**3. Biofilm formation in** *S. aureus* **population**

Biofilm production in *S. aureus* is comprised of three-steps. In each step, there is distinct bacterial physiology with expression of different sets of genes [46]. These steps can be described as follows: (i) initial attachment; (ii) colonization; and (iii) dispersion [47, 48]. In the initial attachment step, bacterial cells attach to the surfaces (6 h–11 h). This step is characterized by active metabolism of the bacterial cells and higher production of adhesion factors. In maturation step, the biofilm production is increased due to bacterial multiplication (18 h). During this step, metabolically active cells and slow metabolism cells both are present and subject to QS signals gene expression changes. At this step, persister cells can be found here. In the third and last step, upon finding the favorable conditions, the metabolically active cells separate from the colonies and begin to function as free cells [49]. Gene expression changes also force the bacteria to decrease the biofilm production [50]. Biofilm production in *S. aureus* is a complex phenomenon that secures this pathogen from environmental stress factors.

#### **3.1 Role of outer surface proteins**

Outer surface proteins play a very important role in initial adhesion and helps bacteria to adhere any surface; playing an important part in beginning of biofilm formation. Previous studies have focused on human isolated bacteria and whole

proteome comparison of biofilm and planktonic states of *S. aureus* [47, 51]. But recent studies have shown that MRSA can also form a varying form of protein based biofilm that is not present in other *S. aureus* bacteria [52]. This difference includes the biofilm components, outer surface protein expression and encoding operons. For example, *S. aureus* can produce two types of biofilms (i) ica-operon dependant/ Polysaccharide intercellular adhesion-based biofilm (ii) ica-operon independent biofilm [13, 53]. Ica-operon independent biofilm is important for persistence in Hospital Associated infections (HA-MRSA) that is structurally different to the former type of biofilm. Hence drugs designed for the former type of biofilm might be not suitable for this type. This implicates that the drugs designed for other *S. aureus* biofilms will not be effective for native or highly antibiotic resistant strains.

Surface proteins are mainly classified into structural based classified groups (i) microbial surface component recognizing adhesive matrix molecule (MSCRAMM) (ii) Near iron transporter (NEAT) motif proteins. (iii) Three-helical bundle proteins (iv) G5–E repeat family (v) Structurally uncharacterized proteins [54]. Among these the last group is least studied and has potentially important proteins such as biofilm associated protein (bap). In this group, SasL and SasD proteins are also included that are expected to have important role in pathogenicity and biofilm formation [55]. But there are still no studies regarding gene mutation and characterization. Furthermore, all the proteins in this fraction are never studied for their role in ica-independent biofilm formation.

#### **3.2 Quorum-sensing regulation system and biofilm formation**

In Staphylococci spp., accessory gene regulator (agr) system acts as a main quorum sensing (QS) regulating system. Another QS regulation system i.e., luxS regulating system is also present but its role is less significant in the physiology of this bacteria [56]. Autoinducing peptide (AIPs) forms basis of agr-mediated QS system and acts as main signal peptides that regulates the biofilm formation and virulence. The main functions of agr mediated QS system are to sense the bacterial cell density in the surrounding environment and to respond with genetic adaptations.

*S. aureus* possess four main genes in the agr-operon such as agrA, agrB, agrC, agrD and divergent transcriptional units, such as RNAII and RNAIII, with promoter-2 (P2) and promoter-3 (P3), respectively. agrD gene in this operon produces a small oligopeptide that further undergoes maturation and transported in extracellular environment via agrB [57, 58]. These mature oligopeptides act as AIs in the extracellular environments. After reaching a certain threshold value, these AIs interacts with extracellular segment of histidine kinase, agrC. This agrC acts as a transmembrane receptor which activates the kinase leading to phosphorylation of agrA response regulator; resulting in the expression of biofilm related genes [59]. This activated agrA regulates the promoters P2 and P3 that further activates or deactivates transcriptional units.

