**2. Antibiotic resistance**

*P. aeruginosa* has a large genome among *Gamma proteobacteria*, which allows it to improve many resistance mechanisms in a versatile way, for example, by transmissible plasmids or integrons. *P. aeruginosa* derepresses the chromosomal AmpC cephalosporinase [12, 13]; it also acquires genes for AmpC enzymes, class A carbenicillinases or *β*-lactamases, class D oxacillinases, and class B carbapenem-hydrolyzing enzymes [14], as it occurs in other bacteria like *E. coli* and *K. pneumoniae*. Other mechanisms include modifying the structure of topoisomerases II and IV to become quinolone resistant [15], decreasing outer membrane permeability by the partial or total failure of OprD proteins [12], overexpressing the active efflux systems with broad substrate patterns [16, 17], or synthesizing aminoglycoside-modifying enzymes as adenylyltransferases, acetyltransferases, and phosphoryltransferases [18].

The range of antibiotic resistance in *P. aeruginosa* is wide, and it represents a major difficulty for health care by its unsuccessful treatment, as a consequence of its low intrinsic antibiotic susceptibility, an effect of the interaction between multidrug

*Pseudomonas aeruginosa - An Armory Within*

target, but bacteria can acquire resistance by modification of drug-binding sites. More than 50 years of studies in *Escherichia coli* have shown that 16S and 23S rRNAs have methylated nucleotides (**Figure 1**). These molecular modifications are performed by methyltransferases (MTases), which take in charge the transfer of a methyl group from a methyl donor S-adenosyl-l-methionine, better known as AdoMet or SAM [2]. These RNA MTases are diverse in posttranscriptional RNA modification, where single RNA nucleosides are chemically transformed. SAM-dependent MTases are involved in biosynthesis, signal transduction, protein repair, chromatin regulation, and gene silencing [3]. More recently, it was shown that aminoglycoside resistance in *E. coli* has its primary target within the decoding

*Ribonucleotides methylated where the methyl moiety is located either in 16S or 23S ribonucleotides: m6*

*G [70], m3*

*using the figures on the next web page, https://mods.rna.albany.edu/mods/modifications/search/. The structure shows* 

*U [67, 71], m5*

*G [66], m7*

*clearly the methyl (CH3-) but in the last structure bottom does not show this methyl.*

*A [66, 67],* 

*U [72], and Ψ [68]. Molecules are designed* 

**52**

*m6*

**Figure 1.**

*2A [68], m5*

*C [67], m4*

*Cm [69], m2*

efflux pumps, like *mexAB*, *mexXY* [19], *AdeABC*, and *AdeDE* genes [17]. Another factor is its efficient capability of acquiring resistance, developed by transfer of horizontal genes, such as specific gene mutations [20], and finally by the low permeability of the cellular membrane [16, 21].


#### **Table 1.**

*P. aeruginosa's rRNA methyltransferases and their point of modifications [23]. The columns are described as follows: first column, the name protein of RNA methyltransferase; second column, the name of its gene; third column, the substrate either 23S or 16S RNA or tRNA; fourth column, the type of nucleotide methylated; fifth column, the electron in nucleotide methylated; sixth column, the ligand for everyone SAM; and seventh column, the UniProt code. Some interesting proteins such as RsmA, RsmG, RsmH, and RsmI are marked in bold.*

**55**

**Figure 2.**

*cefotaxime, erythromycin, and nitrofurantoin.*

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance*

worldwide, being annotated for *P. aeruginosa* in UniProt database [23].

Iran patients with *P*. *aeruginosa*, which the *int1* integron was prevalent [25].

