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Pseudomonas aeruginosa – a phenomenon of bacterial resistance

Tanya Strateva, Daniel Yordanov

  • Department of Microbiology, Medical University of Sofia, 2 Zdrave Street, 1431 Sofia, Bulgaria
  • Correspondence
    Tanya Strateva
    dr.strateva{at}abv.bg

    • Journal of Medical Microbiology 2009; 58(9):1133–1148 · https://doi.org/10.1099/jmm.0.009142-0

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    Abstract

    Pseudomonas aeruginosa is one of the leading nosocomial pathogens worldwide. Nosocomial infections caused by this organism are often hard to treat because of both the intrinsic resistance of the species (it has constitutive expression of AmpC β-lactamase and efflux pumps, combined with a low permeability of the outer membrane), and its remarkable ability to acquire further resistance mechanisms to multiple groups of antimicrobial agents, including β-lactams, aminoglycosides and fluoroquinolones. P. aeruginosa represents a phenomenon of bacterial resistance, since practically all known mechanisms of antimicrobial resistance can be seen in it: derepression of chromosomal AmpC cephalosporinase; production of plasmid or integron-mediated β-lactamases from different molecular classes (carbenicillinases and extended-spectrum β-lactamases belonging to class A, class D oxacillinases and class B carbapenem-hydrolysing enzymes); diminished outer membrane permeability (loss of OprD proteins); overexpression of active efflux systems with wide substrate profiles; synthesis of aminoglycoside-modifying enzymes (phosphoryltransferases, acetyltransferases and adenylyltransferases); and structural alterations of topoisomerases II and IV determining quinolone resistance. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. This review describes the known resistance mechanisms in P. aeruginosa to the most frequently administrated antipseudomonal antibiotics: β-lactams, aminoglycosides and fluoroquinolones.

    Introduction

    Pseudomonas aeruginosa is responsible for 10–15 % of the nosocomial infections worldwide (Blanc et al., 1998). Often these infections are hard to treat due to the natural resistance of the species, as well as to its remarkable ability of acquiring further mechanisms of resistance to multiple groups of antimicrobial agents. P. aeruginosa represents a phenomenon of antibiotic resistance, and demonstrates practically all known enzymic and mutational mechanisms of bacterial resistance (Pechere & Kohler, 1999). Often these mechanisms exist simultaneously, thus conferring combined resistance to many strains (McGowan, 2006). This review describes the resistance mechanisms to the most frequently administrated antipseudomonal antibiotics: β-lactams, aminoglycosides and fluoroquinolones.

    Mechanisms of resistance to β-lactams

    P. aeruginosa is intrinsically resistant to many structurally unrelated antimicrobial agents (Mesaros et al., 2007) because of the low permeability of its outer membrane (1/100 of the permeability of E. coli outer membrane) (Livermore, 1984), the constitutive expression of various efflux pumps with wide substrate specificity (Livermore, 2001) and the naturally occurring chromosomal AmpC β-lactamase (also known as cephalosporinase) (Nordmann & Guibert, 1998). The natural resistance of the species relates to the following β-lactams: penicillin G; aminopenicillins, including those combined with β-lactamase inhibitors; first and second generation cephalosporins. P. aeruginosa easily acquires additional resistance mechanisms, which leads to serious therapeutic problems.

    The susceptible P. aeruginosa phenotype (the so called wild-type) includes susceptibility to carboxypenicillins (carbenicillin, ticarcillin), ureidopenicillins (azlocillin, piperacillin), some third generation cephalosporins (ceftazidime, cefsulodine, cefoperazone), all the fourth generation cephalosporins, the monobactam aztreonam, and the carbapenems imipenem and meropenem (Pechere & Kohler, 1999). There are several basic resistance phenotypes. (i) Often called ‘intrinsic resistance to carbenicillin’, this phenotype is characterized by a fourfold to eightfold increase of MIC for most of the β-lactams, including meropenem but not imipenem. No production of chromosomal AmpC β-lactamase above the basic level is found. This phenotype includes resistance to non-β-lactam antibiotics like quinolones, trimethoprim, tetracycline and chloramphenicol. The cause for the rise in MIC is the low outer membrane permeability combined with activation or derepression of efflux systems (Pechere & Kohler, 1999). (ii) The second phenotype affects resistance to all β-lactams except cephems (cefepime and cefpirome) and carbapenems. The extent of the change is antibiotic-dependent, and is caused by derepression of the AmpC β-lactamase (Livermore, 1995). (iii) In the third phenotype, resistance to penicillins (in particular ticarcillin, azlocillin and piperacillin) is affected more than resistance to cephalosporins, resulting from production of OXA-type β-lactamases (Pechere & Kohler, 1999). These narrow-spectrum oxacillinases determine resistance to carboxypenicillins and ureidopenicillins, but not to extended-spectrum cephalosporins, aztreonam and moxalactam (Bert et al., 2002). (iv) The fourth phenotype is characterized by increased MICs to carbapenems. Resistance to other β-lactams is not affected because strains exhibiting this phenotype have a decreased level of OprD, a carbapenem-specific porin (Livermore, 2001).