It has been determined to maintain a balance between production of virulence factors and biofilm formation. The *agr* based QS system plays a major role in the dispersion step of biofilm formation [60]. Because agr system activation supports the free-living and more mobile lifestyle. On the other hand, its deactivation supports the colonization and sessile lifestyle. Therefore, *agr* mutants are shown to form a higher biofilm production as compared to the wild type. As mentioned, this increased biofilm production and thickness is associated with the inability of cells in dispersion from the mature biofilm. Thus production of factors that stops the bacteria to enter into mobile phase and not due to cell growth or death [61]. However, this agr-QS system needs a deep understanding of pathways and mechanisms.

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

#### **Figure 1.**

*Regulatory networks in biofilm formation. Sigma factor B (SigB) inhibits agr expression, while SarA has been shown to directly enhance it [62].*

#### *3.2.1 Role of alternative sigma B (sigB) operon and agr operon*

Alternative sigma B (sigB) factor-regulated genes include those involved in general stress response, virulence, capsule formation, and biofilm formation (**Figure 1**) [62]. sigB operon is composed of *rsbU, rsbV, rsbW,* and *sigB* genes. It represses the agr operon that is important in depressing the biofilm production. Disruption of any gene from this operon could result in mal-function and enhance the biofilm production. Recently, this operon found to be playing an important role in counterfeiting the oxidation stress in *S. aureus* that are very important risk factors for mastitis infections.

### **4. How genetic diversity affects the biofilm production**

Biofilm forming ability is a variable characteristic of Staph aureus that can categorize the bacteria into different categories such as level of biofilm formation, certain STs with high biofilm formation and types of biofilm formation (discussed earlier).

### **4.1 Relation of MLST with biofilm production**

There is a proposal that genetic diversity could affect the biofilm production. A recent study has demonstrated that MLST types such as ST59 and ST188 isolated from human and canine sources were found to be associated with strong biofilm production [15]. This shows that the biofilm production capacity is strongly affected by evolutionary process that changed the biofilm production among different strain types. Parallel evolution could vary the biofilm production by introducing new mutations. But the genes and pathways in specific sequence types related to biofilm production affected by parallel evolution are not well understood. Further studies are underway to reveal this relation. As discussed previously, parallel evolution could help in emergence of new strains but its relation to biofilm production is still unknown.

#### **4.2 Level of biofilm formation**

Level of biofilm formation is another complex mechanism that shows the diversity among the strains and within the member of strains [46]. There are multiple estimations that can explain these variations [46, 50]. But most importantly these variations in expression of genes are associated with the environmental signals [63]. For instance, some bacterial cells in same colony can produce PNAG to capture water [13]. On the other hand, some pathways like c-di-AMP respond to external environmental chemicals such as glucose and drop in biofilm formation is measured [64]. Biofilm formation and eDNA release from bacterial cells are triggered by significant reduction in c-di-AMP levels and this reduction is related to low *agr* operon expression [65]. Importantly, *gdpP, xdrA* and *apt* genes also play important role in biofilm formation [66]. Although this pathway shifting is notified but environmental factors that drive this reduction in agr operon expression are still under study.

#### **4.3 Types of biofilm**

*S. aureus* biofilms can be classified as ica-dependent and ica-independent based on their matrix composition. Biofilm matrix composition in ica-dependent biofilms is synthesized by the icaADBC operon that is composed of polysaccharide intercellular adhesion (PIA) or polymeric N-acetylglucosamine (PNAG). On the other hand, ica-independent biofilms are further consisting of three types of biofilms based on their biofilm matrix. Protein/e-DNA biofilm, Fibrin biofilm, and Amyloid biofilm are included in this ica-independent classification (**Table 1**). There is an interesting comparison of biofilm types among MSSA and MRSA isolates also exists. It was reported that ica-dependant biofilm was more common in MSSA while icaindependent biofilms were more frequently observed among MRSA isolates [67]. It is possible that multiple types are present at same place [47]. *S. aureus* biofilms can be found everywhere in body after inoculation. These biofilms could of different types with different EPS, places of origin, and genes/operon controlling them.

