genome. The concatenated sequences were subjected to parsimony tree construction using PhyML (Guindon et al., 2010) with HKY85 substitution model and 100 bootstrap replicates. 2.4. Gene prediction and annotation Open reading frames (ORFs) were predicted on the genomes of S. hyicus using Prodigal software (Hyatt et al., 2010) and translated into proteins. Proteins were annotated by comparing them to the sequences of the complete microbial genomes deposited at NCBI using BLAST. In addition, functional protein domains were assigned by comparing the proteins against Pfam version 28.0. Secretory proteins were predicted by SignalP (Petersen et al., 2011), and transmembrane domains were predicted by TMHMM (Krogh et al., 2001). 2.5. Pan-genome tree A Pan-genome tree (Leekitcharoenphon et al., 2012b; Snipen and Ussery, 2010) was reconstructed based on a presence and absence matrix of all gene families of S. hyicus (protein sequences) across S. hyicus genomes. The absence and presence of proteins across genomes are represented by 0’s and 1’s respectively in a matrix. The genomes were clustered using hierarchical clustering of the relative Manhattan distance between genomes. The confidence of branches is shown as bootstrap values.
Fig. 2. Blast atlas of 11 toxigenic strains (red) and 6 non-toxigenic strains (green). The atlas showed the similarity in protein level of S. hyicus strains against the reference genome, S. hyicus ATCC 11249 using BLASTP. The atlas exhibited two regions (region 1 and 2) containing genes uniquely found in toxigenic strains.
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Fig. 3. heatmaps showing similarity of genes from region 1 (A) and region 2 (B) against all S. hyicus genomes. The degree of similarity has been divided into different colors. Any similarities less than 65% were displayed in white. Toxigenic strains of S. hyicus were colored in red and non-toxigenic strains were colored in green. Genes colored in orange (A) were genes found in at least four toxigenic strains, and one non-toxigenic strain (A3793/76) with high similarity (more than 95% similarity) and found in other nontoxigenic strains with low similarity (less than 85% similarity). Genes colored in orange (B) were genes found in toxigenic strains with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) to non-toxigenic strains. Genes colored in purple (B) were genes present only in some toxigenic strains. Genes without function described are hypothetical proteins.
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2.6. Genomic deletions Genomic deletions were determined and visualized by genomic comparisons from BLAST using a reference genome in a BLAST atlas. All predicted proteins from the reference genome were aligned against other S. hyicus genomes using BLASTP. The presence and absence of genes were displayed in a circle, with increasing intensity of color representing higher similarity (Hallin et al., 2008). The homology of genes from genomic deletions against genomes was displayed in a heatmap based on a BLASTN analysis with a cut off at 65% similarity. 2.7. Homology of exfoliative toxins and bacteriophage FETA Exfoliative toxin protein sequences of S. hyicus were retrieved from NCBI by the following GenBank IDs; ExhA (AF515453), ExhB (AF515454), ExhC (AF515455), ExhD (AF515456) (Ahrens and Andresen, 2004), SHETB (AB036767) (Watanabe et al., 2000). Toxins from S. aureus were PSMa1 (Wang et al., 2007), PSMb1 (Wang et al., 2007), Selx1 (Wilson et al., 2011), LukD (Baba et al., 2002; Barrio et al., 2006; Lindsay and Holden, 2006), Sed_Sej (Bayles and Iandolo, 1989; Omoe et al., 2003; Zhang et al., 1998), Ser (Bayles and Iandolo, 1989; Omoe et al., 2003), ETA (M17347), ETB (M17348), ETD (AHC54578), and ExhB from S. chromogenes (AY691553). Eta-encoding bacteriophage FETA genome from S. aureus strain E-1 was retrieved by NCBI with GenBank accession number, AP001553 (Yamaguchi et al., 2000). The homology of the toxin proteins and proteins from FETA phage against S. hyicus genomes was identified by BLASTP. 3. Results 3.1. Phylogeny of toxigenic and non-toxigenic S. hyicus The pan-genome tree based on presence and absence of genes across genomes is shown in Fig. 1A. The tree consisted of toxigenic (red) and non-toxigenic strains (green). The tree showed a cluster of five toxigenic strains, whereas other toxigenic strains were clustered with non-toxigenic strains. The tree composed three subclusters containing non-toxigenic strains but they were close to the toxigenic strains 1289E-88, 842G-88 and 842A-88. In addition, the phylogenetic tree based on SNPs (Fig. 1B) showed two sub-clusters of toxigenic strains. The larger cluster of toxigenic strains was
