**2. Mechanism of genetic diversity in** *S. aureus*

The architecture of *S. aureus* population is mainly based on its genetic markers. Its genome consists of a single chromosome of 2.7–3.1 Mbp [17]; mainly represents the core genome that undergoes the vertical evolution. While accessory genome is dominated by mobile genetic elements (MGE) that include plasmids, transposons, phages and insertion sequences (IS) [18]. Horizontal evolution in MGE is driving the genetic diversity in this fraction of genome. Therefore, diversity in *S. aureus* population include a highly varying accessory/disposable genome with variable distribution of antimicrobial resistance (AMR) genes, virulence factors (VF), sequence types (ST), and clonal complex (CC)-specific pathogenic potentials [19]. The causes of genetic diversity among *S. aureus* strains are: vertical evolution (mutation) [20] and horizontal evolution (transformation, conjugation, transduction, and transposition) [18].

#### **2.1 Vertical evolution and genetic diversity**

The majority of *Staphylococcus aureus* population has highly conserved core genome [21, 22] that has evolved mainly through mutations. This conserved core genome can further undergo to single nucleotide polymorphisms and SCV formation. Such mutations are detected by MLST of selected housekeeping genes (*arc, aroE, glpF, gmk, pta, tpi, yqiL*) or through whole-genome sequencing that helps the researchers to identify the phylogenetic relations in *S. aureus* populations. According to the pubmlst database, there are 632,297 allele sequences in the 35,804 isolates. Furthermore, there are total 6569 MLST types divided into 10 clonal complexes including the untypeable clonal complex [4]. However, mutations in core genome points at the continuous evolution of this bacterial pathogen.

Previous studies have mentioned the synonymous and non-synonymous mutations in bacterial genomes as two main types of mutations [23]. Synonymous mutations are mostly less diversifying and cause least impact due to presence of amino acids against different pairs of codons and introduction of amino acid from same groups [24]. Thus, these mutations are considered as mostly harmless for

#### *Genetic Diversity in* Staphylococcus aureus *and Its Relation to Biofilm Production DOI: http://dx.doi.org/10.5772/intechopen.99967*

bacterial physiology. The other mutation type is non-synonymous mutation that causes gene rupturing by introducing a stop codon that further leads to significant changes in bacterial physiology [25]. Among *S. aureus* population, nonsynonymous mutations generate the irreversible small colony variants (SCVs) that play main role in this genetic diversity [26]. Irreversible mutations introduced in such variants are mainly shaped by parallel evolution and are generated due to environmental stress factors such as cationic peptides, oxidative stress, low pH, bacterial competition [27]. These SCVs have attributes of high biofilm formation, antibiotic resistance and low metabolism that reduce the cure rates [16, 28, 29]. Human joint infections, cystic fibrosis in lungs, and bovine chronic mastitis are some common examples [30]. Studying the underlying mechanisms is important to assess the physiological processes and genetic diversity.

Some recent reports have determined that *S. aureus* also undergo genome reduction similar to mycobacterium spp. [31]. Genome reduction is mainly caused by removal of the ruptured genes and pseudo genes that eventually shortens the genome size [31]. A recent example of such genome reduction in *S. aureus* is isolates from ST-228 that are believe to lose 522 genes in their history of evolution [21]. It can be estimated that persistence of *S. aureus* in an environment for very long time causes genome reduction. The possible reason behind this is the least utilization of genes that are not required in that particular environment [31]. In other words, these proteins could have evolved to fulfill specific nonessential innovations and hence could easily be lost in reductive evolutions. Such complex genetic diversity also points at the continuous evolution of this *S. aureus*. A deep understanding of the mechanisms behind this evolution and genetic diversity is required.

## **2.2 Horizontal evolution and genetic diversity**

The accessory genome is highly diverse among *S. aureus* populations [9]. It mainly encodes proteins necessary for bacteria's adaptation to various environmental conditions via resistance genes or virulence factor [14]. Such exogenous genes are often shared by other bacteria/environment therefore containing different rate of G-C in as compared to the core genome [32]. Generally, these exogenous genes can be obtained through one or multiple ways of horizontal evolution such as transformation, conjugation, transduction, and transposition. The mobile genetic elements (MGEs) are responsible for such kind of genes transfer. MGEs prevalent in *S. aureus* population include plasmids, transposable elements, bacteriophages, and pathogenic islands [18]. A deep knowledge of these MGEs and their mobility methods are of great concern for understanding the horizontal evolution.

### *2.2.1 Plasmids and their role in genetic diversity*

Plasmids are small self-replicating DNA molecules (ranging 1–60 kbp) that can be transferred from one bacterium to other [18]. *S. aureus* has three classes of plasmids based on their sizes and other properties. Class I plasmids include small sized (<4.6 kbp) but multicopy plasmids often with a single resistance determinant [33]. Such type of plasmids is never reported to bear transposons or prophages [34]. Class II plasmids are of intermediate size (15–46 kbp) with lesser number of copies as compared to class I [33]. But some of the plasmids included in this class are antibiotic resistance plasmids such as pencillinase and aminoglycoside resistance plasmids [35]. In addition, there are different resistances genes do present on this kind of plasmids. Class III consisted of large and complex plasmids with determinant of transfer (*tra*) by conjugation along with different combinations of resistant markers [36]. Such plasmids also possess few transposons and insertion sequences.