Bacterial multidrug resistance (MDR) is an important concern in *P. aeruginosa* since this microorganism is capable of mixing several mechanisms, transposons, plasmids, and chromosomally encoded genes, such as methyltransferases or pumps [22]. Methyltransferase genes are spread in bacterial genome ready to trigger antibiotic resistance. **Table 1** compares the reported methyltransferase proteins

One mechanism of adaptation which facilitates natural selection in bacteria is the hypermutation of some genes or chromosomal regions. Previous work in patients with *P. aeruginosa* showed that hypermutation causes a problematic effect during a chronic respiratory infection (CRI) [24], where *P. aeruginosa* was up to 6.5-fold higher in mutator backgrounds. Other elements associated with high antimicrobial resistance are integrons. These elements were found among isolates from

Integrons linked to transposons, plasmids, and chromosome are responsible for bacterial antibiotic resistance [26, 27]. Integrons are composed of three elements: (1) the integrin-associated promoter (Pc), which is required for transcription and expression of gene cassettes (genetic elements that encode antibiotic resistance genes) within the integrin; (2) the *intI* gene, in which coding for the integrase IntI is crucial for site-specific recombination; and (3) the adjacent recombination site *attI*, which is recognized by integrase. On the other hand, *P. aeruginosa* carrying transposon Tn1696 is an element that encodes the *CmlA* gene, an exporter of the major facilitator (MF) superfamily which provides antibiotic resistance, specifically against chloramphenicol [28]. *P. aeruginosa* has a broad spectrum in cephalosporin resistance mechanism, mediated by the extended-spectrum β-lactamases (ESBLs). High prevalence of multidrug resistance in burn patients and production of *oxa-10*, *per-1*, and *veb-1* genes by *P. aeruginosa* isolates confirm the presence of antibiotic-degrading enzymes [29].

The Pathosystems Resource Integration Center (PATRIC) is a massive database that integrates genomic data and analysis tools to support biomedical research on bacterial infectious diseases. The platform provides an interface for biologists to discover data and information and conduct comprehensive comparative genomics and other analyses in a one-step source. PATRIC database provides complete genome information and data regarding susceptibility or resistance [30] to several antibiotics; including aminoglycosides, polymyxin B, colistin, ceftazidime, piperacillin, imipenem, ciprofloxacin, levofloxacin, and meropenem in *P. aeruginosa*. We report

*Resistance and susceptibility profile of P. aeruginosa against a broad spectrum of different types of antibiotics. The data were downloaded from PATRIC database selecting aminoglycosides, beta-lactamases, cephalosporins, licosamides, fluoroquinolones, colistin, doxycycline, ciprofloxacin, nitrofurantoin, and cefazolin. Many strains are resistant to a wide range of antibiotics (red with a larger percentage), and the most strains are susceptible to colistin (green with larger percentage); on the other hand, the overall strains are resistant to ampicillin,* 

*DOI: http://dx.doi.org/10.5772/intechopen.85185*

#### *The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance DOI: http://dx.doi.org/10.5772/intechopen.85185*

Bacterial multidrug resistance (MDR) is an important concern in *P. aeruginosa* since this microorganism is capable of mixing several mechanisms, transposons, plasmids, and chromosomally encoded genes, such as methyltransferases or pumps [22]. Methyltransferase genes are spread in bacterial genome ready to trigger antibiotic resistance. **Table 1** compares the reported methyltransferase proteins worldwide, being annotated for *P. aeruginosa* in UniProt database [23].

One mechanism of adaptation which facilitates natural selection in bacteria is the hypermutation of some genes or chromosomal regions. Previous work in patients with *P. aeruginosa* showed that hypermutation causes a problematic effect during a chronic respiratory infection (CRI) [24], where *P. aeruginosa* was up to 6.5-fold higher in mutator backgrounds. Other elements associated with high antimicrobial resistance are integrons. These elements were found among isolates from Iran patients with *P*. *aeruginosa*, which the *int1* integron was prevalent [25].