clustered with other non-toxigenic strains. The toxigenic S. hyicus strains tended to cluster according to geography of isolation in both the pan-genome tree and the SNP tree as the toxigenic strains from the UK clustered together, as well as certain strains from Germany and Denmark, respectively. 3.2. Comparative genomics The results from protein comparisons of the reference strain S. hyicus ATCC 11249 (Calcutt et al., 2015) with the toxigenic and nontoxigenic strains were visualized in a BLAST atlas (Fig. 2). The atlas contains eleven toxigenic strains in red color intensity and six nontoxigenic strains in green color intensity. The atlas revealed two interesting regions, region 1 (41806-72839) and region 2 (10721061087194), where some genes from toxigenic strains were present but genes from non-toxigenic strains were largely absent, or vice versa. The genes from those regions were extracted and aligned against all 17 S. hyicus genomes. The similarity of genes from region 1 and region 2 against all genomes are shown in heatmaps in Fig. 3A and B respectively. The heatmap constructed from region 1 showed 12 genes (Fig. 3A, genes colored in orange) being present in at least four out of eleven toxigenic strains, and one non-toxigenic strain (A3793/ 76) with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) in non-toxigenic strains. The predicted function of the proteins encoded by those genes are N-6 DNA methylase, type I restriction-modification system, thioesterase, transferase, AMP ligase, equibactin nonribosomal peptide synthase, polyketide synthase, salicylate synthase, DEAD/DEAH box helicase and hypothetical protein (see Fig. S1 and Table S2). There were two polyketide synthase genes in this region. They have different Pfam domains and have different sequence length. The co-location of multiple polyketide synthase have been described (Heather et al., 2008). The heatmap from region 2 showed 5 genes (Fig. 3B, genes colored in orange) found in four to ten toxigenic strains with high similarity (more than 95% similarity) and having low similarity (less than 85% similarity) in non-toxigenic strains. Several of these proteins are hypothetical proteins and one of them is phage tail protein. Furthermore, there were 10 genes (Fig. 3B, genes colored in purple) present only in three to eleven toxigenic strains. One of the 10 genes (NZ_CP008747_CDS_1079564-1078743) encodes for exfoliative toxin ExhA that is an important virulence factor in
Table 1 The presence/absence of exfoliative toxins in S. hyicus genomes. Exfoliative toxin proteins (% sequence similarity) S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S. S.
hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus hyicus
genomes ATCC 11249 toxigenic (Denmark) NCTC10350 toxigenic (Denmark) 1289D-88 toxigenic (Denmark) 1403E-88 toxigenic (Denmark) S3588 toxigenic (Germany) 842A-88 toxigenic (Denmark) 9390-88 toxigenic (Denmark) A2869C toxigenic (Germany) P119 toxigenic (UK) P411 toxigenic (UK) 842G-88 toxigenic (Denmark) 1289E-88 toxigenic (Denmark) 842J-88 non-toxigenic (Denmark) A3793/76 non-toxigenic (Germany) A4596/76 non-toxigenic (Germany) A72/75 non-toxigenic (Germany) SK170 non-toxigenic (UK) 1403B-88 non-toxigenic (Denmark)
ExhA + [100] + [100] – – + [100] – + [100] – + [100] + [100] – – – – – – – –
ExhB – – + [100] + [100] – – – – – – – – – – – – – –
ExhC – – – – – + [100] – – – – – – – – – – – –
ExhD – – – – – – – + [100] – – + [100] + [100] – – – – – –
SHETB – – – – – – – – – – – – – – – – – –
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S. hyicus. The other genes of the 10 genes are hypothetical proteins, glutamyl endopeptidases, a predicted peptidase S8, an epidermal cell differentiation inhibitor protein (ADP-ribosyltransferase) and a putative RNase toxin (polymorphic toxin). Other genes from region 2 (Fig. 3B, genes colored in black) that were found in many toxigenic and some non-toxigenic strains include genes encoding for DNA polymerase, DNA repair protein and bacteriophagerelated proteins (see Fig. S2 and Table S2). Sequences of genes from region 1 and region 2 can be retrieved from File S1 and File S2. The genome sequence of Staphylococcus aureus phage FETA (Yamaguchi et al., 2000) was compared with the whole genome of of S. hyicus strain ATCC 11249. There were 11 genes out of 66 genes from FETA phage found in ATCC 11249. Four genes from the eleven genes were found in region 2 (Table S2). The genes from region 2 that were similar to FETA phage were hypothetical proteins, phage PVL and ExhA. The other seven genes from the phage were found in other regions away from the regions 1 and 2. Using ISfinder (Siguier et al., 2006) to search for insertion sequences, we found ISSep3 in the reference genome, S. hyicus strain ATCC 11249. The insertion site was found from position 1,570,484 to 1,571,023, which is not in the range of region 1 and region 2 or even close to the regions. In addition, we found proteins encoded by the different S. hyicus strains, both toxigenic and non-toxigenic ones, that were not detected in the reference genome S. hyicus ATCC 11249. The number of additional proteins in toxigenic strains (excluding NCTC10350) ranged from 81–203. The numbers in non-toxigenic strains ranged from 39–164. The majority of the additional proteins were phage- and plasmid-related (Table S3). The five exfoliative toxin-encoding genes, ExhA, ExhB, ExhC, ExhD, SHETB, were compared against all S. hyicus genomes. Four of the exfoliative toxins were found in S. hyicus with 100% similarity (Table 1). SHETB was absent from all S. hyicus under study with the cut off 65% similarity. Toxigenic strains encoded for either ExhA, ExhB, ExhC or ExhD. Non-toxigenic strains appear not encode for any of the exfoliative toxins. In addition, toxigenic strains were clustered according to the type of exfoliative toxin genes in heatmaps showing similarity of the genes from region 1 and 2 (Fig. 3A and B). Nine toxin genes from S. aureus and one toxin gene from S. chromogenes were also aligned against S. hyicus genomes. With the cut-off at 65 percent similarity, the exhB gene from S. chromogenes was found in two S. hyicus toxigenic strains, 1403E-88 and 1289D88. The other genes were not detected in any S. hyicus. 