Background

Mastitis is reported as one of the most important diseases for dairy cattle on the basis of great economical losses caused by affecting animal welfare and milk production costs [1]. Staphylococcus aureus is one of the major cause of intra-mammary infection (IMI) in ruminants worldwide, causing mastitis with diverse degrees of severity. In dairy cows, Staph. aureus IMI is almost always subclinical, thus leading to an increasing risk of contagion and diffusion in the herds [2]. The molecular pattern of Staph. aureus isolates in diverse farm animal forms distinct genetic clusters differing in the presence of pathogenic factors that increase their invasiveness, even in the presence of a stronger inflammatory response [3]. In a recent study [4], subtypes of Staph. aureus were associated with high within-herd IMI, compared to other different subtypes that were associated with low within-herd prevalence. This study and previous data [5, 6] confirmed that particular gene patterns, virulence profiles and specific genotypes could be associated with diverse clinical outcomes. More recently, two large European studies [7, 8], demonstrated that the Repetitive-Sequence PCR RS-PCR genotype B (GTB), belonging to the Sequence Type ST8 [9, 10], a high contagious and diffusive Staph. aureus involved in bovine IMI, was the most frequently detected in several European countries (Austria, Belgium, France, Germany, Italy, Switzerland). Conversely, the RS-PCR genotype S (GTS), belonging to ST398 [11, 12], was one of the rare genotypes found in bovine milk samples. The ST8 was previously found in both human and dairy cow mastitis [4], suggesting that, after a human-to-cow host jump, a new bovine adaptation took place. At the same time, the ST398 showed a host transition from human to animal reservoir, becoming the most widely disseminated clonal complex in bovine species and in the milk samples collected in herds with low prevalence of IMI [13–15].

The existence of subtypes of Staph. aureus differing in pathogenic properties emphasizes the need to well define strain characteristics, in order to monitor bacteria dissemination and find potential relevant targets related to their contagiousness. In recent years the advent of next generation sequencing (NGS) technologies has improved the estimate of the correlation of virulence phenotype to genome structure, providing a more detailed picture of gene patterns involved into staphylococcal pathogenesis. High-throughput whole-genome sequencing of Staph. aureus was prevalently used to monitor outbreaks in hospitals [16, 17], to evaluate strains transitioning from carrier to invasive status [18] and to understand aspects of pathogen biology in detailed epidemiological studies in human [19–21]. In livestock community, the molecular basis of virulence in Staph. aureus mastitis was investigated by using an integrated approach that includes NGS, microarray and proteomic data [22] providing the first high-resolution comparison between gene content and gene expression in two Staph. aureus strains. More recently, Peton and co-workers [23] described a fine-tuned characterization of Staph. aureus Newbond 305, a strain belonging to ST115 and associated to bovine mastitis, by genomic and proteomic comparison with the reference strain RF122. Gene expression analysis by microarray techniques has provided, also, information about global transcript changes [24, 25] or molecular basis of virulence [26] in Staph. aureus. Moreover, RNA-seq was recently used to study (i) the gene expression in different Staph. aureus strains [27, 28], (ii) the role of anti-sense transcription [29] and (iii) the identification of small non-coding RNAs [30].

To gain further insight into Staph. aureus features, the aim of this work was the characterization of two groups of Staph. aureus strains differing in their clinical outcome. Each strain was comprehensively studied by comparative genomic and transcriptomic analysis in order to identify staphylococcal factors that can be associated with strain virulence and bacterial diffusion in the herd.