Integrons linked to transposons, plasmids, and chromosome are responsible for bacterial antibiotic resistance [26, 27]. Integrons are composed of three elements: (1) the integrin-associated promoter (Pc), which is required for transcription and expression of gene cassettes (genetic elements that encode antibiotic resistance genes) within the integrin; (2) the *intI* gene, in which coding for the integrase IntI is crucial for site-specific recombination; and (3) the adjacent recombination site *attI*, which is recognized by integrase. On the other hand, *P. aeruginosa* carrying transposon Tn1696 is an element that encodes the *CmlA* gene, an exporter of the major facilitator (MF) superfamily which provides antibiotic resistance, specifically against chloramphenicol [28]. *P. aeruginosa* has a broad spectrum in cephalosporin resistance mechanism, mediated by the extended-spectrum β-lactamases (ESBLs). High prevalence of multidrug resistance in burn patients and production of *oxa-10*, *per-1*, and *veb-1* genes by *P. aeruginosa* isolates confirm the presence of antibiotic-degrading enzymes [29].

The Pathosystems Resource Integration Center (PATRIC) is a massive database that integrates genomic data and analysis tools to support biomedical research on bacterial infectious diseases. The platform provides an interface for biologists to discover data and information and conduct comprehensive comparative genomics and other analyses in a one-step source. PATRIC database provides complete genome information and data regarding susceptibility or resistance [30] to several antibiotics; including aminoglycosides, polymyxin B, colistin, ceftazidime, piperacillin, imipenem, ciprofloxacin, levofloxacin, and meropenem in *P. aeruginosa*. We report

#### **Figure 2.**

*Pseudomonas aeruginosa - An Armory Within*

ability of the cellular membrane [16, 21].

RlmN *rlmN* 23S rRNA

and tRNA

**RsmA** *rsmA* 16S rRNA A1518 and

TrmA *trmA* tm/tRNA U54 in tRNA

TrmH *trmH* tRNA(Leu) Wobble

TrmJ *trmJ* tRNA C32, U32, and

TrmL *trmL\_2* tRNA(Leu) Wobble

**Protein Gene Substrate Nucleotide** 

efflux pumps, like *mexAB*, *mexXY* [19], *AdeABC*, and *AdeDE* genes [17]. Another factor is its efficient capability of acquiring resistance, developed by transfer of horizontal genes, such as specific gene mutations [20], and finally by the low perme-

**methylated**

A2503 in rRNA and A37 in tRNA

A1519

and U341 in tmRNA

nucleotide

A32

nucleotide

*P. aeruginosa's rRNA methyltransferases and their point of modifications [23]. The columns are described as follows: first column, the name protein of RNA methyltransferase; second column, the name of its gene; third column, the substrate either 23S or 16S RNA or tRNA; fourth column, the type of nucleotide methylated; fifth column, the electron in nucleotide methylated; sixth column, the ligand for everyone SAM; and seventh column, the UniProt code. Some interesting proteins such as RsmA, RsmG, RsmH, and RsmI are marked in bold.*

TrmB *trmB* tRNA G46 N7 SAM Q9I6B3 TrmD *trmD* tRNA G37 N1 SAM Q9HXQ1

TrmI *trmI* tRNA A58 N1 SAM A0A2X4FJT8

RsmB *rsmB* 16S rRNA C967 C5 SAM Q9I7A9 RsmC *rsmC* 16S rRNA G1207 N2 SAM A6VC20 RsmD *rsmD\_2* 16S rRNA G966 N2 SAM A0A0F6U8H1 RsmE *rsmE\_2* 16S rRNA U1498 N3 SAM A0A0F6U8B3 **RsmG** *rsmG* 16S rRNA G527 N7 SAM A6VF42 **RsmH** *rsmH* 16S rRNA C1402 N4 SAM A6VB93 **RsmI** *rsmI* 16S rRNA C1402 2'-O-ribose SAM Q9HVZ3 RsmJ *rsmJ* 16S rRNA G1516 N2 SAM Q9HXW0

