**4. Acquired resistance of** *Pseudomonas aeruginosa*

Apart from being resistant to a variety of antimicrobial agents, *P. aeruginosa* develops resistance to anti-pseudomonal drugs as well. This acquired resistance is a consequence of mutational changes or the acquisition of resistance mechanisms via horizontal gene transfer and can occur during chemotherapy [24]. Mutational events may lead to over-expression of endogenous beta-lactamases or efflux pumps, diminished expression of specific porins and target site modifications while acquisition of resistance genes mainly refers to transferable beta-lacta‐ mases and aminoglycoside-modifying enzymes (Table 3).


**Table 3.** Resistance mechanisms of *P. aeruginosa* to anti-pseudomonal drugs.

#### **4.1. Resistance to beta-lactams**

Resistance to beta-lactam antibiotics is multi-factorial but is mediated mainly by inactivating enzymes called beta-lactamases. These enzymes cleave the amide bond of the beta-lactam ring causing antibiotic inactivation and are classified according to a structural [25] and a functional [26] classification.

Among the beta-lactams, carbapenems are the most efficient against *P. aeruginosa*. These agents are stable to the hydrolytic effect of the majority of the beta-lactamases including the Extended Spectrum Beta-Lactamases (ESBLs) [27]. For this reason, the enzymes that possess carbapene‐ mase activity, namely the carbapenemases [28], will be discussed separately in this section.

## *4.1.1. Expression of endogenous beta-lactamases*

Resistance to beta-lactams in clinical isolates is commonly due to the presence of AmpC betalactamases [29-36]. Furthermore, the production of AmpC beta-lactamases in *P. aeruginosa* can be induced by a number of beta-lactam antibiotics such as benzyl penicillines, narrow spectrum cephalosporins and imipenem [37]. In fact, this mutational derepression is one of the most common mechanisms of resistance to beta-lactams in *P. aeruginosa* [29,32,33,36].

AmpC enzymes are not carbapenemases, they posses however a low potential of carbapenem hydrolysis and their overproduction combined with efflux pumps over-expression and/or diminished outer membrane permeability has been proven to lead also to carbapenem resistance in *P. aeruginosa* [38].

#### *4.1.2. Acquired beta-lactamases*

Acquired beta-lactamases are typically encoded by genes which are located in transfera‐ ble genetic elements such as plasmids or transposons [39] often on integrons [40-49]. Integrons are genetic elements that capture and mobilize genes [50]. Other genetic elements associated with transferable resistance in *P. aeruginosa* are the mobile insertion sequences called ISCR elements [49,51-53].

Different types of transferable beta-lactamases have been found in clinical *P. aeruginosa* isolates around the world (Table 4).

Among them, carbapenemases are of major clinical importance because they inactivate carbapenems together with other beta-lactams. Ambler class A ESBLs hydrolyze penicillins, narrow- and broad-spectrum cephalosporins and aztreonam [54]. Some TEM and SHV enzymes do not possess broad-spectrum cephalosporinase activity and are called restrictedspectrum beta-lactamases. Class D OXA beta-lactamases are a heterogenous group of enzymes and not all share the same properties. Generally, most of them show a preference for cloxacillin over benzylpenicillin. They confer resistance to amino- and carboxypenicillins and narrow – spectrum cephalosporins even though some of them are ESBLs and a few members of the class present carbapenemase activity [24].

gene cassettes in integrons and are transferable [42]. Interestingly, more resistance genes for other antibiotic classes can be present in the same integrons contributing thus in the develop‐

IMP and VIM type MBLs were first identified in Japan [81] and Italy [82] respectively and have spread though all continents since then. Other metallo-enzymes are more geographically restricted. SPM-1, after causing outbreaks in Brazil [28], has been found in Basel [83] in a single isolate recovered from a patient previously hospitalized in Brazil. GIM-1 and AIM-1 were

ment of a multi-drug resistant phenotype.

