**3.2 The β-lactamase enzymes**

The β-lactamase enzymes hydrolyze the amide bond of the four-membered βlactam ring. The β-lactamases are classified into four classes: one zinc-dependent or metallo-β-lactamases (class B) and three active-site serine β-lactamases (classes A, C, and D). The β-lactamases of class C are widely distributed in the *P. aeruginosa* chromosome. The overexpression provides resistance to cefoxitin, cefazolin, most penicillins, cephalothin, and β-lactam inhibitor-β-lactam combinations. The AmpC gene encodes the hydrolytic enzyme β-lactamase of class C. The clinical significance of class C enzymes is enhanced by the presence of some family members, such as FOX, DHA, and CMY enzymes, on mobile genetic elements in Enterobacteriaceae and *P. aeruginosa* [20, 21].

#### **3.3 Aminoglycoside-modifying enzymes**

A common mechanism of resistance to aminoglycoside resistance in *P. aeruginosa* is the antibiotic inactivation by chromosome- or plasmid-encoded modifier enzyme. This mechanism involves three enzymes: the aminoglycoside acetyltransferase (AAC), the aminoglycoside nucleotidyl transferase (ANT), and the aminoglycoside phosphoryl transferase (APH) [22]. The AAC (6<sup>0</sup> ) family of enzymes provides resistance to kanamycin, gentamicin, amikacin, netilmicin, and tobramycin.

The APH family confers inactivation by phosphorylation of aminoglycosides such as streptomycin, neomycin, and kanamycin. The *aphA*-encodes an APH (3<sup>0</sup> )-II-type enzyme, and its *aph* (3<sup>0</sup> )-IIb chromosomal aminoglycoside phosphotransferase gene confers resistance to several aminoglycosides, such as neomycin, kanamycin, amikacin, gentamicin, and tobramycin. The *aph* (3')-IIb gene forms an operon with the AraC-type positive transcriptional regulator (hpaA), and in response to the presence of 4-HPA acid, HpaA promotes the expression of *aph* (3')-IIb and that of the hpa regulon promoting the production of metabolic enzymes for the utilization of 4-HPA [22]. The ANTs family provides the adenylation of aminoglycosides such as gentamicin and streptomycin in resistant strains of *P. aeruginosa* [23, 24]*.*

#### **3.4 Modification of antibiotics target**

In *P. aeruginosa*, the target alteration is commonly developed through mutations over the *par*C gene encoding *gyrA* gene that encoding the DNA gyrase subunit A and the DNA topoisomerase IV subunit A, both genes conferring resistance to fluoroquinolone and are homologous. In addition, 16S rRNA methyltransferases confer high levels of aminoglycoside resistance, such as rmtA, gidB, and rsmG, please refer to the methyltransferases and interactome chapter for more information about methyltransferases [25].

#### **3.5 Global response efflux pumps conferring multidrug resistance**

Multidrug efflux is an important mechanism responsible for multidrug resistance, which limits the therapeutic potential of antibiotics, failing to treat the infection caused by *P. aeruginosa.* Multidrug efflux pumps decrease the intracellular concentration of antibiotics through a series of carrier proteins located in the bacterial cell membrane and periplasm. These proteins act by pumping various foreign substrates outside the bacterial cell, including antimicrobials, toxins, and heavy metals. The

efflux pumps also export virulence determinants, including adhesins (biofilm formation) or other proteins, which are important for colonization in host cells.

The pumps can be classified according to the substrate transporting one substrate or a variety of antibiotics and other structurally different substances. In *P. aeruginosa*, there are five main families of efflux transporters: Major facilitator family (MF), ATPbinding cassette (ABC), family of cell division and nodulation of resistance (RND), family of extrusion of toxic compounds and multidrug (MATE), and small family of multidrug resistance (SMR) [26].

The RND-type multidrug efflux pumps are usually present in Gram-negative bacteria. They are integrated into a tripartite complex that includes an outer membrane channel protein, an inner membrane carrier protein, and a membrane fusion protein that joins the inner membrane protein with the outer membrane protein. In *P. aeruginosa*, several Mex efflux systems contributing to antibiotic resistance have been described, such as MexXY-OprM, MexEF-OprN, MexCD-OprJ, and MexAB-OprM.

The MexAB-OprM efflux pump belonging to the RND family is the principal contributor to multiresistance in *P. aeruginosa,* yielding resistance to a diversity of antimicrobial agents: fluoroquinolones, β-lactams, chloramphenicol, cephalosporins, macrolides, carbapenems, tetracycline, and chloramphenicol. The MexAB-OprM efflux pump is constituted by three components: a membrane fusion protein (MexA), a cytoplasmic membrane transporter (MexB), and an External membrane pore (OprM). The mexAB-oprM operon is regulated by nalC, nalD, and mexR repressor genes. Mutations in these repressor genes can lead to overexpressing of the MexAB-OprM pump causing antibiotic resistance [27–30]. The MexXY-OprM (also referred such as AmrAB) regulated by the *par*R-*par*S genes confers resistance to a broad range of antibiotics: tetracyclines, macrolides, glycylcyclines, fluoroquinolones, β-lactams, chloramphenicol, lincomycin, and novobiocin [24].

