Abstract
Multi-drug resistant S. aureus is a leading concern worldwide. Coagulase-Negative Staphylococci (CoNS) are claimed to be the reservoir and source of important resistant elements in S. aureus. However, the origin and evolutionary route of resistant genes in S. aureus are still remaining unknown. Here, we performed a detailed phylogenomic analysis of 152 completely sequenced S. aureus strains in comparison with 7,529 non-S. aureus reference bacterial genomes. Our results reveals that S. aureus has a large open pan-genome where 97 (55%) of its known resistant related genes belonging to its accessory genome. Among these genes, 47 (27%) were located within the Staphylococcal Cassette Chromosome (SCCmec), a transposable element responsible for resistance against major classes of antibiotics including betalactams, macrolides and aminoglycosides. However, the physically linked mec-box genes (MecA-MecR-MecI) that are responsible for the maintenance of SCCmec elements is not unique to S. aureus, instead it is widely distributed within Staphylococcaceae family. The phyletic patterns of SCCmec encoded resistant genes in Staphylococcus species are significantly different from that of its core genes indicating frequent exchange of these genes between Staphylococcus species. Our indepth analysis of SCCmec resistant gene phylogenies reveals that genes such as blaZ, ble, kmA and tetK that are responsible for beta-lactam, bleomycin, kanamycin and tetracycline resistance in S. aureus were laterally transferred from non-Staphylococcus sources. In addition, at least 11 non-SCCmec encoded resistant genes in S. aureus, mostly present in plasmid are laterally acquired from distantly related species. Our study evidently shows that gene transfers played a crucial role in shaping the evolution of antibiotic resistance in S. aureus.

saprophyticus (Grundmann et al. 2006 sciuri, and S. vitulinus were involved in the stepwise assembly of SCCmec elements and it is later transferred to S. aureus (Rolo et al. 2017 ).
However, there are limitations in the existing studies; mainly due to the lack of diverse data used. Most of these studies focused on SCCmec encoded elements, which contribute only 27% of resistance encoding genes in S. aureus. Many efforts have been taken to understand the evolution of antibiotic resistance in S. aureus and ended up with multiple observations, yet an overall picture is still lacking. Since most of the resistant genes in S. aureus located on mobile genetic elements and plasmids, we strongly hypothesize that gene exchange among closely as well as distantly related species, might have shaped the current form of resistant genes distribution in S.
aureus.
In the present study, we performed a detailed phylogenomic analysis of all the resistance encoding genes in 152 completely sequenced S. aureus strains and compared them with 7,529 reference bacterial genomes to construct a better picture of resistance evolution in S. aureus. Our findings reveal that the gene distribution and phyletic pattern of SCCmec elements in Staphylococcus species are significantly different from its core genes and which itself indicating that they are evolved in a different fashion. Our results show that LGT (Lateral Gene Transfers) plays a crucial role in shaping the evolution of SCCmec and non-SCCmec encoded resistant genes in S. aureus.
Materials and Methods