4. Discussion In pigs, the common cause of EE is infection with S. hyicus (Foster, 2012). Differentiation between toxigenic and non-toxigenic S. hyicus strains can readily be obtained by the use of PCR (Andresen and Ahrens, 2004; Futagawa-Saito et al., 2007) but other significant virulence factors with regard to EE have not been examined. In this study, we have sequenced and compared toxigenic and non-toxigenic strains of S. hyicus. The S. hyicus strains exhibited no relationship between phylogeny and virulence according to phylogenetic tree based on single-nucleotide polymorphisms (SNPs). The phylogenetic trees seemed, however, to indicate clustering of toxigenic S. hyicus strains according to geographic location. The protein comparison of S. hyicus genomes against the reference genome revealed two genomic regions containing genes that were predominantly present in toxigenic strains but absent or present with low similarity in non-toxigenic strains. These genes included the exfoliative toxin, exhA. Region 1 appears to encode for non-ribosomal peptide synthetases. It has similarity with equibactin (yersiniabactin) potentially involved in iron-aquisition. The
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genes in the region 2 encode for several toxins/proteins with enzymatic function for example exfoliative toxin (Trypsin-like), two additional Trypsin-like enzymes, EDIN/3C3stau-homolog (ADP-ribosyltransferase), Toxin 46 (polymorphic toxin) (Zhang et al., 2012) and Peptidase S8. The proteins all have predicted signal peptide domains (except Peptidase S8), and hence are likely to be secreted. Peptidase S8 has one TM domain and may be located in the membrane. In addition, region 2 contained four genes from S. aureus phage FETA and we did not identify any insertion sequences in the regions including sequences around the regions. The genes found in the two genomic regions might encode for potential additional virulence determinants for EE. Fig. 3B shows that the strains that carry exhA also harbour the majority of the genes encoded in region 2. Strains carrying exhB, exhD and exhC have 17, 8 and 1 gene(s), respectively, that are highly homologous to the 28 genes from region 2 shown in Fig. 3B. This would indicate that only the gene encoding ExhA of the exfoliative toxins from S. hyicus is associated with region 2. Region 2 is similar to the genomic island previously identified in the genome of S. hyicus strain ATCC 11249T (Calcutt et al., 2015) which is the same strain as NCTC 10350 in this study. These results indicate that strains that carry exhA are very similar with regard to the genes that surround exhA, and gives further support to the indication that exhA may be located on a pathogenicity island or a prophagerelated element as suggested by Calcutt et al. (Calcutt et al., 2015). The strains carrying the other types of exfoliative toxin, ExhB, ExhC and ExhD, have fewer genes that are homologous to the genes from region 2 compared to the ExhA-positive strains, suggesting that these toxin genes have a different genetic background than ExhA. The results of this study adds to the notion that different types of exfoliative toxin from S. hyicus and from S. aureus, although predicted to be part of the same protein family (Ahrens and Andresen, 2004), do have quite different genetic background for the genes of the these toxins, such as bacteriophage (Yamaguchi et al., 2000), plasmid (Yamaguchi et al., 2001) and pathogenicity island (Calcutt et al., 2015; Yamaguchi et al., 2002). In summary, we found that there are other genetic differences between toxigenic and non-toxigenic strains of S. hyicus besides well-known exfoliative toxins. Some of the genes might potentially be virulence determinants. There is however, no apparent relationship between phylogeny and virulence. Other factors are likely involved in S. hyicus colonization and virulence in pigs, such as interactions with other members of the skin microbiota as hypothesized for S. aureus (Yan et al., 2013), as well as host determinants (Daugaard et al., 2007) including immunological factors. Previous investigations in pigs indicated that colonization by a certain strain evokes macroscopic and microscopic signs of disease (EE) to different degrees in different animals (Wegener et al., 1993). However, such differences may also be due to differences in the structure and amino acid composition of the exfoliative toxins (Ahrens and Andresen, 2004). Acknowledgements This work was supported by the Center for Genomic Epidemiology at the Technical University of Denmark funded by grant 09-067103/DSF from the Danish Council for Strategic Research. Sünje J. Pamp was supported by a grant from Carlsbergfondet. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. vetmic.2016.01.018.
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