#### **3.6 Antibiotic resistance by alterations in the cell wall charge**

Polymixin B and colistin are the last antibiotic resources to treat multidrugresistant Gram-negative bacteria, not only *P. aeruginosa*, but also *Klebsiella pneumoniae* and *Acinetobacter baumannii*. The two-component regulatory systems, PhoP-PhoQ and PmrA-PmrB, modulate resistance to cationic antimicrobial peptides, polymyxin B, colistin, and aminoglycosides. This is in response to low Mg2+ concentrations, regulated by an LPS modification operon arnBCADTEF-pmrE that encodes lipopolysaccharide-modifying enzymes. On the other hand, PhoP-PhoQ twocomponent regulatory system contributes to polymyxin resistance. Yang et al. have identified additional PhoP-regulated genes that contribute to the tolerance to polymyxin B [31].

The PhoQ works as a phosphatase that dephosphorylates the regulatory protein PhoP, binding directly to the promoter region of the oprH-phoP-phoQ operon and promoting its transcription. As a result, in the absence of the phoQ gene, genes regulated by PhoP are constitutively expressed. In addition to its operon, PhoP regulates different genes involved in antibiotic resistance, LPS modification, and antimicrobial peptides. In *P. aeruginosa*, the lipid A can be modified by enzymes encoded by the arnBCADTEF operon. These enzymes covalently add 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A, decreasing the negative charge of lipid A and reducing polymyxin and colistin binding. PhoP-PhoQ and other two-component regulatory systems, CprR-CprS, PmrA-PmrB, BqsR-BqsS, and ParR-ParS, directly control the expression of the arnBCADTEF operon [32–34].

#### **3.7 Porins modulating reduction of permeability to antibiotics**

*P. aeruginosa* expresses multiple types of porins, of which OprF (nonspecific porin and structural outer membrane protein OprF) is the most common and has been involved in diverse functions [35]. The OprD porin (outer membrane low permeability porin-basic amino acids, carbapenem uptake) contains the binding sites for carbapenems, and the absence of OprD in *P. aeruginosa,* increases resistance to carbapenems. The overexpression of the OprH porin as a result of Mg2+ starvation is associated with resistance to polymyxin B and gentamicin through modification of LPS.

Other important porins associated with antibiotics resistance encountered in *P. aeruginosa* are the OprB porins (carbohydrate-selective porin), OprE (outer membrane low permeability porin, OprD family), oprM (outer membrane factor lipoprotein ! OprM of MexAB-OprM, multidrug efflux system), oprJ (outer membrane factor lipoprotein ! OprJ of MexCD-OprJ system, multidrug efflux system), oprN (outer membrane factor lipoprotein ! OprN of MexEF-OprN system, multidrug efflux system) [36–38].

#### **4. Methyltransferases and resistome**

Methyltransferase enzymes are fundamental to developing aminoglycoside resistance in *P. aeruginosa*. Some mechanisms were initially well described in *Escherichia coli*. This bacterium has 10 methylations in 16S rRNA and 14 methylations in 23S rRNA [39]. These mechanisms begin to be solved in *P. aeruginosa* [40] and other bacteria [41]. High-throughput omics such as genomics, transcriptomics, and proteomics, together with bioinformatics, data mining, and statistical analysis, are the perfect blend to identify resistome genes and protein networks.

The RNA methyltransferases carry out epigenetics methylation or demethylation on ribosomal RNA to restrict or allow antibiotic resistance development [42]. In *P. aeruginosa*, the Kam and Kgm families of methyltransferases confer resistance to several aminoglycosides [43]. Loss of native methylation may also confer a resistant phenotype. For example, when comparing the RsmG protein of *P. aeruginosa* with that of *Thermus aquaticus*, high structural homology is observed [43]. On the other hand, other methyltransferase genes have been poorly studied *in P. aeruginosa* as *gid*B (glucose-inhibited division gene). It is known that the *gid*B gene is involved in posttranslational modification methylation of 16S RNA. The *gid*B mutants also show a compromised overall bacterial fitness in *Salmonella* [44].

