**2. β-Lactam resistance mechanisms in** *Pseudomonas aeruginosa*

*P. aeruginosa* has intrinsically higher minimum inhibitory concentrations (MICs) against anti-pseudomonal β-lactams when compared to Enterobacteriaceae, even in the absence of specific resistance determinants. For example, typical MICs for the anti-pseudomonal cephalosporin, ceftazidime, for *Escherichia coli*

are 0.06–0.125 mg/L whereas for *P. aeruginosa* isolates, MICs are in the range of 1–2 mg/L. In the main, when *P. aeruginosa* is resistant to β-lactams, specific mechanisms are at play. These include downregulation of outer membrane porins, expression of intrinsic efflux mechanisms, acquisition of β-lactamase enzymes including carbapenamases such as IMP ("imipenemase"), VIM ("Verona imipenemase") and KPC ("Klebsiella pneumoniae carbapenemase"), hyperproduction of chromosomal β-lactamases ("AmpCs or Pseudomonas-Derived Cephalosporinases, "PDCs") and reduced penicillin binding protein affinity for β-lactams that are not considered "anti-pseudomonal" β-lactams. **Table 1** summarizes the mechanism and resistance determinants responsible (adapted from [1]).

Currently, in the clinical microbiology laboratory, susceptibilities are reported to particular antibiotics depending on the specific sample submitted, e.g., urine, blood, sterile body fluids (pleural, joint, cerebrospinal fluid). At least initially, genotypic testing to determine the presence of specific antibiotic resistance determinants is not performed, and it is left to the clinical infectious diseases expert to reason out the most likely resistance mechanisms based on susceptibility patterns, and to select the most appropriate antibiotic(s) for treatment.

#### **2.1 Outer membrane porin loss (OprD)**

The structure, function and regulation of *P. aeruginosa* porins is complex and has been recently reviewed [2]. Porins are involved in structural and signaling tasks in *P. aeruginosa*, as well as passage of nutrients. The Opr D family of porins is the largest and is subdivided into two groups OccD and OccK. These porins are each regulated through their own sigma factors. Porin loss can be related to formation of OprD containing outer membrane vesicles that are also found in high concentrations in biofilms. Resistance to carbapenems in biofilms may be related to this. Mutations in *P. aeruginosa* that cause oprD (occD1) to not be expressed are linked to imipenem resistance. Other occD1 mutations that do not effect transcription also lead to carbapenem resistance [3, 4]. OprD mutations or loss is often associated with overexpression of efflux pumps (see below) leading to high level resistance to carbapenems, other β-lactams and other classes of antibiotics such as aminoglycosides and fluoroquinolones [5].

In the clinical setting, OprD porin loss is often associated with a resistance phenotype in which one observes resistance to carbapenems including imipenem, but *in vitro* susceptibility to anti-pseudomonal pencillins and cephalosporins. In


*OprD, outer membrane porin D; Mex-multidrug efflux; TEM, class A β-lactamase of E. coli, named for patient in which it was discovered; SHV, sulfhydryl variant of TEM; OXA, oxacillinase; GES, German extended spectrum β-lactamase; VEB, Verona extended spectrum; CTX-M, cefotaximase-München; PER, plasmidic extended spectrum β-lactamase; NDM, New Delhi metallo-β-lactamase; SPM, Sao Paolo metallo-β-lactamase.*

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*Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa*

combination with other resistance mechanisms, such as over-expression of the chromosomal PDC enzymes, or presence of other acquired cephalosporinases such as TEM, SHV and OXA β-lactamases, higher level carbapenem resistance is

As with porins, *P. aeruginosa* possesses a large variety of efflux pumps that perform different roles in the bacteria, but mainly function to extrude harmful substances from the cell. These pumps have been reviewed in [6]. Pumps of importance in carbapenem and other antibiotic efflux are in the resistance nodulation or RND type family and include the MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM multidrug efflux pump systems [7]. Increased expression of these pumps leads to high level carbapenem resistance, often in association with OprD loss or modification. Notably imipenem is not a substrate of the multidrug efflux pumps of *P. aeruginosa* while meropenem is [7]. In the clinical setting, if one notes resistance to meropenem and other β-lactams, except for imipenem, then an efflux mechanism is at play. If resistance is noted to both meropenem and imipenem, but not to other β-lactams, OprD loss or modification is responsible. With resistance to carbapenems as well as other β-lactams, multiple resistance mechanisms can be at play including efflux, intrinsic and acquired β-lactamases and decreased perme-