    Other resistance phenotypes are determined mainly by the production of plasmid- or integron-encoded extended-spectrum β-lactamases (ESBLs) from different molecular classes. In P. aeruginosa all possible mechanisms determining resistance to β-lactam antibiotics [enzymic inactivation, active efflux, changes in outer membrane permeability and synthesis of penicillin-binding proteins (PBPs) with lower affinity to β-lactams] may exist simultaneously or in various combinations.

    Resistance to β-lactams due to β-lactamase production

    Enzyme production is the major mechanism of acquired resistance to β-lactam antibiotics in P. aeruginosa. Penicilloyl-serine transferases (usually referred to as β-lactamases) rupture the amide bond of the β-lactam ring, thus the obtained products lack antibacterial activity (Sykes & Mattew, 1976). Molecular classification of β-lactamases is based on the nucleotide and amino acid sequences in these enzymes (Ambler, 1980). To date, four classes are recognized (A–D), correlating with the functional classification defined by enzyme substrate and inhibitor profiles (Bush et al., 1995). Classes A, C and D act through a serine-based mechanism, whereas class B or metallo-β-lactamases (MBLs) need zinc for their action. A significant number of β-lactamases of all four molecular classes are found in P. aeruginosa, including ESBLs of classes A, B and D.

    AmpC β-lactamase.

    P. aeruginosa is naturally susceptible to carboxypenicillins, ceftazidime and aztreonam; however, it can acquire resistance to third generation cephalosporins. The most frequent mechanism by which this occurs is through the constitutive hyperproduction of AmpC β-lactamase (so called stable derepression) (Bagge et al., 2002). Like some species of the Enterobacteriaceae family (Enterobacter spp., Serratia marcescens, Citrobacter freundii, Morganella morganii and Yersinia enterocolitica), P. aeruginosa produces an inducible chromosome-encoded AmpC β-lactamase (cephalosporinase) that belongs to molecular class C, based on Ambler and the first functional group according to Bush (Bush et al., 1995). Usually the enzyme is produced in low quantities (‘low-level’ expression) and determines resistance to aminopenicillins and most of the early cephalosporins (Langaee et al., 2000). However, chromosomal cephalosporinase production in P. aeruginosa may increase from 100 to 1000 times in the presence of inducing β-lactams (especially imipenem) (Bagge et al., 2002). AmpC cephalosporinase activity is not inhibited by β-lactamase inhibitors used in clinical practice, for example clavulanic acid, sulbactam and tazobactam (Nordmann & Guibert, 1998).

    AmpC β-lactamase is encoded by the ampC gene (Lodge et al., 1993). Mechanisms regulating ampC expression have been studied in detail for Enterobacter cloacae. Similar mechanisms regulate the expression of the enzyme in P. aeruginosa. Several genes are involved in ampC induction – a process that is intimately linked to peptidoglycan recycling (Normark, 1995). Of the genes involved, ampR, is contiguous to ampC but divergently transcribed, and it encodes a positive transcriptional regulator that is a member of the LysR family (AmpR). This regulator is necessary for the β-lactamase induction (Lodge et al., 1993). AmpR transcriptional regulatory activity is related to peptidoglycan processing (Jacobs et al., 1994). The second gene, ampG, encodes a transmembrane protein that acts as a permease for 1,6-anhydromurapeptides, which are considered to be the signal molecules involved in ampC induction (Dietz et al., 1997). The third gene, ampD, encodes a cytosolic N-acetyl-anhydromuramyl-l-alanine amidase, which hydrolyses 1,6-anhydromurapeptides, acting as a repressor of ampC expression (Höltje et al., 1994). Mutational inactivation of ampD in P. aeruginosa PAO1 leads to partially derepressed expression of AmpC β-lactamase (Langaee et al., 2000). The fourth gene, ampE, forms the bicistronic ampDE operon and encodes a cytoplasmic membrane protein that is thought to act as a sensory transducer molecule necessary for induction (Honore et al., 1989). Recently, Juan et al. (2006) demonstrated that ampC expression is co-ordinately repressed by three AmpD homologues, including the previously described protein AmpD plus two additional proteins designated AmpDh2 and AmpDh3. The three AmpD homologues are responsible for a stepwise ampC upregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal β-lactam resistance, as shown by the analysis of the three single ampD mutants, the three double ampD mutants and the triple ampD mutant. This analysis was achieved by a three-step escalating mechanism generating four expression states: basal-level inducible expression (wild-type), moderate-level hyperinducible expression with increased antipseudomonal β-lactam resistance (ampD mutant), high-level hyperinducible expression with high-level β-lactam resistance (ampD ampDh3 double mutant) and very high-level (more than 1000-fold compared to the wild-type) derepressed expression (triple mutant). Unlike enterobacteria, P. aeruginosa have not yet been found to contain plasmid-mediated cephalosporinases, although some of the plasmid-encoded cephalosporinases demonstrate a remarkably similar structure to that of the pseudomonal AmpC β-lactamase.

