**2.3 Mechanisms of tetracycline resistance**

Three different tetracycline resistance mechanisms have been described:


All these mechanisms are based on the acquisition of one or several tetracycline resistant determinants, which are widely distributed among bacterial genera [30]. Additionally, mutations in the rRNA, multidrug transporter systems or permeability barriers may be involved in developing resistance to several antibiotics including Tetracyclines [31].

Efflux of the drug occurs through some export proteins from the major facilitator super family (MFS). These export proteins are membrane-associated proteins which are coded for by *tet* efflux genes and export Tetracycline from the cell. Export of Tetracycline reduces the intracellular drug concentration and thus protects the ribosomes within the cell.

Ribosome protection proteins that protect the ribosomes from the action of Tetracyclines [32] are cytoplasmic proteins. They are similar to elongation factors EF-Tu and EF-G that bind to the ribosome and cause changes in ribosomal conformation. This prevents Tetracycline from binding to the ribosome, without altering or stopping protein synthesis. This occurs by a ribosome-dependent GTPase activity, which confers resistance mainly to Doxycycline, Minocycline and a wider spectrum of resistance to tetracyclines than is seen with bacteria that carry tetracycline efflux proteins.

#### *2.3.1 Tetracycline resistance genes*

There are at least 38 different characterized tetracycline resistance (*tet*) genes and three Oxytetracycline resistance genes (*otr*) to date [33]. These genes include 23 genes which code for efflux proteins, 11 genes for ribosomal protection proteins, three genes for an inactivating enzyme and one gene with unknown resistance mechanism. Most environmental *tet* genes encode for transport proteins, which pump the antibiotic out of the bacterial cell and keep the intracellular concentrations low to make the ribosomes function normally [34]. The most common genes found in *S. aureus* are *tet(K), tet(L), tet(M), tet(O).*

*tet (K) gene* is a mobile genetic element originally detected in *S. aureus* plasmids of pT181 family [35]. It is a 4.45-kb plasmid protein consisting of 459 amino acids and belongs to the incompatibility group inc3 [36]. PT181-like plasmids have also been detected either integrated in the large plasmids or in the bacterial chromosome. They are always flanked by directly repeated insertion sequences of the type IS257 [37].

*tet (L) gene* carrying plasmid pSTE1 was identified in *Staphylococcus hyicus* in 1992. In 1996, tet(L) was also found to be carried on the naturally occurring plasmid pSTS7 of *Staphylococcus epidermidis* [38]. It is the second most prevalent tetracycline resistant gene in Streptococci and Enterococci [39]. It consists of 458 amino acids.

*tet (M) gene* is the most widely distributed tetracycline resistant gene in grampositive bacteria [40]. It was first identified in *Streptococcus spp*. Subsequently, it has been isolated in a large number of gram-positive and gram-negative bacteria, including Mycoplasmas and Ureoplasmas [40]. The *tet(M)* gene is frequently associated with conjugative transposons of the Tn916-Tn1545 family [41, 42]. which also carry additional antibiotic resistance genes. According to the study of Schmitz et al. [34], *tet(M)* is the most prevalent single tetracycline resistance determinant in MRSA (Methicillin Resistant *Staphylococcus aureus*). The majority of *tet(M)*-positive *S. aureus* isolates also carry *tet(K).* Hence, MRSA isolates are typically of *tet(M) or tet(K,M)* genotype [43].

*tet (O) genes* also have been detected very rarely in Staphylococci.