As in other organisms, of which *Enterobacter cloacae* is the most well-known example, *P. aeruginosa* possesses a chromosomal AmpC β-lactamase also called PDC. Chromosomal β-lactamases likely play a role in cell wall maintenance, as well as degradation of β-lactam antibiotics. As characterized in *E. cloacae* [8], the AmpC cephalosporinases are under the regulation of *amp*R, a LysR type regulatory system [9]. Under normal circumstances, there is low level constitutive expression of the AmpC protein. Upon exposure to β-lactam antibiotics, muramyl pentapeptides are released that displace a repressor protein encoded by *amp*R from the promoter of AmpC. This leads to increased expression of AmpC cephalosporinase. The increased expression of AmpC can occur with exposure to cephamycins like cefoxitin for example. Increased expression of AmpC in *E. cloacae* occurs via a pathway involving NagZ, a N-acetyl-β-D-glucosamindase, or independent of NagZ [8]. The muramyl pentapeptides are also degraded by a cytosolic amidase, Amp D. This leads to re-association of the repressor to the promoter and resumption of normal levels of Amp C expression. There are also insertion sequence mutations in AmpR that can lead to increased expression of AmpC, as well as mutations in AmpD amidases that reduce degradation of muramyl pentapeptides. The regulation of Amp Cs differs somewhat in *P. aeruginosa*, involving 2 pathways that include the lytic transglycosylases Slt, SltB1, MltB and MltF, and PBP 4 in the generation of muramyl peptides [10]. Mutations in PBP4 are associated with higher levels of AmpC expression. Finally there are specific AmpC mutations that can lead to a carbapenemase phenotype in these enzymes, although the significance of this in

terms of clinically relevant carbapenem resistance is unclear [11].

β-lactamases from all four Ambler classes have been described in *P. aeruginosa*, including Class A extended spectrum β-lactamases (ESBLs) of the TEM, SHV,

**2.4 Acquired β-lactamases in** *P. aeruginosa*

observed as well as resistance to other classes of β-lactam antibiotics.

*DOI: http://dx.doi.org/10.5772/intechopen.85151*

**2.2 Efflux pumps in** *P. aeruginosa*

ability (porin loss).

**2.3 Hyperproduction of PDC β-lactamases**

#### **Table 1.**

*β-Lactam resistance determinants in P. aeruginosa.*

*Bulgecins as β-Lactam Enhancers Against Multidrug Resistant (MDR) Pseudomonas aeruginosa DOI: http://dx.doi.org/10.5772/intechopen.85151*

combination with other resistance mechanisms, such as over-expression of the chromosomal PDC enzymes, or presence of other acquired cephalosporinases such as TEM, SHV and OXA β-lactamases, higher level carbapenem resistance is observed as well as resistance to other classes of β-lactam antibiotics.

### **2.2 Efflux pumps in** *P. aeruginosa*

*Pseudomonas aeruginosa - An Armory Within*

determinants responsible (adapted from [1]).

**2.1 Outer membrane porin loss (OprD)**

and to select the most appropriate antibiotic(s) for treatment.

classes of antibiotics such as aminoglycosides and fluoroquinolones [5].