Data:
Completely sequenced 7,681 bacterial genomes (protein sequences) and its feature tables were downloaded from National Center for Bioinformatics Information (NCBI) Genbank database (July 2017)(ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/bacteria/). This includes 152 S. aureus strains from different hosts (Human, Bovin, Chicken, Swine) isolated from different geographic regions and 7,529 bacterial reference genomes ( Supplementary table 1) .
Pan-genome reconstruction and functional annotation of genes:
Homologous genes were obtained from a total of 424,469 genes encoded in the chromosomes and plasmids of 152 S. aureus genomes. An all-against-all genomes BLASTp (Altschul et al. 1997 ) was performed with e-value <1e-05, ≥30% amino acid BLAST identity and ≥70% query coverage followed by reciprocal best BLAST hits families. There were 1,764 genes, which don't have any significant homolog in 152 strains were classified as unique cases. 192 paralog genes, which skipped the MCL clustering pipeline, were manually added back to the existing clusters using a best cluster match criteria. Altogether 4,565 protein families and 1,764 unique genes were converted into a binary matrix of presence/absence patterns (PAPs). Based on the distribution pattern of clusters within S. aureus strains, its pan-genome is classified into three categories (genes present in >=90% strains as core genome, gene presence in >1%-<90% of strains as auxiliary genome and presence of a gene in a single strain as unique). The S. aureus pan-genome is summarized as a presence absence matrix where x-axis represents protein families and unique elements; where y-axis represents 152 S. aureus strains. If a protein family or unique gene is present in any of the S. aureus strain, its corresponding entry is represented with "1"; otherwise, a "0". The presence absence matrix was sorted in ascending order of its distribution and 
Identification of resistance related clusters (SCCmec encoded and non-SCCmec encoded)
Based on the classification proposed by International Working Group on 
Classification of MRSA, MSSA and SCCmec typing
Reference MecA sequence was BLASTed against 152 S. aureus strains and based on presence or absence of homolog (e-value <1e-05, query coverage ≥70 and BLAST identity ≥90) strains were classified as resistant and sensitive. Similarly, ccr allotypes of known SCCmec reference strains were BLASTed and the presence or absence of homologs based on best hits (BLAST identity ≥80% and query coverage of ≥70%, evalue <1e-05), strains were classified into 12 SCCmec types (I to XII).
Phylogenetic tree reconstruction and comparison:
16S rRNAs, SCCmec and antibiotic resistant genes with their homologs were aligned separately using MAFFT (Katoh et al. 2002) with the options -localpair, -maxiterate = 1000 and the alignment confidence score is evaluated using GUIDANCE2 server LGT inference Any topological discordance between gene trees and 16S rRNA reference tree at the species level is considered as potential LGTs. In 177 resistant gene trees, we condensed OTUs at species-level and compared the nearest neighbor relationship of each OTU with the reference tree ( Supplementary table 4 ). A gene is considered as acquisition in S. aureus if it is absent in nearest neighbors and widely distributed in distantly related species, which are different from its reference phylogeny. Taxa that are branching within S. aureus clade are considered as exports from S. aureus.
16SrRNA phylogeny used for the comparison is provided in Figure 3 and its newick tree (including all OTUs) is provided in Supplementary table 4 Results
Characteristics of S. aureus pan-genome
A total of 428,070 genes encoded in the genomes of S. aureus strains were clustered into 4,565 protein families and 1,764 unique genes using Markov Chain Clustering procedure (Enright et al. 2002) . The S. aureus pan-genome is open (α<1) and appears to be moderately expanding with the inclusion of new genomes (Figure 1 , Figure S1 ). The openness of pan-genome and presence of mobile genetic elements in S. aureus is similar to many other bacterial species, for example E. coli that is known to have an essential capability of acquiring new genes into its genome via inter- aureus was found to have a different functional distribution as compared to that of the core genome. Unlike unique and core genomes, the auxiliary genome of S. aureus is more involved in cellular processing and signaling functions, which is similar to other bacterial species (Ozer et al. 2014 ). Also, a large proportion (12%) of auxiliary and unique genes in S. aureus have been identified as mobile genetic elements like transposons and prophages genome (replication, recombination, and repair) ( Supplementary table 2 ). S. aureus showed an extensive genetic diversity, with an average 27% of genes being specific for a single strain. were nearly universally distributed among the selected 152 S. aureus strains ( Figure   2 , Figure S2 ). The cassette flanking gene viz. orfX was found to be universally (99%) present in S. aureus strains, clearly indicating its importance in the integration of these elements. The mec-box genes (MecA-MecR-MecI), which are the decisive elements in SCCmec, are predominantly distributed among 152 S. aureus strains (73%, 73%, and 64% distribution respectively). The ccr-box genes that code for recombinases are widely distributed within resistant strains of S. aureus (66%). Some of its allotypes such as ccrAB4 and ccrB1 occur at a lower frequency (1.3%) when compared to other ccr allotypes namely ccrA1 and ccrB2. In addition to the mec-box and ccr-box genes, various IS elements and genes associated with the mobility of SCCmec were also found to be widely distributed within S. aureus (Figure 2) . Figure S7a ).
Evolution of SCCmec elements in S. aureus

As expected, frequent
LGTs were detected on plasmid-encoded genes in S. aureus which includes important genes such as mupA, aadA5, tetL that are responsible for resistance against mupirocin, aminoglycoside and tetracycline respectively ( Figure S7b Figure   S7b ). Moreover, some genes such as dfrA and mupA were acquired it from the distantly related lineages in Bacillales.
Conclusions
To better understand the evolution of antibiotic resistance-related genes in S. 
Discussion
For better insights on the resistance evolution in S. aureus, we have considered completely sequenced bacterial genomes (all publically available) in our analysis that is a limiting factor in the previous studies. The gene sharing patterns of S. aureus strains were not uniform between different resistant types; for example, Type IV strains are sharing more genes each other, but having less number of genes in its SCCmec element than others ( Figure S1 ). Interestingly, we noticed that the 16S rRNA reference phylogeny of S. aureus strains shows no monophyletic structure in terms of its resistant gene presence or SCCmec type, indicating that S. aureus lineages were not primarily evolved toward the resistant trait (Figure 2a) T1  T2  T3  T4  T5  T8  T9  T11   Unchar   T12 non-S. aureus species 
Figure legends


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