## **2.4 Mechanisms of macrolide resistance**

Macrolides inhibit protein synthesis by stimulating dissociation of the peptidyl-tRNA molecule from the ribosomes during elongation. This results in polypeptide chain termination and a reversible stoppage of protein synthesis. The first described mechanism of Macrolide resistance was due to post-transcriptional modification of the 23S rRNA by the adenine-N6 methyltransferase. These enzymes add one or two methyl groups to a single adenine (A2058 in *Escherichia coli*) in the 23S rRNA moiety. Over the last 30 years, a number of adenineN6-methyltransferases from different species, genera, and isolates have been described. In general, genes encoding these methylases have been designated *erm* (erythromycin ribosome methylation), although there are exceptions, especially in the antibioticproducing organisms. As the number of *erm* genes described has increased, the nomenclature for these genes has varied and has been inconsistent. In some cases, unrelated genes have been given the same letter designation, while in other cases, highly related genes (90% identity) have been given different names [33].

#### *2.4.1 Macrolide resistance genes*

Although structurally unrelated to each other, Macrolides, Lincosamide, and Streptogramin, are often investigated simultaneously for microbial resistance, as some Macrolide resistance genes (*erm*) encode for resistance to two or all three of these compounds. In total, more than 60 different genes conferring resistance to one or more of the MLS antibiotics have been identified, including genes associated with rRNA methylation, efflux and inactivation.

The *erm (A) gene* is associated with the transposon, Tn554. It is integrated into SCCmec II elements, and is a non-conjugative or conjugative transposon. It is mostly seen in Methicillin resistant staphylococci [43].

The *erm (B) gene* is seen in transposons Tn917/Tn551. It is 2.3 and 4.4 kb in size and does not carry additional resistant genes [44].

The *erm (C) gene* is commonly located on small plasmids. It is widely spread in Methicillin susceptible strains [45].

The **msr (A)** gene is efflux- pump mediated, codes for 488 amino acids, ABC transporters system and is encoded by plasmid borne *msr (A)* genes [46]. It is an ATP-binding transport protein which mediates the active efflux of 14-membered ABC transporters system and confers resistance to Macrolides and B-compounds of the Streptogramins.

#### **2.5 Aminoglycosides resistant** *S. aureus*

Aminoglycosides are broad spectrum antibiotics that inhibit protein synthesis of the bacteria. They were first isolated from the Actinomycetes spp. namely

*Antibiotic Resistant* Staphylococcus aureus *DOI: http://dx.doi.org/10.5772/intechopen.100057*

*Streptomyces griseus* and introduced for clinical use in 1944. They were used as the first-line drugs worldwide but were replaced by Cephlaosporins, Carbapenems and Flouoroquinolones due to lesser toxicity and broader coverage than Aminoglycosides [47]. Members of these groups include Neomycin, Amikacin, Gentamicin, Netilmicin, Tobramycin, Kanamycin etc. The novel Aminoglycosides recently developed, namely Arbekacin and Plazomicin were meant to overcome the Aminoglycoside resistance mechanisms [48]. Clinical studies reported a higher incidence of nephrotoxicity in patients on Aminoglycosides. Hence, screening the patients for serum urea and creatinine after injection of Aminoglycosides is important to monitor the severity of the toxic effects. Aminoglycosides have got a substantial activity against *S. aureus* infections including MRSA, VISA, and VRSA [47].

Entry of Aminoglycosides inside the bacteria mostly comprises of three distinct stages [49]:


Aminoglycoside resistance mostly occurs by


The major mechanism of aminoglycoside resistance among both gram negative and gram positive clinical isolates is the enzymatic modification of amino or hydroxyl group of these antibiotics. Three families of enzymes are responsible in performing co-factor dependent drug modification:


These are further subdivided into many types (designated by Roman numerals). AAC (6′)-I enzymes are aminoglycoside acetyltransferases, modifying the antibiotic at position 6′ [50, 51].

Aminoglycoside resistance in clinical strains of *S. aureus* is due to the acquisition of cytoplasmic Aminoglycoside Modifying Enzyme (AME) by plasmids. For example, Gentamicin and Neomycin resistance is confered by bifunctional Acetyl Transferase –Phosphotransferase (aac-aphD) encoded by Tn4001.

Neomycin resistance occurs by aphA encoded adenyl transferase which is encoded by PUB 110 or Tn 5405. It is seen in SSC II mec [52].