*Staphylococcus aureus* strains commonly resist against Penicillin and glycopeptides such as vancomycin [15]. The resistance to methicillin is commanded by the *mecA* gene, responsible of the 76 kDa penicillin binding protein (PBP) synthesis. This protein with a low affinity to β-lactams is called PBP 2′ or PBP 2a [37]. The *blaZ* gene encodes for β-lactamase in *S. aureus* strains and both the two adjacent regulatory genes *blaI* (repressor) and *blaR1* (antirepressor) control this gene [38]. There are five different phenotypes of resistance genes (*vanA*, *vanB*, *vanC*, *vanD* and *vanE*) to vancomycin in enterococci [39]. *vanA* and *vanB* resistance operons in the plasmids possess the Tn1546-like and Tn1547 transposon elements [40].

#### *2.2.2 Transposable elements (Tn) and insertion sequences (IS)*

The genome of *S. aureus* carries heterogeneous MGE. The mobile genetic elements contain insertion sequences (IS), transposons (Tn), and transposon-like elements [40]. These mobile genetic elements are involved in evolution of bacteria and these can be found on chromosomes as single or multiple copies. MGE can also be found in association of other genetic elements.

IS sequences are the segments of DNA which can be transposed from one site of genome to another [18]. The genetic information required for their transposition is carried by these transposable elements. They are responsible for the recombination and stabilization of some genes which are responsible for resistance, though they do not code for resistance. These IS sequences are responsible for inducing changes in the expression levels of chromosomal genes and thus are very important in the process of evolution of the bacterial genome [41]. IS sequences can affect the transcription of other genes which are nearby, either by direct insertion or by polar effect, in order to inactivate them. IS sequences which also contain some other genes are called as composite transposons i.e. Tn4001 and Tn4003 which are composite transposons are known to contain IS256 and IS257 respectively which mediate resistance to gentamycin (Gmr), kanamycin (Kmr), and tobramycin (Tmr) [18]. IS256 and IS257 on styphylococcal chromosome have been observed in both contiguous and independent form. It suggested that these genetic elements in the genome may have a role in molecular rearrangements. The circular chromosome of *S. aureus* contains two copies of IS257. The recombination of either of IS257s of the plasmid (pJ3356) mediating ertgromycin resistance, in the pOX7 has been observed.

Transoposons present in staphylococcal genome are relatively small and they carry genes for resistance. Tn552 carries 'bla' gene for pencillinase and Tn554 carries gene for resiatance against spectinomycin, erythromycin and mactolidelincosamide-streptogramin B [18, 40]. These elements are present in staphylococal cassette chromosome, plasmids or on the chromosome in multiple copies. Two copies of transposon 554 (Tn544) are commonly observed in N315, Mu50 and MRSA252 genome, while three addition copies were reported in N315 genome [33]. A unique conjugative transposon i.e., Tn5801 that carries '*tetM*' gene mediating resistance to tetracycline and minocycline was found in Mu50 genome. The single copies of transposons which are larger than 18 kbp are rare to find relatively. They encode genes mediating resistance to tetracycline, trimethoprim, aminoglycosides, or vancomycin. A specific transposon is present on the penicillinase plasmid (pl524) which carries methicillin resistance gene.

#### *2.2.3 Bacteriophages and* S. aureus *diversity*

The presence of mobile genetic elements, especially prophages, help to determine the diversity of *S. aureus* species [34]. Both the horizontal and vertical

#### *Genetic Diversity in* Staphylococcus aureus *and Its Relation to Biofilm Production DOI: http://dx.doi.org/10.5772/intechopen.99967*

evolutions are closely linked to phages. In horizontal evolution, the phages being a mobile genetic element can be transferred to the recipient bacterial cell present in the environment. The prophages carry the many accessory genes in their genomes that are responsible for staphylococcal virulence factors and help in the survival of certain *S. aureus* strains [34, 42, 43]. The phages aid in the genomic island induction and its transfer. Additionally, phage transduction also transfers plasmids and chromosomal markers. Phages, in this way, diversify the *S. aureus* population and directs the horizontal evolution.

Currently, *S. aureus* strains isolated from non-human mammals are being sequenced and studied. Such strains have been shown adaptation to different host species through mutations in the core genome and through potential phage-encoded virulence genes [34]. Recent examples are the cattle-associated strains that were shown to originate in humans [43]. Furthermore, isolates from birds were shown to possess Sa3int phages with unique genes [44]. Therefore, phages are believed to be the one of the tools for host-diversification.

The phages are often regarded as selfish elements even though bacteria are utilizing them for their own survival. In this context, lysogeny could only serve as a short-term strategy of evolution. There are many reports indicating that phages provide *S. aureus* with additional genes that allow them to survive and persist. Several genes are the examples of introduction such as Panton-Valentine leukocidin (*lukSF*), exfoliative toxin A (*eta*), cell wall anchored *SasX* protein and the immune evasion group (IEC) composed of enterotoxin S (*mar*), staphylokinase (*sak*), the chemotaxis inhibitor protein (*chp*) and the inhibitor of the staphylococcal complement (*scn*) [45]. Such gene transfers between species and between different strains is limited due to receptor modifications in restriction barrier and phage exclusion. These effects most likely play an important role in species diversification of staphylococci. Hence, deeper insights into phage biology will be beneficial in understanding bacterial evolution.