Methods

Bacterial strains

Six bacterial strains, originally isolated from subclinical cases of bovine IMI in six different Holstein herds (A-F, Table 1) located in Lombardy region in the northern of Italy, were used in this study. The average size of the herds was 106 milking cows (range 38 to 285 cows). Milk samples were collected aseptically. Samples were kept at 4 °C and bacteriological assays were performed within 48 h. Isolates were classified into two groups: Staph. aureus belonging to low within-herd mastitis prevalence (herds A, B, C) or high within-herd mastitis prevalence (herds D, E, F). As described in Table 1, and reported by Cremonesi and colleagues [4], Staph. aureus isolates had been previously characterized by RS-PCR, Multi Locus Sequence Type (MLST) [31], for presence of mecA gene and for different virulence genes. The strains collected in three different herds with low mastitis prevalence (between 2 and 4%) were identified by RS-PCR as genotype S (GTS) and by MLST as ST398 (hereinafter referred as GTS/ST398). Two out of three were positive for mecA gene. The strains isolated from three herds with high IMI prevalence (between 49 and 62%) were identified by RS-PCR as genotype B (GTB) and ST8 (hereinafter referred as GTB/ST8) and none of them harboured the mecA gene coding for methicillin resistance.

Table 1 Characteristics of the bacterial strains used in this study
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Growth conditions

The strains were isolated and grown on Blood Agar plates and a single colony of the third passage in culture was transferred into 5 ml of Brain Heart Infusion medium (BHI). Bacteria were grown overnight at 37 °C. Cultures were subsequently diluted 1/100 into 40 ml of BHI and grown at 37 °C. Optical density at 600 nm (OD600) was performed hourly until mid-exponential phase (OD600 = 0.4) was achieved. At the appropriate OD600, bacteria were pelleted by centrifugation at 10 000 g for 2 min; after surnatant removing, the pellet was resuspended in 500 μl of saline solution (NaCl 0.9%) and centrifuged at 10 000 g for 2 min. The pellet was immediately used for RNA extraction and stored at −20 °C for DNA extraction.

Bacterial DNA and RNA extraction

Genomic bacterial DNA was extracted using the protocol previously described [32], starting from step 2. Total RNA was isolated using the NucleoSpin® mRNA kit (Macherey-Nagel, Germany), according to the manufacturer protocol, in combination with TRIzol® lysis. DNAs and RNAs were quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and RNAs quality was checked using the Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA). Only RNA samples with RNA Integrity Number (RIN) values higher than 6.5 were used for the analysis. The isolated DNAs and RNAs were stored at −20 and −80 °C until use, respectively.

Library preparation and Miseq sequencing

DNA

Libraries were constructed using TruSeq PCR free Kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions, sequenced in one 2 × 300-cycles Miseq run (Illumina, San Diego, CA, USA).

RNA

RNA was processed as previously described [27] with some variations. Briefly, bacterial rRNA was depleted with RiboZero rRNA removal kit for gram-positive organisms (Epicentre Illumina, Madison, WI, USA). RNA quality was assessed for each passage by the Agilent Bioanalyser 2100 (Agilent, Santa Clara, CA). Libraries were prepared using TruSeq® RNA Sample Preparation v2 Kit (Illumina). Samples were sequenced on a Miseq Instrument (Illumina) in a 1 × 50-cycles run.

Bioinformatics analysis

Staph. aureus NCTC 8325 core gene evaluation and De-Novo Assembly of GTB/ST8 and GTS/ST398 strains. The quality of the raw sequencing reads was assessed by using FastQC software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adapter removal and quality trimming has been performed using Trimmomatic [33], with default parameters and nucleotide PHRED quality > 30.

High-quality reads were mapped against the reference genome of NCTC 8325. BWA has been used as mapping software to detect common genes between GTS/ST398, GTB/ST8 and NCTC 8325. GTS/ST398 and GTB/ST8 reads were cross-mapped against NCTC 8325 genome. We applied quality filters by excluding those reads with more than four mismatches or those with mapping quality score (MAPQ) less than 15 in the resulting BAM files. To select genes present in the three genomes, we applied filters on coverage and depth. Briefly, only those genes that present 100% length coverage and a minimum 10X mean depth in the two mapping strains have been selected (Additional file 1). 2478 genes were selected and used in RNASeq analysis for quantification and differential expression. To perform the assembly the short-read assembly tool SPAdes 3.1.1 [34] was used. To obtain a reference assembly (ra) for each group of strains (genotype GTS/ST398 and GTB/ST8), the single assemblies (three for each group), were merged with CISA [35]. GTB/ST8 reference assembly (GTB/ST8ra) and GTS/ST398 reference assembly (GTS/ST398ra) were annotated with RAST [36]. To overcome false protein duplication and misassembly issues, we performed a reciprocal BLASTp within GTB/ST8ra set and GTS/ST398ra set separately. Whether a protein presented a perfect match (100% sequence identity and 100% length identity) with another one, only one of them was selected.