    Class A carbenicillin hydrolysing β-lactamases.

    Four carbenicillin hydrolysing β-lactamases of Pseudomonas-specific enzyme (PSE) type were found in P. aeruginosa: PSE-1 (CARB-2), PSE-4 (CARB-1), CARB-3 and CARB-4 (Bert et al., 2002). Their substrate profile includes carboxypenicillins, ureidopenicillins and cefsulodine. These enzymes belong to molecular class A and functional group 2c (Bush et al., 1995). PSE-1, PSE-4 and CARB-3 are closely related (they differ by just 1 or 2 amino acids), but they are only 86.3 % homologous with CARB-4 (Sanschagrin et al., 1998). The blaCARB-4 gene is likely to have been acquired from other bacterial species, as the mol% G+C in this gene is 39.1 % unlike the mol% G+C of genes that are typical for P. aeruginosa, which is 67 %. Carbenicillinase producers show variable susceptibility to cefepime, cefpirome and aztreonam, and 100 % susceptibility towards ceftazidime and carbapenems.

    Class A ESBLs.

    Unlike PSEs, ESBLs of molecular class A and functional group 2b’ (Bush et al., 1995) lead to the development of resistance not only to carboxypenicillins and ureidopenicillins, but also to extended-spectrum cephalosporins (ceftazidime, cefepime, cefpirome) and aztreonam (Weldhagen et al., 2003). They show low affinity to carbapenems. Their in vitro activity is inhibited by clavulanic acid and tazobactam (Nordmann & Guibert, 1998). Discovery of class A ESBLs in clinical isolates of P. aeruginosa occurred after 1990. Apart from the TEM and SHV types of enzyme that are well known in the Enterobacteriaceae family, in P. aeruginosa other enzymes that were identified are PER (mostly in clinical isolates from Turkey), VEB (from South-East Asia, France and Bulgaria), GES/IBC (France, Greece and South Africa) and BEL types (Table 1⇓). These six types have low identity at the genetic level, and yet they have similar hydrolysis profiles.

    Table 1.

    Epidemiology of molecular class A ESBLs produced by P. aeruginosa

    SHV-2a was originally detected in France (in 1995) (Naas et al., 1999a), and later in Thailand and Poland (Chanawong et al., 2001). This enzyme vigorously hydrolyses fourth generation cephalosporins (Weldhagen et al., 2003). SHV-5 and SHV-12 producing P. aeruginosa strains were also found in Thailand (Chanawong et al., 2001). Afterwards, SHV-5 and SHV-12 ESBLs were identified in clinical P. aeruginosa isolates from Greece as well (Poirel et al., 2004a; Neonakis et al., 2003). SHV-5 determines high level of resistance to ceftazidime and monobactams.

    Between 1992 and 1998 in France, P. aeruginosa strains producing the following TEM enzymes were consecutively isolated: TEM-42, TEM-4, TEM-21 and TEM-24 (Mugnier et al., 1996; Poirel et al., 1999; Dubois et al., 2002a; Marchandin et al., 2000). The hydrolytic spectrum of TEM enzymes in P. aeruginosa is similar to that of the classical ESBLs in Enterobacteriaceae and includes: narrow-spectrum penicillins, extended-spectrum cephalosporins and aztreonam (Weldhagen et al., 2003).

    It is likely that the genes for the TEM- and SHV-type ESBLs in P. aeruginosa originate from Enterobacteriaceae, from which genes are passed by gene transfer. This has been shown for the sequence of TEM-24 (Marchandin et al., 2000) and the downstream-located chromosomal DNA sequences of P. aeruginosa RP-1, producing SHV-2a, which were found to be identical to those reported to be plasmid encoded in a Klebsiella pneumoniae isolate (Naas et al., 1999a). Several P. aeruginosa strains, including respiratory and urinary isolates producing TEM-24 ESBL, were isolated from a long-term-hospitalized woman (Marchandin et al., 2000). TEM-24-producing isolates of Enterobacter aerogenes recovered from wound, venous catheter and faeces, and TEM-24-producing wound E. coli isolate were cultured from the same patient. TEM-24 and the resistance markers for aminoglycosides, chloramphenicol and sulfonamides were encoded by a 180 kb plasmid transferred by conjugation into E. coli HB101. The multiplicity of TEM-24-producing bacteria recovered from the same patient strongly suggests the in vivo horizontal transfer of this plasmid-mediated ESBL from Enterobacteriaceae to P. aeruginosa.