RlmB *rlmB* 23S rRNA G2251 2'-O-ribose SAM Q9HUM8 RlmD *rlmD* 23S rRNA U1939 C5 SAM Q9I525 RlmE *rlmE* 23S rRNA U2552 2'-O-ribose SAM A6VCK9 RlmF *rlmF* 23S rRNA A1618 N6 SAM A6V0S3 RlmG *rlmG* 23S rRNA G1835 N2 SAM A6VC08 RlmH *rlmH* 23S rRNA Ψ1915 N3 SAM A6V0A6 RlmJ *rlmJ* 23S rRNA A2030 N6 SAM Q9HUF0 RlmK/L *rlmL* 23S rRNA G2445 N2 SAM A6V328 RlmM *rlmM* 23S rRNA C2498 2'-O-ribose SAM A6V7T6

**Position of methylation**

C2 Radical

SAM

N6 SAM Q9I5U5

C5 SAM A6VCH5

2'-O-ribose SAM A0A0H2ZHL8

2'-O-ribose SAM A0A0H2ZF87

2'-O-ribose SAM A0A0G5X8M9

A6V0V7

**Ligand UniProt**

**54**

**Table 1.**

*Resistance and susceptibility profile of P. aeruginosa against a broad spectrum of different types of antibiotics. The data were downloaded from PATRIC database selecting aminoglycosides, beta-lactamases, cephalosporins, licosamides, fluoroquinolones, colistin, doxycycline, ciprofloxacin, nitrofurantoin, and cefazolin. Many strains are resistant to a wide range of antibiotics (red with a larger percentage), and the most strains are susceptible to colistin (green with larger percentage); on the other hand, the overall strains are resistant to ampicillin, cefotaxime, erythromycin, and nitrofurantoin.*

#### **Figure 3.**

*Strains with P. aeruginosa's genomes showing susceptibility (green) and resistance (red) against some aminoglycosides (amikacin, gentamicin, and tobramycin) and other antipseudomonal antibiotics. Many strains are resistant to a wide range of antibiotics (red with a larger percentage), and most strains are susceptible to doxycycline or colistin (green with larger percentage).*

in **Figure 2** antibiotic resistance or susceptibility from different *P. aeruginosa* strains as well as in **Figure 3** with those antibiotics mentioned.

### **3. Resistance to antibiotics through rRNA methylation**

Kgm and Kam families are two different groups of SAM-dependent RNA methyltransferases, which modify nucleotides of 16S rRNAs in the specific drug-binding site to confer self-resistance in aminoglycoside-producing bacteria [31]. The Kgm and Kam families have been distinguished based on their nucleotide targets, G1405 and A1408, respectively. The *kgmB* and *armA* genes (Kgm family kanamycin gentamicin methyltransferase) methylate m7 G1405(N7) position that confers a high level of resistance against gentamicin, kanamycin, and tobramycin. The addition of a methyl group in this position interferes directly with the binding to the antibiotic, inducing a steric hindrance between the modified base and the structure of the antibiotic, causing electrostatic repulsions derived from the positive charge in the modified base [32]. On the other hand, the *kamA* and *npmA* genes (Kam family kanamycin-apramycin methyltransferase) methylate m1 A1408(N1) position conferring a high level of resistance to kanamycin, apramycin, and neomycin [5].

Another interesting non-aminoglycoside resistance related to RNA methylation is the macrolide-lincosamide-streptogramin-B (MLSB) antibiotics, which is strongly associated with the expression of the methyltransferase of ErmC RNA that causes the dimethylation of the N-6 atom of adenine and interacts with the nucleotide 2058 in the 23S rRNA. Such antibiotics bind to overlap sites within the 50S ribosomal subunit tunnel near the peptidyl transferase center, either by inhibiting the catalysis directly at the peptidyl transferase site or by acting as a physical barrier to the extension of the peptide chain inside the tunnel [33]. Many more *erm*-type methyltransferase genes have been identified in a wide range of Gram-positive and Gram-negative bacteria. Among them, the *ermB*, *ermF*, and *ermA* genes are transferred by transposons, and the *ermC* gene transferred by plasmids. The family of Erm methyltransferases that mediate the mono- or dimethylation of A2058 consists of approximately 40 different classes of methylases [34, 35].