The protein sequences comparison between the strains (GTS/ST398, GTB/ST8) and the definition of the “core” (set of genes shared between GTB/ST8ra and GTS/ST398ra) and “accessory” (set of unique genes for both genotypes) genomes was performed by using In Paranoid 4.1 software [37], a BLAST-based algorithm to compute protein homology analysis between two or more species. For GTB/ST8 and GTS/ST398 analysis, a cut-off of 0.9 for sequences overlap and the default values for the other options were used. The functional enrichment analysis was performed using the Fisher’s test on the functional categories after Bonferroni multiple testing correction using R software version 3.0.3.

Genomic comparison with other Staph. aureus reference strains

For comparative analysis, reference genome sequences of 22 strains available in NCBI were used (Additional file 2). The genome similarities based on phylogenetic distances were analyzed using the Gegenees software [38]. A fragmented alignment in TBLASTX mode was performed with settings 500/500 and dendrogram was produced in SplitsTree 4 [39]. Visualization of genome comparisons was performed using BLAST Ring Image Generator [40].

Transcriptomic analysis

Transcriptome reads were mapped against the reference sequence of Staph. aureus NCTC 8325 genome with BWA aligner [41]. To generate a high-quality mapping for each sample, we applied quality filters by excluding those reads with more than four mismatches or those with mapping quality score (MAPQ) less than 15. Read counts for gene relative abundance, differential expression analysis and statistical analysis were calculated as previously described [42, 43].

Differential expression analysis was performed on the gene set belonging to core genome of Staph. aureus NCTC8325 and the calculation of differential expression genes was performed with DESeq [44]. Differential expressed genes were selected with specific filters: 1) p-value less than 0.01 after Bonferroni correction (padj) 2) log2FC >1.5 or < -1.5. Functional categories annotation for each gene was extracted from COG database [45] and the Fisher’s test was used for enrichment analysis after Bonferroni correction. All the statistical analyses were performed using R version 3.0.3. DNA-Seq and transcriptomic data were visualized using the Integrated Genomics Viewer IGV [46]. Transcriptomic data are available in Sequence Reads Archive (SRA) accession number SRX965931.

Real Time PCR qRT-PCR

Primers used for real-time PCR were designed using Primer Express software V2.0 (Applied Biosystems, Foster City, CA) and are listed in (Additional file 3). Pyrroline-5-carboxylate reductase (proC) was used as a reference gene [47]. Each sample was treated with DNAse and cDNAs were synthesized using GoScript™ Reverse Transcription System (Promega, Madison, WI) with random primers following manufacturer instruction. Real-time PCR was performed with 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA) using Power SYBR® Green PCR Master Mix (Applied Biosystems) according to manufacturer protocols. Data were analyzed with Sequence Detection Systems SDS Software (version 2.3).

Pathway analysis

Protein sequences from unique GTB/ST8 and GTS/ST398 and differential expressed genes (DEGs) were used as queries in KOALA (KEGG Orthology And Links Annotation) tool for pathways reconstruction [48].

fnb B partial re-sequencing

Primers fnbB-F1 (5′-TTCTGCATGACCTTCTGCAC-3′) and fnbB-R1 (5′-AGCAAGCGAAACACAAACAA-3′) were used to amplify a portion from 1222 up to 2656 bp of fnbB genes (NCBI accession number: CP000253, region: 2577879......2580632) in all the six strains. PCR was performed in a final volume of 25 μl, containing ~60 ng of DNA, 0.8 μM of each primer, 12.5 μl of GoTaq® Long PCR Master Mix (Promega, Italy), with the following cycling parameters: 95 °C for 2 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 2 min; and then 72 °C for 10 min. PCR products were loaded in 1.5% agarose gel. PCR products were purified with Wizard Clean-up (Promega, Italy), following manufacturer′s instructions. Purified PCR products were sequenced bi-directionally (GATC Biotech, Konstanz, Germany) with primer fnbB-F1 and fnbB-R1. fnbB partial sequences determined in this study have been submitted to GenBank with accession numbers KY024702 and KY024703 for GTB/ST8 and GTS/ST398, respectively.