    PER-1 was the first identified and fully characterized ESBL in P. aeruginosa. It was found in 1991 in France in an isolate from the urine culture of a Turkish citizen (Nordmann & Naas, 1994) and was chromosome encoded. Later, plasmid encoded PER-1 enzymes were reported as well (Nordmann & Guibert, 1998). Currently, there is widespread dissemination of blaPER-1 among nosocomial P. aeruginosa isolates in Turkey (Vahaboglu et al., 2001; Kolayli et al., 2005). Other geographical regions where PER-1 producing P. aeruginosa strains were isolated were Italy, Belgium and Poland (Luzzaro et al., 2001; Pagani et al., 2004; Claeys et al., 2000; Empel et al., 2007). PER-1 exhibits the substrate profile typical of classical ESBLs. It is moderately inhibited by β-lactamase inhibitors and imipenem (Weldhagen et al., 2003).

    Another type of molecular class A ESBLs are the VEB enzymes. The first isolation of a VEB-1 β-lactamase was in 1998 in France (Naas et al., 1999b); later Girlich et al. (2002) found a high prevalence of blaVEB-like genes (93 %) in ceftazidime-resistant clinical isolates of P. aeruginosa in the University Hospital in Thailand. During that study, a new blaVEB-2 gene was identified. VEB-2 differed from VEB-1 by just a single amino acid outside the active centre of the enzyme. In 2007 high dissemination (56.8 %) of VEB-1 ESBL among ceftazidime-resistant nosocomial P. aeruginosa isolates from Bulgaria was reported (Strateva et al., 2007). The substrate profile of VEB enzymes was identical with that of PER-1 (Weldhagen et al., 2003).

    At the very end of the 20th century, a novel family of ESBLs was described, referred to as Guiana extended spectrum (GES), named after the country of origin of the first isolate, French Guiana. GES-1 and GES-2 were found in France and Brazil (Dubois et al., 2002b; Castanheira et al., 2004a), and South Africa (Poirel et al., 2002b), respectively. GES-1 has an unusually low level of catalytic activity, low affinity to the most of the substrates, and an unusual inhibition profile that includes clavulanic acid and imipenem. Unlike most of class A ESBLs, GES-1 has strong affinity to the second generation cephalosporin cefoxitin (Weldhagen et al., 2003). GES-2 (discovered in 2000) possesses carbapenemase activity (Weldhagen & Prinsloo, 2004). This enzyme originated through a point mutation of GES-1. Like GES-1, GES-2 β-lactamase has cysteine residues in positions 69 and 238 that may form a disulphide bridge, which explains the imipenem-binding properties. GES-2 demonstrates 100 times higher catalytic activity towards imipenem than GES-1; but this is still a much lower activity than that of the metallo-enzymes of molecular class B (Nordmann & Poirel, 2002). New variants of GES-1 enzyme have been reported: GES-5 (Poirel et al., 2005a) and GES-9 (Poirel et al., 2005b). In comparison with GES-1 identified in 1999 (in France) the newly discovered GES-5 hydrolyses penicillins to a greater extent, as well as extended-spectrum cephalosporins and aztreonam. It is distinguished from GES-1 by a Gly242Ser amino acid substitution. Enzyme activity is suppressed by clavulanic acid, tazobactam and imipenem. The blaGES-5 gene is located on a class 1 integron simultaneously with aacA4, encoding resistance to aminoglycosides. GES-9 enzyme also differs from GES-1 by a single amino acid substitution (Gly243Ser). It is inactivated by clavulanic acid and imipenem and hydrolyses aztreonam. The blaGES-9 gene encoding it is localized within a class 1 integron containing two copies of an insertion sequence from the IS1111 family.

    IBC-2 (now called GES-8) ESBL was discovered in a clinical P. aeruginosa isolate in Greece (Mavroidi et al., 2001). It is a variant of GES-1 with a single substitution (Ala125Leu) compared to GES-1. Production of this β-lactamase determines resistance to ceftazidime and other oxyiminocephalosporins, and is inhibited by imipenem, clavulanic acid and tazobactam.

    Recently, in Belgium, a discovery of a new type of ESBL of Ambler class A was made – BEL-1. The new ESBL was identified in a clinical strain of P. aeruginosa isolated at a hospital in Flanders, Belgium (Poirel et al., 2005c). The enzyme had a hydrolysis profile comprising extended-spectrum cephalosporins and aztreonam, and its activity was suppressed by clavulanic acid, tazobactam, cefoxitin, moxalactam and imipenem. The encoding gene, blaBEL-1, is a part of a class 1 integron, In120, localized in a chromosome transposon that also contains three other gene cassettes. Later on, between May and November 2006, BEL-1-producing P. aeruginosa isolates were discovered in several hospitals located in different geographical areas of Belgium (Bogaerts et al., 2007).

