**2.3 Hyperproduction of PDC β-lactamases**

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].

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

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,

CTX-M, GES, PER and VEB types; Class A carbapenemases such as KPC variants; metallo-β-lactamases such as the VIM, IMP, NDM and SPM B1 di-Zn2+ enzymes: and OXA carbapenemases [9, 12]. Weak imipenemases in the so-called Class C AmpCs have already been discussed above. In combination with OprD loss and/or upregulation of MEX efflux pumps, high level carbapenem resistance can be seen in *P. aeruginosa* due to acquired β-lactamases. Traditional class A β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam have *in vitro* activity versus the Class A ESBLs but not against other β-lactamases, e.g., the anti-pseudomonal combination ceftolozane-tazobactam is not effective against KPC, metallo-blactamases, or OXA enzymes [1]. New β-lactam-β-lactamase inhibitor combinations such as ceftazidime-avibactam and meropenem-vaborbactam will be active against isolates with KPC enzymes, and Class C β-lactamases, as long as they lack other resistance mechanisms that increase the β-lactam MIC beyond what is caused by the β-lactamase enzyme [13, 14].

#### **2.5 Penicillin binding proteins (PBPs) of** *P. aeruginosa*

PBPs of *P. aeruginosa* have high affinities for so called anti-pseudomonal β-lactams namely piperacillin, ticarcillin, ceftazidime, cefepime, ceftolozane, meropenem, imipenem, doripenem and aztreonam [1]. PBP 3 is the most important target of inhibition as it is essential for growth of the bacteria [15]. PBP3 is the primary target for ceftazidime whereas PBP2 is the target of carbapenems. Mutations in PBPs have not been described in *P. aeruginosa* leading to β-lactam resistance. The interactions of specific PBPs with specific lytic transglycosylases in the maintenance of cell wall will be further discussed below.

#### **2.6 Current therapeutic strategies to treat infections with resistant** *P. aeruginosa*

Given that 15–33% of *P. aeruginosa* isolates are multidrug resistant (have at least one resistance mechanism) [16, 17] and that resistance is associated with up to fivefold greater mortality [18, 19], choosing the right antibiotic combinations have a tremendous impact on patient outcomes. Advances in the rapid diagnosis of *P. aeruginosa*, and use of both rapid phenotypic tests such as CARBA NP [20] or rapid molecular diagnostics to identify specific ESBL and carbapenemase enzymes, have enhanced the clinician's ability to get patients on the right therapy sooner. Identification of patient risk factors, including prior antibiotic exposure, and knowledge of local trends in resistance patterns are useful in selection of empiric antibiotics, until antimicrobial susceptibilities and genotypic results are available for guidance. Carbapenems (meropenem or imipenem) and anti-pseudomonal cephalosporins in combination with colistin, an aminoglycoside or fosfomycin, versus ceftolozane/tazobactam or meropenem/vaborbactam or ceftazidime/avibactam are all good empiric choices for critically ill patients [16], provided multidrug resistance is not present. However, clearly more therapeutic options are needed for infections with extensively drug resistant and pan-resistant *P. aeruginosa*. Lytic transglycosylases represent a new target for bacterial inhibition.