#### **5. Role of biofilm environment itself in SCV generation**

Biofilm acts like a micro-environment with its own conditions and stressors. There are many studies demonstrated that chronic cases with biofilm formation for a certain period of time also cause the mutation in genomes via natural selection or parallel evolution [68–71]. This reshaping of genome could result in non-synonymous mutations or shortening of genome. In chronic mastitis, *S. aureus*


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

#### **Table 1.**

*Comparison of different types of* S. aureus *biofilms. Polysaccharide type biofilm is only considered as icadependent biofilm while all the remaining are considered as ica-independent biofilms.*

also form biofilm and remains sub-clinical for very long time that could be helpful in causing non-synonymous mutations. Non-synonymous mutations often also involve the introduction of stop codons that disrupt the gene leading to nonfunctional or pseudogene formation. Loss in gene function irreversibly changes the phenotype of bacterium. This newly formed phenotype could be more antibiotic resistant, highly biofilm forming or reduced metabolic form of persisters [72–74]. This phenotypic variation should be considered during therapeutic developments and treatment regimes. Hence, it is necessary to study and mimic those conditions to understand which genes undergo mutation formation.

#### **6. Role of SCVs in persistence**

Biofilm formation helps the *S. aureus* to persist and multiply sub-clinically in inhospitable environment. As mentioned earlier, the Small Colony Variants (SCV) phenotype are found potentially responsible for the sub-clinical and chronic infections. Such SCVs phenotypes share some common features of slow-growth and quasi-dormancy with low virulence potential [75]. SCVs further express some distinctive features such as small colony formation, a dormant metabolism, less enzymatic activities, and elevated antibiotic resistance [76]. Such SCVs are mostly point mutations in the important genes. Therefore, during proof-reading mechanisms, SCVs can be return to a wild-type (WT) or converted to a different phenotype. Later, clinically observed phenotypes are stable and permanent genetic changes showing irreversible SCVs. Such irreversible SCVs are examples of parallel evolution or evolution with-in population [77]. External environmental stress factors can also trigger the emergence of SCVs such as reactive oxygen species, low pH, cationic peptides, limited nutrition and bacterial biofilm competition [78, 79].

SCVs can be generated spontaneously under any sub-clinical and chronic disease condition. Considering bovine mastitis as an example, SCVs will be discussed now. The detection of mastitis origin SCVs, especially permanent genetic changes within population, in routine laboratories and their accurate studies in research laboratories are challenges not overcome yet. Among these mastitis studies, such isolates were also found positive for biofilm producing genes i.e., ica operon, adhesive proteins, bap operon [80]. According to a study based on different food samples, approximately 72% of the isolates produced biofilms. As discussed above, biofilm producing *S. aureus* are really important in chronic and sub-clinical mastitis infection. Moreover, a few studies have also studied the SCVs formed and found that SCVs formed can cause different level of mastitis based on their severity. Another study has also pointed out the isolation of *S. aureus* irreversible mutation variant from dairy cows in Yunnan province that was responsible for chronic mastitis [26]. This mutation was found in thymine related pathway that promotes the resistance against Sulfamethoxazole (SXT) and helps the bacterium to develop in fibrotic conditions. Similarly, a Beijing based study described the slow growth, antibiotic resistance and chronic mastitis as features of isolated irreversible thymidine SCV [81]. Most of the studies have focused on the antibiotic resistance profiles of *S. aureus* isolated from mastitis infection. On the other hand, there are very few studies that determined the SCVs and relation of chronic sub-clinical mastitis. Additionally, in Austria, a study related to chronic mastitis also revealed that irreversible mutations in rsbU, one of sigB genes, generated from SCVs caused the bacterium to persist and resist the therapy [16]. Further, SCVs related to regulatory circuits have also been revealed such as agr genes, hemin (hemB), menadione (menD), α-Toxin (hla), γ-Hemolysin (hld), Coagulase (coa), L-lactose dehydrogenase (ldh), Alcohol dehydrogenase (adh), Arginine deiminase (arcA), Capsular biosynthesis (capA) and Alkaline shock Proteins (Asp) [82]. Some experimental studies have reported the induction of SCVs by growing *S. aureus* with antimicrobial peptides, and magnesium ions (Mg+2). Further these studies have also mentioned the need of in vivo experiments for complete understanding. This indicates that there is lack of animal experiments based comprehensive studies explaining the factors of this within population and parallel evolution.
