**3.1 Resistance to β-lactam antibiotics**

In early 1940's introduction of penicillin improved the outcome cases due to *Staphylococcus* infections but soon penicillin resistance *Staphylococcus* were recognized in early 1942 [20] which among late 1960's reaches to 80% in both community and hospital-acquired staphylococcal isolates with well-established pattern of resistance [21]. Furthermore, blaZ gene is responsible for resistance in *Staphylococcus aureus*, that encodes for β-lactamase an enzyme which is synthesized when *Staphylococcus aureus* is exposed to β-lactam antibiotics by hydrolyzing the β-lactam ring, rendering the β-lactam inactive. blaZ is regulated by the two adjacent genes blaR1 and blaI. The gene blaR1 is anti-repressor and blaI is repressor [22]. For the synthesis of β-lactamase, the signaling pathway involves the sequential cleavage of these regulatory proteins such as blaR1 and blaI where on exposure to β-lactams, blaR1 which is a transmembrane sensor transducer cleaves itself [23, 24], cleaved protein acts as protease that directly or indirectly cleaves the repressor blaI and thus allowing the blaZ to synthesize enzyme [23]. Furthermore, Methicillin, the first semisynthetic penicillin which was resistance to penicillinase, introduced in 1961 and soon followed by the reporting of methicillin-resistance isolates [25]. The spread of Methicillin-resistant *Staphylococcus aureus* (MRSA) has been critical and the infections resulting from MRSA is worse than the infections outcome of methicillin ensitive strains [26]. MRSA isolates like the penicillin resistance strains too carried resistance genes for other antimicrobial agents [27]. For the resistance to methicillin, requires chromosomally localized mecA gene [28, 29], which is a part of large unique mobile genetic element, SCC mec found in all MRSA strains may contain additional genes for antimicrobial resistance [30, 31] is responsible for the synthesis of PBP2a/PBP2′ a 78-kDa protein which binds to penicillin (penicillin-binding protein 2a) [32–34]. Transpeptidation which is necessary for the cross-linkage of peptidoglycan chains is catalyzed by these membranes bound enzymes-PBPs, thought to have appeared and works similar as serine proteases. PBP2a blocks the binding of all β-lactams but allows transpeptidation and because of its low affinity it allows staphylococci to survive even in the high concentration exposure of β-lactam antibiotics. Isolates Resistance to methicillin shows resistance to all β-lactam agents, including cephalosporins [34–36]. In some MRSA strains its resistance mechanism by mecA via the mecI and mecR1 genes is regulated in the manner similar to the regulation of blaZ by the genes blaR1 and blaI when exposed to penicillin [37]. Fem genes (factor essential for resistance to methicillin resistance, also play a role in cross-linking the peptidoglycan strands and contribute in methicillin resistance [38]. Ceftaroline the fifth-generation cephalosporin according to the U.S. Food and Drug Administration (FDA) in 2010 has been considered

superior among other comparator drugs for the treatment of complicated skin and soft tissue infections as well as pneumonia [39]. β -lactam antibiotics bind to other PBPs, named PBP1, −2, −3, and − 4 but in the presence of PBP2a they are unable to bind effectively to their PBP targets. Ceftaroline on other hand is active against MRSA strains because of its high binding affinity for PBP2a as comparison to other β -lactam [40]. Binding of PBPs by ceftaroline block these enzymes to catalyze the transpeptidase function that is important for the synthesis of staphylococcal cell wall [41]. Ceftaroline is generally considered safe and successfully used to treat wide infections alone and in combination with other active drugs often with daptomycin [42]. Several studied over MRSA clinal strains showed these were susceptible to ceftaroline in wide range such as >98.4% in North America [43], >83.3% in Latin America [44], >83% in Europe [45], 78.8% in Asia/South Pacific countries [46] the variation in resistance among MRSA may be due to the variation in geographical distribution of strains around the world [47, 48]. MRSA strains carry mobile genetic element known as SCCmec, which carries mecA gene [40]. Ceftaroline resistance is usually due to the nonsense mutations in mecA, resulting in amino acid sequence change in PBP2a hence a target protein mutation [49]. Glu447Lys mutation in mecA in presence of ceftaroline on SF8300 USA300 MRSA strain yields low level resistance isolates whereas COL common laboratory strain showed high ceftaroline resistance due to mutations in pbp2, pbp4 and gdpP not due to mecA [50]. There are strains developing resistance with no change in mecA [51].

#### **3.2 Resistance to vancomycin**

Vancomycin, a lipopeptide antibiotic approved by Food and Drug Administration of the United States in 1958 found in recent years that the MRSA isolates are resist to it [52]. Vancomycin works by binding to bacterial cell envelopes and inhibiting their cell wall synthesis instead of targeting protein like other antibiotics [53]. It binds to C-terminal D-Ala–D-Ala residue of the pentapeptide to inhibit the cross-bridge formation between pentapeptide and pentaglycine preventing cell wall synthesis [54]. MRSA strains shows different ranges of resistance against vancomycin according to their MIC and are named accordingly such as MRSA showing complete resistance to vancomycin is termed vancomycin-resistant *Staphylococcus aureus* (VRSA), showing medium resistance is termed as vancomycin intermediateresistant *Staphylococcus aureus* (VISA) and least resistance as VSSA [55].