Results

In this study six strains of Staph. aureus previously described [4] as strictly associated with high and low within-herd IMI prevalence, respectively, were analysed in order to discover, thanks to comparative genomics and transcriptomics, potential pathogenic factors associated with the different clinical outcome found in the herds.

Sequencing of GTB/ST8 and GTS/ST398 genotypes

Genome assembly and Comparative Genomics

Genomic diversity between the Staph. aureus GTB/ST8 and GTS/ST398 genotypes was assessed analyzing three DNA samples for each genotype that were deeply sequenced with an average production of 2,908,485 (max 4,630,318 and min 1,399,737) reads per sample. The sequencing reads from every sample were assembled obtaining an average of 95 number of large contigs (>500 nt) from all the samples (mean GTB/ST8: 50; mean GTS/ST398: 139) (Additional file 4).

Three GTB/ST8 and three GTS/ST398 assemblies showed a high level of within group similarity, ranging from 91 to 99% and 95 to 98% respectively, whereas only a partial similarity (from 78 to 86%) was observed between the two groups (Fig. 1a).

Fig. 1
figure 1

a Percentage of similarity between the three GTB/ST8 and the three GTS/ST398strains. b Phylogenesis distances and percentage of similarity between the two reference assemblies GTB/ST8ra (blue) GTS/ST398ra (red) and other Staph. aureus strains. In brown the NCTC_8325 strain used as reference for RNASeq data analysis

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To create a single reference genome representing each group, all the single genotype-specific assemblies were merged together producing two reference assemblies, one for GTB/ST8 and one for GTS/ST398 with 19 (3.366.835 nt) and 291 (3.284.103 nt) large contigs, respectively. These reference assembly genomes, named GTB/ST8ra and GTS/ST398ra, respectively, were used for the genomic analysis in comparison with the genomes of other 22 Staph. aureus reference strains, fully sequenced and available in GenBank (Fig. 1b). The two genotypes GTS/ST398ra and GTB/ST8ra here analysed clustered better with the Staph. aureus ST398 prototype and Staph. aureus TCH1516, respectively, the latter being a human ST8 reference strain.

GTB/ST8ra and GTS/ST398ra genome comparison

Two thousand six hundred sixty-seven and 2712 Coding DNA Sequences (CDS) were annotated from the prediction tool for GTB/ST8ra and GTS/ST398ra, respectively. Predicted proteins were functionally categorized using the COGs database. As expected, about 50% were annotated with a functional role (48.74% GTB/ST8ra and 48.48% GTS/ST398ra, respectively). Protein homology analysis revealed that the majority of CDSs (n = 2247) was shared between the two groups of strains, since up to 84.25% and 82.85% of the CDSs belonged to the core genome of GTB/ST8ra and GTS/ST398ra, respectively (Additional file 5). The COGs distributions were similar in the two genomes: both GT8/ST8ra and GTS/ST398ra presented a set of unique genes belonging to “Phages, Prophages, Transposable elements, Plasmids” and “Virulence, Disease and Defence” categories that were significantly enriched (p-value < 0.01) compared to the core genome. In addition, GTB/ST8ra was significantly enriched also in “Membrane Transport” genes (Fig. 2, Additional file 6). For both groups of strains, unique genes associated to virulence were prevalently attributed to “Adhesion function” and “Resistance to antibiotics and toxic compounds and toxin production” (Table 2).