    Dissemination of class A ESBL-encoding genes plays an important role in antibiotic resistance dissemination, and may limit the possibilities for the choice of antibiotic regimen in the treatment of life-threatening infections caused by P. aeruginosa. Plasmids and integrons are important factors for this dissemination. In this regard, plasmid localization was proven for most of the genes encoding TEM and SHV enzymes in P. aeruginosa (Chanawong et al., 2001; Mugnier et al., 1996). Whereas genes encoding β-lactamases of Ambler class B (metalloenzymes) and Ambler class D (oxacillinases) were usually located in class 1 integrons, genes encoding VEB- and GES-type enzymes were the only class A ESBL-encoding genes that are associated with these genetic determinants (Girlich et al., 2002; Poirel et al., 2002b). Localization of some genes on transposons provides an additional route for the mobilization of antimicrobial-resistance genes, and this fact can explain the simultaneous localization of the same ESBL-encoding genes on plasmids, as well as on the chromosome of P. aeruginosa (Weldhagen et al., 2003).

    Class D β-lactamases (oxacillinases).

    Oxacillinases (OXA type enzymes) belong to molecular class D and functional group 2d (Bush et al., 1995). Classical OXA enzymes (OXA-1, OXA-2, OXA-10) determine resistance to carboxypenicillins and ureidopenicillins but not to ceftazidime (Bert et al., 2002). Resistance to ticarcillin and piperacillin resulting from production of OXA-2 enzymes is lower than the resistance that develops when OXA-10 and OXA-1 oxacillinases are produced (Bert et al., 2003). Ceftazidime hydrolysing extended-spectrum oxacillinases have the greatest clinical importance. Their hydrolysis spectrum also includes: cefotaxime, cefepime, cefpirome, aztreonam and moxalactam (Bradford, 2001). With the exception of OXA-18, the activity of these enzymes is not suppressed by β-lactamase inhibitors (clavulanic acid and tazobactam). This fact hampers their identification by routine laboratory practices (Naas & Nordmann, 1999). Sanschagrin et al. (1995) described five different groups of oxacillinases in P. aeruginosa. OXA group I integrates OXA-5, OXA-7, OXA-10 and its derivates (OXA-11, OXA-14, OXA-16 and OXA-17), and OXA-13 and its derivates (OXA-19 and OXA-28) (Couture et al., 1992; Scoulica et al., 1995; Mugnier et al., 1998a, b; Hall et al., 1993; Danel et al., 1995, 1998, 1999; Poirel et al., 2001a). In the last few years OXA-13 and its derivates (OXA-19 and OXA-28) were defined as an OXA-10-related subgroup (Bert et al., 2002). OXA-11, OXA-14 and OXA-19 affect mostly ceftazidime activity (Aubert et al., 2001) while OXA-17 attacks mainly cefotaxime (Danel et al., 1999). Generally, extended-spectrum variants of OXA-10 determine low-level resistance to fourth generation cephalosporin cefepime, in contrast to third generation ceftazidime (which is ‘high level’) (Aubert et al., 2001). OXA group II includes OXA-2, OXA-3, OXA-15 and OXA-20 (Sanschagrin et al., 1995; Dale et al., 1985; Danel et al., 1997; Naas et al., 1998). OXA-15 is an extended-spectrum variant of OXA-2 β-lactamase (Danel et al., 1997). Recently, Poirel et al. (2002a) found one more derivate of OXA-2 (OXA-32) that is an ESBL. The OXA group III includes OXA-1 and its derivates – OXA-4, OXA-30 and OXA-31 (Aubert et al., 2001). The OXA group IV comprises just a single enzyme – OXA-9; OXA group V is represented by LCR-1 (Couture et al., 1992; Sanschagrin et al., 1995).

    Apart from OXA-15 and OXA-32, the rest of the extended-spectrum oxacillinases derive from OXA-10 β-lactamase. Most of the class D ESBLs were found in clinical isolates from Turkey (Bradford, 2001). It is known that all extended-spectrum variants of OXA-10 have one of the following two amino acid substitutions: Ser73Asn or Gly157Asp. The latter determines high-level resistance to ceftazidime (Bradford, 2001).

    OXA-18 enzyme is encoded by the chromosomal blaOXA-18 gene and has low amino acid identity with the other class D oxacillinases produced by P. aeruginosa (the highest identity is with OXA-9 and OXA-12 – 45 and 42 %, respectively) (Philippon et al., 1997). This enzyme does not belong to any of the groups introduced by Sanschagrin et al. (1995). Its hydrolytic properties are like these of class A ESBLs – it affects amoxicillin, ticarcillin, cefalotin, ceftazidime, cefotaxime and aztreonam, but not imipenem. OXA-18 activity is totally inhibited by clavulanic acid. Recently, at the National Centre of Bone Marrow Transplantations in Tunisia the first outbreak in the world of a nosocomial infection (1998–2000) caused by OXA-18-producing P. aeruginosa strains was reported (Kalai Blagui et al., 2007).

    In 2003, a new class D ESBL – OXA-45 – was identified in a multidrug-resistant clinical P. aeruginosa isolate from Texas, USA. Its substrate profile was similar to that of OXA-18, and clavulanic acid inhibited its activity. The enzyme revealed highest amino acid identity with OXA-18 (65.9 %) and OXA-9 (42.8 %). blaOXA-45 is located on a 24 kb plasmid (Toleman et al., 2003).