However, it is not only the development of aminoglycosides resistance by *P. aeruginosa* that has been significant, being a crucial case to be studied. Other methyltransferases have been associated with other antibiotics, such as macrolides comprise erythromycin and azithromycin [45]. The macrolide resistance in *Mycobacterium abscessus* complex (MABc) can develop through chromosomal mutations on the 23S rDNA (rrl) gene that promotes a high resistance level. This process goes through the induction of the *erm* gene, which causes inducible resistance in the presence of a macrolide [46]. For example, in *E. coli* studies, mobile resistance genes encoding Mph phosphotransferases or Erm methyltransferases expressed by their *mph* and *erm* genes, which were discovered in clinical settings, but their origins remain unknown. The *erm* gene from *P. aeruginosa* belongs to the large family UGF311 and confers

**Figure 2.** *Network of interactions of methyltransferases RsmH, RsmG, RsmE, RsmI, and RsmC, among others.*

resistance to erythromycin and azithromycin (3,8 μg ml1 and 3,2 μg ml1, respectively) [45].

The RsmG, RsmH, and RsmI methyltransferases were described initially in *E. coli* with antibiotic resistance. The *rsmG* mutations confer streptomycin resistance in *E. coli*. This methyltransferase methylates the N7 of nucleotide G527 within the 530 loop of 16S rRNA [42]. This methyltransferase is associated not only with aminoglycoside resistance and the collective importance of these rRNA modifications but even involved in protein synthesis [47–49]. However, RsmG was associated with oxidative stress [50] and redox imbalance processes in *Deinococus geothermalis* [51] and *Bacillus subtilis*, promoting low-level resistance to streptomycin, but *rsmG* mutants emerged spontaneously at a relatively high frequency, 106 [52]. In our studies, we did not find evidently relationships between resistance to antibiotics from *rsmG, rsmH,* and *rsmI* mutants [53].

The methyltransferase activity is diverse: antibiotic resistance, mobile genetic elements, and oxidative stress, among others. The resistome for antibiotic resistance and therefore, its interactome encloses a broad genome plasticity [54] and a high diversity of extensive proteomic interactions. For example, RsmG, RsmH, and RsmI methyltransferases interact with other proteins shown in **Figure 2**. It will be complex to establish the wide bacterial resistome; however, omics analysis is going in the right direction.

#### **5. Interactome**

The interactome is defined as the complex network of biomolecules used by a living cell or organism to interact with another living cell or organism [55]. This interaction occurs through proteins essentially, which are the signaling

#### *Searching for the Resistance Interactome of* Pseudomonas aeruginosa *DOI: http://dx.doi.org/10.5772/intechopen.108245*

molecules most commonly used by cells and unicellular organisms such as bacteria. Advances in technology and bioinformatics have made it possible to map the interactome of several species, especially microorganisms, and to find out about their habitats and host adaptation mechanisms [56, 57]. There are different kinds of interactome networks: within the same species, which include protein-protein interactions, and also within different species, as in the case of interactions between bacteria. Bacteria can develop resistance and adaptation capabilities that give them advantages over their hosts due to mechanisms such as horizontal gene transfer [58, 59].

The pathogen-host interactome has been well studied from genome-wide analysis. For instance, it has been found that the versatility of many microorganisms, such as viruses, to adapt to host conditions, depends on the sequestration of endogenous host protein-protein interfaces. Like in many microorganisms, it tends to mimic or hijack host interfaces rather than generate new ones due to the limited innovative nature of microorganisms. For this reason, evolutionary studies of the interactome between species have revealed that depends on the antagonistic interactions between the pathogen and its host and the pathogen's limited capacity for innovation [60].

The study of interactome interspecies listed model organisms, such as *E. coli*, *Drosophila melanogaster*, *Saccharomyces cerevisiae*, *Caenorhabditis elegans,* and *Arabidopsis thaliana*. These species were the main tools to analyze, within a systemic approach, complex macromolecular networks, designing topological and dynamic properties that reflect biological phenomena [12]. The main networks of proteinprotein interactions were built to a system-level understanding of cellular processes from genomics and proteomics data. The best example today is STRING (*Search Tool for the Retrieval of Interacting Genes/Proteins*), which contains multiple metadata from different sources, such as experimental and computational data, or statistical predictor identification and metadata of scientific collections [61]. String also contains the networks of antibiotic-resistant genes and proteins from the well-known *P. aeruginosa* PAO and ATCC 10145 strains and other strains and bacteria species.

*P. aeruginosa* has been used to investigate interactome too. Trouillon et al. analyzed three different strains of *P. aeruginosa* to identify Hfq-interacting RNAs, which are one the most relevant systems in the mRNA translation and degradation in bacteria. They identified growth phase-specific and strain-specific Hfq targets, including previously undescribed sRNAs, and found that the accessory Hfq interactome includes most mRNAs encoding Type III Secretion System (T3SS) components, secreted toxins, and a cluster of CRISPR guide RNAs [62].

Interactomics has a problem when the studies tend to focus on single reference strains and assume that the discoveries can be applied to the entire species, forgetting the critical intra-species genetic diversity. It will be vital to focus the interactomics studies including or enriching the number of strains to be correlated with other species. In *P. aeruginosa*, it is true that the best studies are developed with reference PAO strain; however, interesting studies have begun to change this point of view [62, 63].