Fig. 2
figure 2

Gene ontology (GO) categories comparison between annotated genes belonging to the core genome (shared GTB/ST8ra&GTS/ST398ra genes, in green) and unique gene for GTB/ST8ra (blue) and GTS/ST398ra (brown). X axis indicates the percentage value of genes belonging to each categories reported

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Table 2 Genes associated to virulence resulting different between the two reference genotypes (GTB/ST8ra and GTS/ST398ra)
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GTB/ST8 and GTS/ST398 transcriptomic comparison

Transcriptomic analysis and qRT-PCR validation

RNA-Seq data covering the Staph. aureus genome were used to quantitatively compare gene expression levels between the two groups of strains grown in the exponential phase. Transcriptome reads were aligned against Staph. aureus NCTC8325 reference genome with an high read-mapping rate and high coverage for both genotypes (average: 93.25% ± 1.39%) (Additional file 7). Among the 2479 genes that were in common between the three genomes (GTS/ST398ra, GTB/ST8ra and NCTC8325), 237 differential expressed genes (DEGs) were found between the two groups with a distribution of 56.1% of the DEGs up-regulated in GTB/ST8 strains and 43.9% in GTS/ST398 strains (Additional file 8).

Four of these differentially expressed genes were validated by RT-PCR: SAOUHSC_00773, the Lys M domain-containing protein; SAOUHSC_01181 an hypothetical proteins; SAOUHSC_01314, DNA-binding response regulator; SAOUHSC_01450the basic amino acid/polyamine antiporter, APA family protein. For each test, qPCR results confirmed RNA-Seq data: SAOUHSC_01314 not expressed in one of the two genotypes resulted in undetermined CT value, whereas the three differentially expressed genes SAOUHSC_00773, SAOUHSC_01181, SAOUHSC_01450 showed fold changes of 0.0538, 9.0972 and 0.1297 in qPCR respectively, comparable with the fold changes of 0.0694, 91.4000 and 0.0265 obtained from RNA-Seq (Additional file 9a, b).

Functional analysis of transcriptomic data

The comparison between the number of DEGs and the total number of genes present in the NCTC8325 reference genome, revealed that genes belonging to “Amino acid transport and metabolism” category varied significantly between the two genotypes (corrected p-value 3.86E-05). Other two categories, “Defence mechanism” and “Inorganic ion transport and metabolism” indicated a trend toward enrichment in DEGs list (Fig. 3).

Fig. 3
figure 3

Gene ontology (GO) categories comparison between significant DEGs (in grey) and genes expressed in the Staph. aureus NCTC_8325 strain (in black). ****P ≤ 1e-4

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Within the 42 annotated genes related to “Aminoacid transport and metabolism” pathway, only 9 were up-regulated in GTB/ST8 (Table 3). Concerning “Defence mechanism” we found 34 annotated genes, within 14 and 20 up-regulated in GTB/ST8 and GTS/ST398, respectively. Finally, among the 24 DEGs belonging to “Inorganic ion transport and metabolism”, 4 and 20 were up-regulated in GTB/ST8 and GTS/ST398, respectively (Table 3).

Table 3 DEGs between GTB/ST8 and GTS/ST398 strains
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Virulence and defence pathways analysis: comparison between genomic and transcriptomic data

Functional and pathway enrichment analysis for integrated regulatory network of the two groups of strains was performed considering annotated genes from DNA-Seq and RNA-Seq experiments, grouped in the three dataset, i.e. gene exclusively present in GTB/ST8ra, gene exclusively present in GTS/ST398ra and DEGs (Additional files 5 and 8).

Considering the functional categories belonging to bacterial invasion of epithelial cells and infection pathways, we found genes that were detected by using or (i) strain genotyping comparison or (ii) transcriptomic analysis or (iii) by the two combined approaches, as reported in Fig. 4. For example, from this analysis Fibronectin-Binding Protein B gene (fnbB), an important adhesin involved not only in adhesion to cells but also in internalization by cells, was detected in both groups by means of a partial alignment between GTB/ST8ra and GTS/ST398ra protein sequence. In parallel, transcriptomic analysis revealed a variation in fnbB expression, over-expressed in GTB/ST8 and down regulated in GTS/ST398 (Additional file 10 a, b, c). The nucleotide sequence between the two groups of strains was verified by sequencing a 1285 bp portion of fnbB gene (Additional file 11). The nucleotide sequence was conserved within each group of strains and was high polymorphic between the two genotypes. Overall, GTS/ST398 showed a high polymorphism compared to reference NCTC8325 strain sequence (83% of identity), whereas no differences were observed for GTB/ST8 (100% of identity). Further, the protein translation for GTS/ST398 resulted in a truncated protein form.