Failure in vancomycin treatment of MRSA results due to formation of intermediate-resistant isolates namely hetero resistant vancomycin-intermediate *Staphylococcus aureus* (hVISA) and vancomycin intermediate *Staphylococcus aureus* (VISA) [56] which includes features such as cell wall thickening, reduced autolytic activit and reduced growth rates [57]. Several studies found that the mutation in genes VraS(S329L), MsrR(E146K), GraR(N197S), RpoB(H481Y), Fdh2(A297V) and Sle1(67aa) were also responsible for vancomycin resistance in VISA strain Mu50 [58]. Other genes involving in high- and low-level resistance to vancomycin includes vanA, vanB, vanD, vanF, vanI, vanM, encodes for D-Ala:D-Lac ligases whereas vanC, vanE, vanG, vanL, and vanNgenes encoding D-Ala:D-Ser ligases (**Figure 2**) [59, 60].

#### **3.3 Resistance to lipopeptide based antibiotic daptomycin**

The only approved and available lipopeptide in the US in the year 2003 with in vitro bactericidal activity and an alternative to vancomycin for various MRSA infections, is daptomycin [61]. However, during the treatment, the emergence of non-susceptible MRSA strains for daptomycin has been reported [62, 63]. Even

*Mechanistic Insights of Drug Resistance in* Staphylococcus aureus *with Special Reference... DOI: http://dx.doi.org/10.5772/intechopen.100045*

#### **Figure 2.**

*Molecular mechanism of* Staphylococcus aureus *resistance toward penicillin and vancomycin.*

before the approval of drug, Silverman et al. observed daptomycin non-susceptible mutants and identified number of changes such as increase in membrane fluidity, increase in net positive charge over the surface, decrease in susceptibility to daptomycin-induced depolarization and low in surface binding of daptomycin in the cytoplasmic membrane of non-susceptible strains [64, 65]. Though the basis for reduction in susceptibility to daptomycin in MRSA strains has not been fully clarified [66]. The transfer and addition of positively charged lysine molecules to phosphatidyl glycerol in the cell membrane associated with the activity of enzyme lysyl-phosphatidyl glycerol synthetase is encoded by mprF gene [67], Mutation in mprF gene causes an increase of lysyl-phosphatidyl glycerol in the outer layer of the cell membrane, leading to an increased positive charge resulting in reduced susceptibility to daptomycin [68]. mprF mutations are the most common type of mutation in MRSA strains with reduced susceptibility to daptomycin (**Figure 3**) [69]. Several more genes are also identified which are associated with the reduced susceptibility to daptomycin such as dsp1 or asp23. The inactivation of these genes leads to reduced daptomycin susceptibility and the overexpression of single or both of the genes leads increase in susceptibility [70] whereas expression of dltA gene contributes to the staphylococcal net positive surface charge [71]. Kanesaka et al. using transmission electron microscopy, found that the some of the strains which were exposed to daptomycin which shows resistance developed an increase in the thickness of their cell wall and their thickness decreases on revert to daptomycin susceptible [72].

#### **3.4 Resistance to aminoglycosides**

Aminoglycosides works by mistranslation and changing the conformation of tRNA during bacterial protein synthesis by binding to A-site present on 16S rRNA of the 30S ribosome. Some even acts by inhibiting initiation /or elongation phase thereby blocking bacterial protein synthesis [73]. Most common mechanism of resistance to aminoglycosides especially in *Staphylococcus aureus* includes Aminoglycoside modifying enzymes which works by acetylating, phosphorylating, or adenylating amino or hydroxyl groups therefore inactivating aminoglycosides. Hundreds of aminoglycosides modifying enzymes are known encoded by genes which are commonly found on plasmids and transposons [74]. On clinical practising with some

#### **Figure 3.**

*Molecular mechanism of* Staphylococcus aureus *resistance toward daptomycin via mprF.*

aminoglycosides such as gentamicin, tobramycin, and amikacin these three among Aminoglycoside modifying enzymes such as ANT(4=) nucleotide transferase, bidomain AAC(6=)le-APH(2=)la acetyltransferase and phosphotransferase, and APH(3=)IIIa phosphotransferase which are common in MRSA isolates with varied appearance, shows resistance [75]. Plazomicin, a synthetic aminoglycoside showed in vitro activity against 55 MRSA isolates that expressed one or more aminoglycosidemodifying enzymes [76] and has no protection against other resistance mechanism such as 16 s rRNA methyltransferases that modifies the aminoglycoside target site but these enzymes are not reported in S. aureus (**Figure 4**) [77].