    Most of the extended-spectrum oxacillinases are encoded by plasmid- or integron-located genes (Nordmann & Guibert, 1998), and this contributes to their easy dissemination and to the increased prevalence of class D ESBLs, producing P. aeruginosa isolates throughout Europe.

    Class B MBLs.

    Another group of ESBLs occurring in P. aeruginosa are the carbapenem-hydrolysing enzymes, which are also known as carbapenemases or MBLs due to the presence of Zn2+ in their active centre (Nordmann & Guibert, 1998). They belong to molecular class B (Bush et al., 1995). Carbapenemase production determines resistance to all β-lactams including the carbapenems imipenem and meropenem. Only the monobactam aztreonam is not influenced by the hydrolytic features of MBLs. The activity of class B carbapenem hydrolysing enzymes is not inhibited by clavulanic acid and tazobactam, but is suppressed by bivalent ionic chelators, e.g. EDTA (Nordmann & Poirel, 2002). IMP, VIM, SPM and GIM type MBLs were identified in P. aeruginosa (Table 2⇓).

    Table 2.

    Molecular class B MBLs found in P. aeruginosa

    The first carbapenemase proven in P. aeruginosa was IMP-1. It was found in Japan in a large-scale study of carbapenem-resistant clinical isolates during 1992–1994 (Senda et al., 1996). A total of 11 % of the strains studied harboured blaIMP-1. The gene was localized to a large plasmid (36 kb) and found to be part of a gene cassette within a class 1 In31 integron. Recently, IMP-1 MBL was reported among carbepenem-resistant P. aeruginosa isolated in two hospitals in Singapore (Koh et al., 2004).

    From 2000 until 2001 other IMP variants of MBLs were found in various Gram-negative bacteria worldwide. blaIMP-7 were identified among P. aeruginosa clinical isolates in Canada (Gibb et al., 2002; Parkins et al., 2007) and Singapore (Koh et al., 2004), and blaIMP-9 was found in China (Xiong et al., 2006), and blaIMP-13 in Italy (Pagani et al., 2005). In 2002, IMP-16 MBL was found in a P. aeruginosa strain from Brazil (Mendes et al., 2004a). Its encoding gene is chromosomal and is located in a class 1 integron that also carries genes for aminoglycoside-modifying enzymes. Currently, the most recent IMP MBL (IMP-18) was found in a P. aeruginosa clinical isolate in the USA (Hanson et al., 2006).

    VIM-1 carbapenamase, found in a nosocomial P. aeruginosa strain isolated at the Verona University Hospital, Italy, in 1997, is the first representative of a new family of acquired MBLs (Lauretti et al., 1999). Although VIM-1 shows less than 30 % amino acid identity to IMP enzymes, it has the same extended spectrum of hydrolysis (Nordmann & Poirel, 2002). Like blaIMP genes, blaVIM-1 is a part of a gene cassette inserted in the In70 class 1 integron, which carries the following genes: the integrase-encoding gene, blaVIM-1, and the aminoglycoside resistance encoding gene, aacA4 (Riccio et al., 2001). In 2003–2004 a new nosocomial infection outbreak was registered in two departments of the same Italian hospital. It was caused by VIM-1 producers of P. aeruginosa (Mazzariol et al., 2005a). In 2004–2005 Corvec et al. (2006) detected four P. aeruginosa clinical isolates producing VIM-1 from different French hospitals.