Acquired ESBLs (TEM, SHV,OXA, GES, VEB, CTX-M, PER)

Acquired carbapenemases (KPC, OXA, metallo-β-lactamases like NDM, VIM, IMP, SPM types)

*β-Lactam resistance determinants in P. aeruginosa.*

*β-lactamase; NDM, New Delhi metallo-β-lactamase; SPM, Sao Paolo metallo-β-lactamase.*

In the clinical setting, OprD porin loss is often associated with a resistance phenotype in which one observes resistance to carbapenems including imipenem, but *in vitro* susceptibility to anti-pseudomonal pencillins and cephalosporins. In

**Resistance determinant Antibiotics affected**

*OprD, outer membrane porin D; Mex-multidrug efflux; TEM, class A β-lactamase of E. coli, named for patient in which it was discovered; SHV, sulfhydryl variant of TEM; OXA, oxacillinase; GES, German extended spectrum β-lactamase; VEB, Verona extended spectrum; CTX-M, cefotaximase-München; PER, plasmidic extended spectrum* 

OprD loss Carbapenems, some cephalosporins, penicillins Efflux pumps (e.g., MexA-B/OprM) Meropenem, some cephalosporins, penicillins Chromosomal Amp C of *P. aeruginosa* Anti *P. aeruginosa* penicillins, anti *P. aeruginosa*

cephalosporins except ceftolozane

ceftolozane, cefepime

and carbapenems

anti *P. aeruginosa* cephalosporins except

anti *P. aeruginosa* pencillins, cephalosporins

are 0.06–0.125 mg/L whereas for *P. aeruginosa* isolates, MICs are in the range of 1–2 mg/L. In the main, when *P. aeruginosa* is resistant to β-lactams, specific mechanisms are at play. These include downregulation of outer membrane porins, expression of intrinsic efflux mechanisms, acquisition of β-lactamase enzymes including carbapenamases such as IMP ("imipenemase"), VIM ("Verona imipenemase") and KPC ("Klebsiella pneumoniae carbapenemase"), hyperproduction of chromosomal β-lactamases ("AmpCs or Pseudomonas-Derived Cephalosporinases, "PDCs") and reduced penicillin binding protein affinity for β-lactams that are not considered "anti-pseudomonal" β-lactams. **Table 1** summarizes the mechanism and resistance

Currently, in the clinical microbiology laboratory, susceptibilities are reported to particular antibiotics depending on the specific sample submitted, e.g., urine, blood, sterile body fluids (pleural, joint, cerebrospinal fluid). At least initially, genotypic testing to determine the presence of specific antibiotic resistance determinants is not performed, and it is left to the clinical infectious diseases expert to reason out the most likely resistance mechanisms based on susceptibility patterns,

The structure, function and regulation of *P. aeruginosa* porins is complex and has been recently reviewed [2]. Porins are involved in structural and signaling tasks in *P. aeruginosa*, as well as passage of nutrients. The Opr D family of porins is the largest and is subdivided into two groups OccD and OccK. These porins are each regulated through their own sigma factors. Porin loss can be related to formation of OprD containing outer membrane vesicles that are also found in high concentrations in biofilms. Resistance to carbapenems in biofilms may be related to this. Mutations in *P. aeruginosa* that cause oprD (occD1) to not be expressed are linked to imipenem resistance. Other occD1 mutations that do not effect transcription also lead to carbapenem resistance [3, 4]. OprD mutations or loss is often associated with overexpression of efflux pumps (see below) leading to high level resistance to carbapenems, other β-lactams and other

**70**

**Table 1.**

*Pseudomonas aeruginosa*

As with porins, *P. aeruginosa* possesses a large variety of efflux pumps that perform different roles in the bacteria, but mainly function to extrude harmful substances from the cell. These pumps have been reviewed in [6]. Pumps of importance in carbapenem and other antibiotic efflux are in the resistance nodulation or RND type family and include the MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM multidrug efflux pump systems [7]. Increased expression of these pumps leads to high level carbapenem resistance, often in association with OprD loss or modification. Notably imipenem is not a substrate of the multidrug efflux pumps of *P. aeruginosa* while meropenem is [7]. In the clinical setting, if one notes resistance to meropenem and other β-lactams, except for imipenem, then an efflux mechanism is at play. If resistance is noted to both meropenem and imipenem, but not to other β-lactams, OprD loss or modification is responsible. With resistance to carbapenems as well as other β-lactams, multiple resistance mechanisms can be at play including efflux, intrinsic and acquired β-lactamases and decreased permeability (porin loss).