### **3.5 Resistance to oxazolidinones**

Oxazolidinones, the synthetic antibiotics blocks the formation of functional 70S initiation complex thereby preventing bacterial protein synthesis. Linezolid and tedizolid types of drugs from Oxazolidinones works interrupting transitional RNA positioning by binding to the bacterial 23S rRNA at the ribosomal peptidetransferase center. Even with the similarity in both of the structure tedizolid still

*Mechanistic Insights of Drug Resistance in* Staphylococcus aureus *with Special Reference... DOI: http://dx.doi.org/10.5772/intechopen.100045*

shows increased and better interactions at the binding site with increased potency [78]. All these resistance mechanisms make alteration to oxazolidinone binding site, most common are the point mutations occurring in the genes encoding for 23S rRNA mostly in the central loop of domain V [79]. S. aureus has four to seven copies of 23S rRNA gene collection of which determines the effect and degree of linezolid resistance [80, 81]. This kind of mutation, G2576T, in all five copies of its 23S rRNA gene has been found in the first clinical isolates of linezolid-resistant MRSA [82] are most common. Mutations in the genes which are encoding for L3 and L4 similar to mutation in 23S rRNA, induces a change in the linezolid binding site shows linezolid resistance. Studies showed structural rearrangement of the linezolid binding site due to deletion of one amino acid in L3 causing change in the position of several of the 23S rRNA bases as targeted by point mutations. Gene cfr (chloramphenicol-florfenicol resistance) linked with various mobile genetic elements also shows resistance to linezolid and other antibiotics by change in the drug binding site at the ribosomal peptide-transferase center by encoding a rRNA methyltransferase that causes change in position A2503 [83–85]. Several bacterial species port the cfr gene, a reservoir for drug resistance. MRSA isolates with cfr genes are more likely have additional antibiotic resistance genes as compared to non-cfr gene isolates. Another gene, optrA found commonly symbiosis with cfr gene in MRSA isolates also shows resistance to oxazolidinones [84]. Acts as an ATP-binding cassette transporter, which mediate the influx and efflux of drugs. Another optrA structurally similar gene poxtA first identified in MRSA isolates, shows in vitro resistance to oxazolones [86–89].

### **3.6 Resistance to quinolones with a focus on novel antibiotic delafloxacin**

The fluoroquinolones (FQ ) were first introduced into clinical practice in the year 1962 along with the development of Nalidixic acid. Fluoroquinolones (FQ ) are class of fully synthetic antibiotics which are active against a broad range of gram positive and gram-negative bacteria and have a pivotal role in multidrug resistance therapy in Mycobacterial infection (Tuberculosis and non-tuberculosis). To treat acute bacterial skin and skin structure infections (ABSSSIs) with both enteral and intravenous preparations FDA approved non zwitter ionic FQ delafloxacin in 2017 [90]. Due slower MICs against S. aureus than other FQs delafloxacin has a higher barrier to resistance, it can serve as ant staphylococcal drug as monotherapy. Delafloxacin is found to be effective against multiple like Streptococcus pneumoniae, anaerobic bacteria Legionella, *Chlamydia pneumoniae*, *Neisseria gonorrhoeae*, Mycoplasma spp., in addition to *Staphylococcus aureus*. Its activity against the enterococci is variable [91]. Delafloxacin shows a property of "dual-targeting" in which it can form complexes with DNA and topoisomerase IV or DNA gyrase. Double strand break can be produced by the inhibiting the one or both the enzymes which results in the death of bacterial cell as they lack enzymes that can repair double strand break in DNA. Delafloxacin shows more potency against Gram positive bacteria as it shows anionic behavior at neutral pH due to the substitution of the R7 position (3-hydroxy-1-azetidinyl) [90, 92]. An anionic behavior of delafloxacin makes diffusion and accumulation of drug within the bacteria more readily as it is retained in bacterial cell for longer duration at neutral intracellular pH [93]. These characteristics makes antibiotics more effective in acidic environments [94]. Depending upon the ambient pH it shows activity against biofilm related infections and intracellular infections [91]. Estimated concentration of Delafloxacin selecting resistant mutant is 8 to 32 times lesser than for other Fluoroquinolones. This difference is due to the drugs dual targeting mechanism of action. Point mutations are method by which resistance is shown by bacteria, resistance occurs due to point mutations in target enzyme or by the action of efflux pump. Point mutation in ParC

subunit of topoisomerase IV results in resistance in case of *Staphylococcus aureus*. Delfatoxin resistance occurs due to various mutations in the target regions of topoisomerase IV [92–95]. Resistance to the FQs, including delafloxacin, often involves point mutations in the target enzymes or the action of efflux pumps in bacterial cells. In S. aureus, resistance is usually mediated by point mutations in the ParC subunit of topoisomerase IV. Delafloxacin often retains potency against S. aureus resistant to other FQ drugs due to target gene mutations or modifications. This relative resistance seems related to the structure of delafloxacin (perhaps due to C-7 and C-8 substitutions); delafloxacin resistance occurs only with several mutations in the target regions of topoisomerase IV. NorA, NorB, NorC, MdeA, QacA, and QacB includes a resistant phenotype of Common S. aureus efflux pumps active against Fluoroquinolones. The antiseptic chlorhexidine gluconate is also removed from cells by the plasmid-encoded efflux pumps QacA and QacB, sometimes called antiseptic resistance genes and their acquisition in a S. aureus population is co-selected by use of chlorhexidine or FQs. Delafloxacin is not as active substrate for typical *Staphylococcus aureus* efflux pumps compared to other drugs in the class [96–99].