Fig. 4
figure 4

Bacterial invasion of epithelial cells and infection pathways analysis of Staph. aureus. In grey highlighted DEGs or unique genes for GTB/ST8ra or GTS/ST398ra. Fibronectin-Binding Protein B FnBPB, clumping factor B ClfB, iron-regulated surface determinant protein A IsdA, serine-aspartate repeat-containing protein C/D/E SdrC/D/E, staphylococcal complement inhibitor (SCIN) and eap/map protein EaP/Map and leukocidin/hemolysin Hlg/Luc. Numbers define the dataset from which the genes were found: 1) unique GTB/ST8ra, 2) unique GTS/ST398ra and 3) DEGs from transcriptomic analysis, and their combination: (1-2: genes with differences in protein sequence; 1-3: genes expressed and present only in GTBra; 1-2-3: genes differentially expressed and different in protein sequence between the two reference genotypes)

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Moreover as shown in Fig. 4, clumping factor B gene (ClfB) and iron-regulated surface determinant protein A gene (IsdA) showed differentially expression by transcriptomic profiling (over-expressed and down regulated in GTB/ST8 and vice versa for GTS/ST398, respectively) (Additional file 12 a, b). Furthermore, the serine-aspartate repeat-containing protein C/D/E gene (SdrC/D/E) and eap/map protein gene (EaP/MaP) differed in protein sequence exclusively by DNASeq comparison (Additional file 13 a, b). Finally leukocidin/hemolysin gene (Hlg/Luc) was seen to be exclusively present and expressed in the GTB/ST8 strains, whereas the staphylococcal complement inhibitor SCIN (scn) was differentially expressed between the two groups, over-expressed in GTB/ST8 and down-regulated in GTS/ST398.

Discussion

Staph. aureus IMI clinical outcomes are highly variable and depend on several factors, including animal genetics, environmental conditions and strain-dependent factors. All these conditions should be correctly evaluated in order to predict the spread of bacterial strains within the herd. Here we achieved an in-depth characterization through NGS of six Staph. aureus strains previously genotyped as GTB/ST8 and GTS/ST398, differing in virulence properties such as within-herd Staph. aureus IMI prevalence [4].

These six strains belonged to two distinct clonal complexes and sequence types (CC8/ST8 for GTB/ST8, and CC398/ST398 for GTS/ST398), with a high intra-group similarity among the three strains associated to the same clonal complex (about 97% and 95% for GTB/ST8 and GTS/ST398, respectively), significantly supporting the creation of the reference assemblies as representative of these two lineages. Both genotypes clusterized in groups that include strains of human origin: GTS/ST398 showed high similarity with the prototype of Staph. aureus ST398, SO385, isolated from human endocarditis [49], whereas GTB/ST8 to Staph. aureus TCH1516, a methicillin susceptible ST8 strain, isolated from an adolescent patient with severe sepsis syndrome [50]. Recent studies on these CCs [4, 13] showed a closed genetic relationship between CC8 isolated from dairy cow mastitis and human CC8, suggesting human-to-bovine jump. On the other hand, the presence of CC398 strains was described only in herds with IMI prevalence lower than 5% [4]. In the present study, a comparison of the genome sequences of these strains with a core genome (set of genes shared between GTB/ST8ra and GTS/ST398ra) revealed about 17% of differences for their gene content, with a relevant enrichment in genes associated with virulence properties. Both groups of strains showed differences in several genes associated to virulence factors and some of them were present in only one of the two genotypes.

Furthermore, the transcriptomic profiling for both groups of strains confirmed the functional enrichment for genes related to adaptation and propensity to chronicity. Interestingly, GTB/ST8ra showed higher expression of signal transduction Target of RNAIII-activating Protein TRAP, that leads to the activation of agr system, resulting in the expression of several virulence factors. As previously described [51, 52], the protein TRAP activates RNAIII synthesis by RNAIII-activating protein (RAP) system, increasing the pathogenic potential of the bacteria.