**Table 4.** Beta-lactamases found in *P. aeruginosa* isolates.

D 2d OXA

**Ambler molecular class Bush-Jacoby-Madeiros**

**group**

A 2b TEM-1, -2, -90,

2be PER-1, -2

**Enzymes References**

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[10] [53] [59-62]

[10] [63]

[10] [47] [66-76]

[10] [12] [54] [57] [78-80]


VEB-1, -2, -3 TEM-4, -21, -24, -42, -116 SHV-2a, -5, -12 GES/IBC-1, -2, -5,


(CARB-1), CARB-3, CARB-4, CARB-like, AER-1

2f KPC-2, -5 [64,65]


SPM-1 GIM-1 AIM-1 NDM-1

LCR-1 NPS-1

VIM-1, -2, -3, -4, -5, -7, -8, -11, -13, -15, -16,-17, -18

2c PSE-1 (CARB-2), PSE-4

B 3 IMP-1, -4, -6, -7, -9, -10,

C 1 AmpC [77]

#### *4.1.3. Carbapenemases*

*P. aeruginosa* is the species in which all types of transferable carbapenemases, except SIM-1 [55], have been detected. The class B carbapenemases that bear Zn2+ in their active center [56] are the most frequent around the world in *P. aeruginosa* isolates and are called metallo-betalactamases (MBLs). They hydrolyse *in vitro* all beta-lactams except aztreonam and are the major cause of high-level carbapenem resistance. Genes that encode MBLs are commonly found as



**Table 4.** Beta-lactamases found in *P. aeruginosa* isolates.

Among the beta-lactams, carbapenems are the most efficient against *P. aeruginosa*. These agents are stable to the hydrolytic effect of the majority of the beta-lactamases including the Extended Spectrum Beta-Lactamases (ESBLs) [27]. For this reason, the enzymes that possess carbapene‐ mase activity, namely the carbapenemases [28], will be discussed separately in this section.

Resistance to beta-lactams in clinical isolates is commonly due to the presence of AmpC betalactamases [29-36]. Furthermore, the production of AmpC beta-lactamases in *P. aeruginosa* can be induced by a number of beta-lactam antibiotics such as benzyl penicillines, narrow spectrum cephalosporins and imipenem [37]. In fact, this mutational derepression is one of the most

AmpC enzymes are not carbapenemases, they posses however a low potential of carbapenem hydrolysis and their overproduction combined with efflux pumps over-expression and/or diminished outer membrane permeability has been proven to lead also to carbapenem

Acquired beta-lactamases are typically encoded by genes which are located in transfera‐ ble genetic elements such as plasmids or transposons [39] often on integrons [40-49]. Integrons are genetic elements that capture and mobilize genes [50]. Other genetic elements associated with transferable resistance in *P. aeruginosa* are the mobile insertion sequences

Different types of transferable beta-lactamases have been found in clinical *P. aeruginosa* isolates

Among them, carbapenemases are of major clinical importance because they inactivate carbapenems together with other beta-lactams. Ambler class A ESBLs hydrolyze penicillins, narrow- and broad-spectrum cephalosporins and aztreonam [54]. Some TEM and SHV enzymes do not possess broad-spectrum cephalosporinase activity and are called restrictedspectrum beta-lactamases. Class D OXA beta-lactamases are a heterogenous group of enzymes and not all share the same properties. Generally, most of them show a preference for cloxacillin over benzylpenicillin. They confer resistance to amino- and carboxypenicillins and narrow – spectrum cephalosporins even though some of them are ESBLs and a few members of the class

*P. aeruginosa* is the species in which all types of transferable carbapenemases, except SIM-1 [55], have been detected. The class B carbapenemases that bear Zn2+ in their active center [56] are the most frequent around the world in *P. aeruginosa* isolates and are called metallo-betalactamases (MBLs). They hydrolyse *in vitro* all beta-lactams except aztreonam and are the major cause of high-level carbapenem resistance. Genes that encode MBLs are commonly found as

common mechanisms of resistance to beta-lactams in *P. aeruginosa* [29,32,33,36].

*4.1.1. Expression of endogenous beta-lactamases*

resistance in *P. aeruginosa* [38].

38 Infection Control

*4.1.2. Acquired beta-lactamases*

called ISCR elements [49,51-53].

present carbapenemase activity [24].

*4.1.3. Carbapenemases*

around the world (Table 4).

gene cassettes in integrons and are transferable [42]. Interestingly, more resistance genes for other antibiotic classes can be present in the same integrons contributing thus in the develop‐ ment of a multi-drug resistant phenotype.

IMP and VIM type MBLs were first identified in Japan [81] and Italy [82] respectively and have spread though all continents since then. Other metallo-enzymes are more geographically restricted. SPM-1, after causing outbreaks in Brazil [28], has been found in Basel [83] in a single isolate recovered from a patient previously hospitalized in Brazil. GIM-1 and AIM-1 were reported from Germany [41] and Australia [84] and did not spread elsewhere. Finally, the only report for NDM-1 in *P. aeruginosa* was made from Serbia [76].