#### **6. Methyltransferases and interactome**

DNA methylation is a major epigenetic factor influencing gene expression in prokaryotes and eukaryotes. DNA methylation is carried out by the enzymes known as methyltransferases, which involve the addition of a methyl group to cytosines at the

5'-position to form 5-methylcytosines [64] and also, the addition of a methyl group to adenines at the 6'-position to assemble N6-methyladenine (6mA) [65]. Dcm (DNA cytosine methyltransferase) is one of the methyltransferase enzymes discovered in prokaryotes. This, as first depicted in *E. coli*, is highly conserved in the different strains of this bacterium and reduces the expression of ribosomal protein genes during the stationary phase [66].

Methyltransferases are part of the network of biomolecules that make up the bacterial interactome. In the case of 6mA, which is part of the defense systems against phages and plasmids, it allows bacteria to distinguish between their own DNA and that of the invader, thus generating a complex network of biomolecules that allow the bacterial cell to mark (methylate) its DNA and protect it from the digestion carried out by restriction enzymes, which degrade the invading DNA that is not methylated [67].

Likewise, methyltransferases are among the molecules that play a relevant role in the interaction of bacteria with their hosts. Some studies reported that *Helicobacter pylori*, a pathogen related to the development of gastric cancer, can induce DNA hypermethylation in human gastric mucosal epithelial cells by manipulating some gene pathways associated with early steps of oncogene expression [68, 69]. Ali et al. found a link between the Rv1523 methyltransferase of *Mycobacterium tuberculosis* and its contribution to virulence, related to antibiotic resistance. In this study, they found that Rv1523 methyltransferase catalyzes the transfer of methyl group from S-adenosyl methionine (SAM) to the cell wall components of *M. tuberculosis*, modifying their components and conferring antibiotic resistance and survival under stress and acidic conditions [70].

In *P. aeruginosa*, the involvement of methyltransferases in the bacterial interaction with the host and its ability to adapt to host conditions as antibiotic pressure was largely reported. For example, the 16S rRNA methyltransferase enzyme, along with other enzymes, has been shown to be responsible for aminoglycoside resistance, acting through the irreversible transfer of methyl groups to the amino or glycoside groups of these antibiotics [71].

Bacterial epigenetic marks are currently generating a lot of interest from researchers as they are part of the bacterial interactome responsible for antibiotic resistance, for instance. The interaction of multiresistant bacteria with their hosts can cause molecular alterations in the transcriptional programs of host cells involving epigenetic mechanisms such as non-coding RNAs, DNA methylation, and histone modifications conferring survival to the bacteria [72]. However, more studies are still needed to understand in depth the epigenetic mechanisms that are part of the bacterial interactome to find new therapeutic targets to address public health problems such as bacterial multiresistance to antibiotics.

#### **7. Relationship resistome-pangenome**

What is the size of the resistome inside the pangenome? And what is the set of resistome genes? These questions will be the future challenge in the antibiotic's resistance panorama and its relationship with other omics such as pangenomics. The term "pangenome"/pangenome was created by Tettelin et al. in 2005 [73] to describe the complete set of genes of a given species by sequencing the complete genomes of several strains of that species. The pangenome is composed of the core genome, accessory genome, and unique genome.

The core or central genome refers to the set of genes common to all the genomes of the species studied. The accessory or variable genome refers to the collection of genes that are in one or various genomes but not in all of them in each species studied. While the core genome is highly conserved, the second serves to understand the variations in the genomes of the different species studied; thus, their specific lifestyles and evolutionary trajectories. Finally, the unique genome refers to the set of genes with no homologous copy within the genomes under study [16, 74, 75].

The resistome is part of the pangenome and is the set of genes that confer resistance to a bacterial species and that are transmitted from one strain to another by different mechanisms known as part of the global set of genes of that bacterial species. Indeed, according to Gillings, the genes that confer antibiotic resistance are ancient components of the bacterial pangenome, having been recovered from 30,000-year-old permafrost and from a cave microbiome of 4 million years that has been isolated [76]. Therefore, pangenomics analysis is one of the tools available today for in-depth and broad studies of the resistome in pathogenic microorganisms [77].

However, pangenomics faces some fundamental concerns: actually, today it is impossible to solve all the questions using classical molecular biology or descriptive genomics. The core and accessory genome help to figure out the role of some genes inside species. Not all resistance genes are in the core genome, and some strains are part of HGT genes as unique genes, never incorporated in the core genome, giving plasticity to the resistome. For this reason, the study of the resistome through pangenomics analyses remains a challenge for current research that requires new developments to better realize the complex landscape of resistance genes [78, 79].