    VIM-2 was originally identified in a P. aeruginosa bloodstream isolate from a patient with neutropenia in Marseille (South France) (Poirel et al., 2000). It was closely related to VIM-1 MBL reported from Italian P. aeruginosa clinical isolates (90 % amino acid identity). The blaVIM-2 was located on a 45 kb plasmid that, in addition, conferred resistance to sulfonamides. Also, blaVIM-2 was the only gene cassette located within the variable region of a novel class 1 integron, In56 (Poirel et al., 2000). Two clonally unrelated P. aeruginosa clinical strains expressing VIM-2 enzyme were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris (Poirel et al., 2001b). In both isolates, the blaVIM-2 cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes (AMEs): AAC(6′)-29a and AAC(6′)-29b. These aminoglycoside acetyltransferases (AACs) conferred resistance to amikacin, isepamicin, kanamycin and tobramycin, but not to gentamicin, netilmicin and sisomicin. A retrospective epidemiological study in the Marseille hospital where the first VIM-2 producer was isolated found 20 more genetically indistinguishable P. aeruginosa isolates producing VIM-2 from several departments during 1996–1998 (Nordmann & Poirel, 2002). At the same time, VIM-1 and VIM-2-positive P. aeruginosa were reported as causes for numerous nosocomial infections in Italy and Greece (Cornaglia et al., 2000; Lagatolla et al., 2004; Tsakris et al., 2000; Mavroidi et al., 2000). Besides these VIM-2 metalloenzymes were found in P. aeruginosa clinical isolates in Spain (Prats et al., 2002; Peňa et al., 2007), Germany (Henrichfreise et al., 2005), Portugal (Pena et al., 2005), Poland (Patzer et al., 2005), Russia (Toleman et al., 2007a), Ireland (Walsh & Rogers, 2007), Turkey (Yakupogullari et al., 2008), Venezuela (Mendes et al., 2004b), Korea (Lee et al., 2002), Japan (Yatsuyanagi et al., 2004), Saudi Arabia (Guerin et al., 2005), China (Yu et al., 2006), India (Toleman et al., 2007b), the USA (Lolans et al., 2005), Columbia (Villegas et al., 2006) and Canada (Parkins et al., 2007), i.e. in the territories of four continents. In P. aeruginosa, VIM-2 is now the most widespread MBL that is associated with the localization of its encoding gene. The blaVIM-2 allele was found to be carried on mobile elements known as gene cassettes. They are inserted into class 1 integrons (Poirel et al., 2000, 2001b; Yu et al., 2006). Integron-located resistance genes provide them with an increased potential for expression and dissemination. Several class 1 integrons have been found in transposons (Yu et al., 2006), which enables the integrons to be transposed. This increases the threat of the blaVIM-2 gene being disseminated among diverse genera of bacteria.

    VIM-3 metalloenzyme was identified in a P. aeruginosa isolate in Taiwan (Yan et al., 2001). VIM-3 differs from VIM-2 by two amino acid substitutions and blaVIM-3 is a chromosomal gene. The following discoveries of VIM-type MBLs in P. aeruginosa isolates were made: VIM-4 in Greece (Pournaras et al., 2002), Hungary (Libisch et al., 2004), Poland (Patzer et al., 2004) and Sweden (Giske et al., 2003); VIM-5 in Turkey (Bahar et al., 2004); VIM-7 in the USA (Toleman et al., 2004); VIM-8 in Columbia (Crespo et al., 2004); VIM-11 in Argentina (Pasteran et al., 2005) and Italy (Mazzariol et al., 2005b); VIM-13 in Spain (Juan et al., 2008); VIM-15 in Bulgaria (Schneider et al., 2008); and VIM-16 in Germany (Schneider et al., 2008).

    In 2002 Toleman et al. (2002) detected a plasmid blaSPM-1 gene determining production of a new Ambler type class B MBL – SPM-1 – in a clinical P. aeruginosa isolate from Brazil. This enzyme is significantly distinct from IMP and VIM types MBLs (it has just 35.5 % amino acid identity with IMP-1), and is considered to be a representative of a new subfamily of class B MBLs (Poirel et al., 2004b). SPM-1 has a significantly higher molecular mass due to a unique loop containing 23 amino acid residues, which is not present in IMP and VIM-metalloenzymes. Generally, this carbapenemase binds cephalosporins more tightly than penicillins, which results in relatively large Km values (Walsh et al., 2005). Zavascki et al. (2000) reported the first nosocomial infection caused by P. aeruginosa producing carbepenem-resistant SPM-1 strains at the University Hospital in Porto Allegre, South Brazil (Zavascki et al., 2005).

    In 2002, Castanheira et al. (2004b) found a new MBL subclass – GIM-1 – in five multidrug-resistant P. aeruginosa strains isolated from different patients at a medical centre in Düsseldorf, Germany. The GIM-1 enzyme contains 250 amino acid residues and has a pI of 5.4. In respect to the amino acid identity with currently known molecular class B carbapenemases it differs from IMP, VIM and SPM-1 by 39–43, 28–31 and 28 %, respectively. GIM-1 does not hydrolyse aztreonam and serine-β-lactamase inhibitors. blaGIM-1 can be located on a plasmid (22 kb plasmid) and an integron (takes first position on a 6 kb class 1 integron, In77, which also includes aacA4, aadA1 gene cassettes and blaOXA-2).

    Resistance to β-lactams due to active efflux

    Generally, P. aeruginosa clinical isolates are less susceptible than Enterobacteriaceae to most classes of antimicrobials. For a long time the main reason for this natural resistance was considered to be the low outer membrane permeability due to the presence of proteins with high molecular mass – about 50 kDa (Livermore, 1984). According to modern concepts, these proteins (OprM, OprJ, OprN) act as components of active efflux systems with wide substrate specificity. Thus, the inherent resistance level of P. aeruginosa is to a great extent determined by the interplay between low membrane permeability and efflux of antimicrobial agents (Livermore, 2001). Active efflux is an important non-enzymic mechanism of β-lactam resistance in P. aeruginosa. Efflux also contributes to the development of multiple resistances to all strategic antipseudomonal antibiotics and is mediated by four genetically different three-component efflux systems that belong to the resistance–nodulation–division (RND) family (Livermore, 2001, 2002): MexA–MexB–OprM, MexC–MexD–OprJ, MexE–MexF–OprN and MexX–MexY–OprM. The structure of these efflux systems is shown in Table 3⇓. The first component is a protein located in the cytoplasmic membrane (MexB, MexD, MexF and MexY) that operates as an energy-dependent pump with wide substrate specificity. The second component is a gated outer membrane protein (OprM, OprJ, OprN and OprM). The third protein (MexA, MexC, MexE and MexX) is located in the periplasmic space and links the other two (Livermore, 2002).