Diminished expression or loss of the OprD porin is a frequent phenomenon during imipenem

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High-level resistance to fluoroquinolones is mediated by target site modifications. Efflux plays

Gyrase and topoisomerase are comprised by two subunits each. DNA gyrase (GyrA and GyrB) is the main target of fluoroquinolones in *P. aeruginosa*. Consequently, mutations are most common for this enzyme rather than for topoisomerase IV (ParC and ParE) [98-102]. Highly resistant isolates have multiple mutations in *gyrA* and/or *parC* [98,101-103] while mutations

Four efflux pumps contribute to fluoroquinolone resistance: MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM [105] as a consequence of mutational events in their repressor genes [24]. Among these, MexAB-OprM, MexCD-OprJ, and MexEF-OprN have been associ‐ ated to fluoroquinolone resistance in clinical isolates [31,105-107] whereas MexXY-OprM has

Acquired resistance to aminoglycosides is mediated by transferable aminoglycosidemodifying enzymes (AMEs), rRNA methylases and derepression of endogenous efflux

Modification and subsequent inactivation of aminoglycosides is achieved by three deferent mechanisms: (1) acetylation, by aminoglycoside acetyltransferases (AACs), (2) adenylation, by aminoglycoside nucleotidyltransferases (ANTs), and (3) phosphorylation, by aminoglycoside

Genes encoding AMEs are typically found on integrons together with other genes responsible for transferable resistance for other antibiotic classes. This way AMEs be‐ come important determinants for the development of multi-drug resistance in *P. aerugino‐*

Enzymatic families that acetylate the 3 and 6' position of the antibiotic are the most common. Five subfamilies of AAC(3) and two of AAC(6') have been described for *P. aeruginosa,* each

one presenting different preferences for aminoglycoside substrates (Table 6).

a contributing role as well [96,97] and the two mechanisms often coexist [32,98-100].

regarding the other subunits are less frequently encountered [100-102,104].

treatment [95].

**4.2. Resistance to fluoroquinolones**

*4.2.2. Efflux pumps contribution*

**4.3. Resistance to aminoglycosides**

*4.3.1. Aminoglycoside-modifying enzymes*

posphoryltransferases (APHs) [108].

*sa* and other species [24,108,109].

systems [24,108,109].

*4.2.1. DNA gyrase and topoisomerase IV mutations*

only been linked rarely to such type of resistance [106].

Ambler class A carbapenemase KPC was first reported in *P. aeruginosa* isolates in Colombia [64] but KPC-producing *P. aeruginosa* isolates have not been reported from other continents except Latin America. KPCs present high rates of carbapenem hydrolysis and inactivate all other beta-lactams including aztreonam.

Enzymes GES/IBC belong to the same enzymatic class but their carbapenemase activity is not as high as that of the KPCs. It may become important however if combined with diminished outer membrane permeability or efflux over-expression. For *P. aeruginosa*, GES-2 has been reported in South Africa [85] and IBC-2 in Greece [86].

Class D carbapenemases like OXA-198 have been found in *P. aeruinosa* isolates although such findings are rather rare for this species [87]. The most clinically important carbapenemases are summarized in Table 5.


**Table 5.** Clinically important carbapenemases found in *P. aeruginosa* isolates.

#### *4.1.4. Efflux systems over-expression*

Among the various efflux systems of *P. aeruginosa*, MexAB-OprM, MexXY-OprM and MexCD-OprJ play an important role in developing beta-lactam resistance [88]. Between these three, MexAB-OprM accommodates the broadest range of beta-lactams [24], is by far the better exporter of meropenem [24] and is most frequently related to beta-lactam resistance in clinical *P. aeruginosa* isolates [33,89]. The efflux pumps may be over-expressed in some isolates [90] contributing thus, together with other mechanisms in the development of multi-drug resist‐ ance [24].

#### *4.1.5. Diminished permeability*

OprD is a specific porin of the outer membrane of *P. aeruginosa* through which carbapenems (mainly imipenem) enter into the periplasmic space [91]. Diminished expression [92] or mutational loss [93] of this porin is the most common mechanism of resistance to carbapenems [24,94] and is frequently associated with efflux pumps and/or AmpC over-expression [36,38]. Diminished expression or loss of the OprD porin is a frequent phenomenon during imipenem treatment [95].
