Pseudomonas aeruginosa: Multi-Drug-Resistance Development and Treatment Options

Antibiotic resistance is a worldwide problem of major importance. Isolations in somecountries of multi-drug-resistant (resistant to three or more classes of antimicrobials),extensively-drug-resistant (resistant to all but one or two classes) or even pan-drug-resistant (resistant to all available classes) Gram-negative pathogens are causing therapeu‐tic problems and- in the same time- are posing infection control issues in many hospitals.In fact, numerous studies highlight the link between multi-drug-resistance and increasedmorbidity and mortality, increased length of hospital stay and higher hospital costs [1-4].Pseudomonas aeruginosa is a Gram-negative opportunistic nosocomial pathogen responsi‐ble for a wide range of infections that may present high rates of antimicrobial resistance.The genome of this microorganism is among the largest in the bacterial world allowingfor great genetic capacity and high adaptability to environmental changes. In fact, P.aeruginosa has 5567 genes encoded in 6.26 Mbp of DNA while Escherichia coli K12 forexample has 4279 genes encoded in 4.46 Mbp and Haemophilus influenzae Rd has 1.83 Mbpencoding 1714 genes [5]. This large genetic armamentarium- that can be further enrichedwith the addition of genes acquired by transferable genetic elements via horizontal genetransfer- is a major contributing factor to its formidable ability to develop resistance againstall known antibiotics.Generally, antibiotic resistance mechanisms of P. aeruginosa can be divided in intrinsic andacquired. Intrinsic refers to resistance that is a consequence of a large selection of genetical‐ly-encoded mechanisms and acquired refers to resistance that is achieved via the acquisi‐


Introduction
Antibiotic resistance is a worldwide problem of major importance. Isolations in some countries of multi-drug-resistant (resistant to three or more classes of antimicrobials), extensively-drug-resistant (resistant to all but one or two classes) or even pan-drugresistant (resistant to all available classes) Gram-negative pathogens are causing therapeutic problems and-in the same time-are posing infection control issues in many hospitals. In fact, numerous studies highlight the link between multi-drug-resistance and increased morbidity and mortality, increased length of hospital stay and higher hospital costs [1][2][3][4].
Pseudomonas aeruginosa is a Gram-negative opportunistic nosocomial pathogen responsible for a wide range of infections that may present high rates of antimicrobial resistance. The genome of this microorganism is among the largest in the bacterial world allowing for great genetic capacity and high adaptability to environmental changes. In fact, P. aeruginosa has 5567 genes encoded in 6.26 Mbp of DNA while Escherichia coli K12 for example has 4279 genes encoded in 4.46 Mbp and Haemophilus influenzae Rd has 1.83 Mbp encoding 1714 genes [5]. This large genetic armamentarium-that can be further enriched with the addition of genes acquired by transferable genetic elements via horizontal gene transfer-is a major contributing factor to its formidable ability to develop resistance against all known antibiotics.
Generally, antibiotic resistance mechanisms of P. aeruginosa can be divided in intrinsic and acquired. Intrinsic refers to resistance that is a consequence of a large selection of genetically-encoded mechanisms and acquired refers to resistance that is achieved via the acquisi-

Quinolones
Quinolones are synthetic antimicrobials that block DNA replication by inhibiting the activity of DNA gyrase and topoisomerase IV [16]. The fluorquinolones with anti-pseudomonal activity are ciprofloxacin, levofloxacin and ofloxacin.

Aminoglycosides
Aminoglycosides inhibit protein synthesis by binding to the 30S or 50S ribosomal subunit [22]. Drugs of this antibiotic class that can be used against P. aeruginosa are tobramycin, amikacin and gentamicin. Aminoglycosides are associated with ototoxicity and nefrotoxicity [23]. Because of these adverse effects and because of their narrow therapeutic range, aminoglycosides are used in combination with agents belonging to other antibiotic classes. The only treatment in which aminoglycosides are recommended as monotherapy is that of urinary tract infections due to P. aeruginosa [14].

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-lactamases and aminoglycoside-modifying enzymes (Table 3).

Resistance to Resistance mechanism
Beta-lactams Endogenous beta-lactamases  Table 3. Resistance mechanisms of P. aeruginosa to anti-pseudomonal drugs.

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 carbapenemase activity, namely the carbapenemases [28], will be discussed separately in this section.
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].
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 narrowspectrum cephalosporins even though some of them are ESBLs and a few members of the class present carbapenemase activity [24].

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 Zn 2+ 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 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 development 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].
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.

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 resistance [24].

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].

Resistance to fluoroquinolones
High-level resistance to fluoroquinolones is mediated by target site modifications. Efflux plays a contributing role as well [96,97] and the two mechanisms often coexist [32,[98][99][100].