**57**

linezolid [42].

**in** *P. aeruginosa*

*The Role of Pseudomonas aeruginosa RNA Methyltransferases in Antibiotic Resistance*

The *rsmG* gene encodes a 16S rRNA mRNA which methylates the N7 of nucleotide G527 within the 530 loop of 16S rRNA; one of the main examples is the loss of native methylations that confers a resistance phenotype to streptomycin. Streptomycin interacts with the rRNA in the adduced region (loop 530), and the loss of methylation correlates with a low level of resistance. Although this resistance is at a low dose of antibiotic, the mutation of *rsmG* apparently has a mutator effect which promotes the appearance of a high number of mutants resistant to high doses of streptomycin [36]. Another interesting aspect is that not only methylation generates resistance; cases have been reported where demethylation also promotes resistance. The first and best characterized example is *ksgA* gene (RsmA protein), which encodes the native methyltransferase KsgA or RsmA, responsible for the N6 dimethylation of A1518 and A1519 in the 3′-terminal fork of the 16S rRNA in the 30S rRNA. It was the first resistance to aminoglycosides (kasugamycin) associated with demethylation in the 16S rRNA [37]. It was found that adenine methylated by MTase is far from the binding site of kasugamycin, so this demethylation should lead to a conformational rearrangement which would be associated with the acqui-

Another research showed that preventing adenine methylation from occurring, resistance to kasugamycin can be induced; the base U793 fills the site usually occupied by the methylated adenines and the adjacent bases, A792 and A794, [39]. The phenomena mentioned above give place to a conformational change, causing the union site of Ksg to be blocked by the U793. Accordingly, it can be assured that

Likewise, it was found that the *tlyA* gene in *Mycobacterium tuberculosis* encodes the MTase 2'-O-ribose TlyA responsible for the C1409 methylations in the 16S rRNA and C1920 in the 23S rRNA. The loss of such methylations confers resistance to capreomycin and viomycin, two antibiotics which bind at the interface of the ribosome subunit and are used to help define their binding site. Another example of the absence of methylations in the 23S rRNA is the lack of methylation in U2584 (*E. coli* numbering), which causes resistance to sparsomycin in 23S rRNA

Recent findings regarding intrinsic resistance refer to the Ψ at position 2504 of the 23S rRNA in *E. coli*, where inactivation of the *rluC* gene confers significant resistance to clindamycin, linezolid, and tiamulin [41]. The *cfr* gene was originally discovered in an isolate of a multiresistant plasmid during a follow-up study of chloramphenicol resistance in *Staphylococcus* spp. isolates. The molecular characterization of the resistance led to the gene encoding a methyltransferase that methylated the nucleotide A2503 in the 23S rRNA. In *E. coli* and *S. aureus*, there is a natural methylation of A2503 mediated by the methyltransferase encoded by the *yfgB* gene (*rlmN*). The lack of natural methylation in A2503 confers a slight increase in susceptibility to tiamulin, hygromycin A, sparsomycin, and

**5. rRNA methyltransferases associated with aminoglycoside resistance** 

Methyltransferases have been intensely studied in *P. aeruginosa*, but this is not the case for RNA methyltransferases, particularly those conferring aminoglycoside resistance. Nowadays, we focus our study in *P. aeruginosa* methyltransferases using

this structural change in the helix 24 causes resistance to Ksg [39].

**4. Resistance or susceptibility to antibiotics through rRNA** 

*DOI: http://dx.doi.org/10.5772/intechopen.85185*

sition of antibiotic resistance [38].

*Halobacterium salinarum* [40].

**demethylation**