Contemporary, the integrated pathway analysis between the two genotypes of Staph. aureus genes involved in pathogenicity showed an interesting variation in the microbial surface component recognizing adhesive matrix molecule (MSCRAMM), whose function includes adhesion to and invasion in host cells and tissues, evasion of immune responses and biofilm formation [53]. As well known, the fnbB gene is a multifunctional MSCRAMM, which recognizes fibronectin, fibrinogen and elastin and promoting the internalization of Staph. aureus into epithelial and endothelial cell mediating bacterial invasion [54, 55]. Most Staph. aureus strains can express two distinct fibronectin-binding proteins (FnBPA and FnBPB), which both mediate adhesion to fibrinogen, elastin and fibronectin. The GTB/ST8 and GTS/ST398 strains, analysed in this study, presented the two fibronectin-binding proteins but only fnbB showed changes in genomic and transcriptomic analyses between these two groups. The fnbB gene showed high variability between the two genotypes, revealing a high level of polymorphisms that lead to a premature stop codon and a truncated form of the protein for GTS/ST398 strains. Similar results were previously published by McCarthy and colleagues [56], which postulated that the truncated FnBPB form could affect Staph. aureus colonisation and infection. Also Burke and co-workers found different FnBPB isotypes in diverse STs Staph. aureus strains, revealing an association between this gene and the invasiveness [57]. And more, the presence of protein variations for both groups of strains suggests a different affinity for fibronectin, necessary for the internalization of Staph. aureus into host cells [23].

In addition, ClfB and SdrC/D/E genes encoding for MSCRAMMs proteins, whose functions are related to adhesion and colonization [58, 59], showed differences by transcriptomic and genomic analyses in both genotypes, respectively, indicating that surface adhesins are not only present/absent, but also variable amongst lineages of Staph. aureus [56], such as GTB/ST8 and GTS/ST398. Polymorphisms in these genes are well known and used to Multilocus Variable Number Tandem Repeat Fingerprinting (MLVF), a genotyping method for epidemiological studies [60]. Other multi-functional proteins, such as the “Staph. aureus surface G Sas G protein the iron-dependent adhesion IsdA, IsdB, IsdC”, over-expressed in GTS/ST398, play a role in biofilm formation [61, 62]. The adhesive properties displayed by MSCRAMM proteins reside within the cell surface; however, several important adhesins are also formally secreted from the bacterial cell. The “Secreted Expanded Repertoire Adhesive Molecules (SERAM) extracellular adherence protein” (eap) is nearly ubiquitously distributed amongst Staph. aureus strains and appears to function as a virulence determinant in animal models of chronic infection [63]. Eap gene was proposed as novel target for specific identification of Staph. aureus [64]. According to our results the sequence alignment of eap gene from all Staph. aureus genomes published to date revealed a significant polymorphism in this gene [65]. As stated by McCarthy et al. [56], the genetic variation in Staph. aureus surface and immune evasion genes is lineage associated and carries a range of unique variants in order to improve the adaptation of this microorganism to different host species.

Finally, the staphylococcal complement inhibitor (scn), over-expressed in GTB/ST8 and down-regulated in GTS/ST398, produced by the Staph. aureus during the early phase of infection, helped the microorganism to survive into the host [66], preventing both chemotaxis and phagocytosis. The fact that surface and immune evasion proteins are different between lineages suggests that they are essential for virulence, opening a window for further investigations.

Conclusions

In conclusion, our analysis proves that integration of RNA-Seq and DNA-Seq data well depicts Staph. aureus strains associated with different within-herd IMI prevalence in dairy cows. Our results disclosed congruent patterns of genetic variation in colonization and invasion factors between GTB/ST8 and GTS/ST398 strains. Notwithstanding, results highlight a high number of unknown genes differing between genotypes, whose unknown functionality lacks a direct association with virulence function. Overall, the fine genomic characterization of these strains was a first step towards developing strategies able to provide new insights into mechanisms associated to Staph. aureus mastitis, including genomic comparison of a larger set of high and low diffusive strains, improvement of Staph. aureus reference strains annotation and new ad hoc bioinformatic tools.