Resistance to aminoglycosides
Acquired resistance to aminoglycosides is mediated by transferable aminoglycosidemodifying enzymes (AMEs), rRNA methylases and derepression of endogenous efflux systems [24,108,109].
Genes encoding AMEs are typically found on integrons together with other genes responsible for transferable resistance for other antibiotic classes. This way AMEs become important determinants for the development of multi-drug resistance in P. aeruginosa and other species [24,108,109].
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).
Among the nucleotidyltransferases, ANT(2')-I is the most frequently encountered in P. aerugiosa. This enzyme is present in isolates showing resistance to gentamicin and tobramycin but not to amikacin [109].
Almost all phosphoryltransferases of P. aeruginosa act in the 3' position of the aminoglycoside molecule [24]. However, they have less clinical importance because of the fact that they inactivate aminoglycosides that are not routinely used for the treatment of P. aeruginosa infections such as kanamycin and neomycin [109]. The enzymes of this family that inactivate anti-pseudomonal aminoglycosides are APH(3')-VI [110][111][112], APH(3')-IIb-like [113] and APH(2'') [110]. Despite being reported in some cases, these enzymes remain rare for clinical P. aeruginosa isolates [24].

Efflux systems
Resistance to aminoglycosides in P. aeruginosa can occur independently of aminoglycosidemodifying enzymes in cystic fibrosis patients. This type of resistance has been reported in several studies [99,[118][119][120] and is attributable to over-expression of the MexXY-OprM efflux pump.

16S rRNA methylases
Methylation of the 16S rRNA of the A site of the 30S ribosomal subunit interferes with aminoglycoside binding and consequently promotes high-level resistance to all aminoglycosides [24]. Different 16S rRNA methylases have been described for P. aeruginosa: RmtA [112,121], RmtB [122], ArmA [122,123] and RmtD which is commonly found together with the MBL SPM-1 in Brazil [124,125].

Treatment options for MDR Pseudomonas aeruginosa
Different combinations of the aforementioned mechanisms may be present in a single P. aeruginosa isolate leading to simultaneous resistance to various anti-pseudomonal compounds. The most potent combination is obviously that of a carbapenemase producing isolate usually enriched by resistance to quinolones and aminoglycosides leaving very limited options for antimicrobial treatment.
As far as newer carbapenem compounds are concerned, data suggest that doripenem does not offer advantages over other carbapenems against carbapenemase producing strains [126].
Tigecycline is an option for Gram-negative MDR pathogens but it cannot be used against P. aeruginosa, Morganella morganii, Proteus spp. and Providencia spp. because it is intrinsically vulnerable to their chromosomal-encoded efflux pumps [127].
Furthermore, time-kill studies on 12 MBL-producing P. aeruginosa isolates performed with aztreonam alone and in combination with ceftazidime and amikacin, showed bactericidal activity against one and eight isolates respectively. In the same study, colistin was bactericidal against all 12 isolates [128].
In fact, polymyxins and colistin in particular, are quite effective in the treatment of MDR P.
aeruginosa infections [129,130]. The target of colistin is the bacterial cell membrane. More precisely, colistin interacts with the lipid A of lipopolysaccharides, allowing penetration through the outer membrane by displacing Ca 2+ and Mg 2+ . The insertion between the phospholipids leads to loss of membrane integrity and consequent bacterial cell death [131]. There are reports of resistance to polymyxin B [132][133][134] and colistin [135][136][137] in clinical isolates but they remain to date relatively rare for P. aeruginosa [24]. While in many cases the mechanism of clinical polymyxin resistance is unknown, substitution of the lipopolysaccharide lipid A with aminoarabinose has been shown to contribute to polymyxin resistance in vitro [138] and  cystic fibrosis isolates [139]. Colistin is frequently associated with nephro-and neurotoxicity but both these adverse effects seem to be dose-dependent and reversible [140].
Another interesting option for the treatment of MDR P. aeruginosa is fosfomycin, an old antibacteial that has regained attention because of its in vitro activity against such isolates [140]. Fosfomycin inactivates the enzyme pyruvil-transferase, which is required for the synthesis of the cell wall peptidoglycan. In a review of the existing fosfomycin studies, 81.1% of 1529 patients were successfully treated for infections caused by P. aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterobacter spp. and Klebsiella spp. Fosfomycin was administered together with aminoglycosides, cephalosporins and penicillines [141]. More studies are needed however to determine the future role of fosfomycin against MDR P. aeruginosa isolates.

Combination therapy
The application of combination therapy instead of monotherapy in cases of non-MDR P. aeruginosa remains to date a controversial issue [14]. Combination treatment against MDR strains instead seems to be some times necessary (for example in cases of pan-resistance or resistance to all except a single agent). In such cases better results are expected by the additive or subadditive activity of a combination or by the enhancement of a single active agent by an otherwise inactive drug [142].
Several old and newer studies have showed the increased activity in vitro of various antibiotic combinations against MDR P. aeruginosa (Table 7) even though, the mechanisms of positive interaction between the various agents are rarely known [142].

Conclusion
P. aeruginosa is a nosocomial pathogen of particular clinical concern not only because of its extraordinary resistance mechanisms armamentarium but also for its formidable ability to adapt very well to the hospital environment. There are important challenges in the treatment of MDR P. aeruginosa strains and their isolation in healthcare settings poses serious infection control issues. For these reasons, the prudent use of antibiotics, mainly those used as last resort treatment like carbapenems is of outmost importance in order to prevent evolutionary pressure that may lead to the emergence of highly resistant clones.