    Table 3.

    Structure and substrate specificity of the three-component active efflux systems in P. aeruginosa

    MexA–MexB–OprM and MexX–MexY–OprM efflux systems participate simultaneously in natural and acquired antimicrobial-resistance mechanisms of P. aeruginosa, while MexC–MexD–OprJ and MexE–MexF–OprN act only in acquired resistance (Llanes et al., 2004; Poole et al., 1996; Kohler et al., 1999). Substrate specificities of the active three-component efflux systems operating in P. aeruginosa are also presented in Table 3⇑. The substrate profiles include various classes of antimicrobials (Masuda et al., 2000a).

    MexA–MexB–OprM overproduction often occurs in clinical isolates of P. aeruginosa and usually it is a result of increased transcription of the mexA–mexB–oprM operon due to mutations in the chromosomal gene encoding the MexR repressor protein, i.e. mutations at the mexR locus (Saito et al., 1999). nalB mutants are characterized by increased MICs and corresponding clinical resistance to most of the β-lactams (penicillins, cephalosporins, monobactams, meropenem to some extent, but not imipenem), quinolones, tetracyclines, chloramphenicol and trimethoprim (Livermore, 2001). They can be selected in vitro or during treatment with fluoroquinolones, penicillins or cephalosporins (Ziha-Zarifi et al., 1999). There are also other mutants called nalC that have intact mexR genes (Srikumar et al., 2000). nalC mutants originate from the wild-type P. aeruginosa PAO1 and are characterized by a mutation in the PA3721 gene (Cao et al., 2004). The protein encoded by this gene is a repressor of a two-gene operon; its function is unclear and its overexpression in nalC mutants leads to overproduction of the MexAB–OprM efflux system. Recently, nalD mutants were found. They have a mutation in the PA3574 gene that leads to MexA–MexB–OprM overexpression (Sobel et al., 2005). Masuda & Ohya (1992) were the first to report that MexA–MexB–OprM overexpression in P. aeruginosa determines decreased susceptibility to meropenem, but does not affect the activity of the other carbapenems – imipenem and panipenem (compared to wild-type P. aeruginosa). This is due to the different molecular structure of carbapenems – meropenem has a hydrophobic side-chain at the second position, which makes it a substrate for this efflux system, while imipenem and panipenem are not substrates as their side-chains are strongly charged and hydrophilic.

    The mexC–mexD–oprJ operon cannot be expressed constitutively, but is overexpressed in P. aeruginosa mutants possessing mutations in the nfxB gene, which encodes a transcriptional repressor (Poole et al., 1996). This efflux system predominantly exports extended-spectrum cephems (cefepime and cefpirome) from the bacterial cell, as well as quinolones, macrolides, tetracycline and chloramphenicol (Li et al., 2000).

    The third known efflux operon, mexE–mexF–oprN, determines resistance to quinolones, chloramphenicol and trimethoprim, and is overexpressed by the so called nfxC P. aeruginosa mutants (having a mutation at the mexT locus) (Kohler et al., 1999). nfxC mutants also show cross-resistance towards carbapenems (predominantly imipenem) as these have decreased expression of OprD outer membrane proteins. Unlike the rest of the efflux operons, mexE–mexF–oprN is subject to positive regulation by MexT protein, which belongs to the LysR family of transcriptional activators (Kohler et al., 1999; Li et al., 2000).

    Masuda et al. (2000b) found that MexX and MexY proteins export aminoglycosides, tetracycline and erythromycin from bacterial cells, and cooperate closely with spontaneously expressed OprM outer membrane proteins; thus taking part in the so called ‘intrinsic resistance’ of P. aeruginosa to antimicrobial agents. Like MexAB–OprM, MexXY proteins may be constitutively overproduced due to mutations in the mexZ repressor gene, which is located nearby and transcribed independently from the mexXY operon (Llanes et al., 2004; Vogne et al., 2004). Upregulation of MexXY–OprM affects aminoglycosides and fluoroquinolones (Mao et al., 2001).

    Overexpression of efflux systems with wide substrate profiles is an important mutational mechanism in P. aeruginosa. Its impact on the resistance to antipseudomonal antibiotics (β-lactams, fluoroquinolones, aminoglycosides and polymyxin B) is summarized in Table 4⇓ (Livermore, 2002).

    Table 4.

    Impact of overexpression of the active efflux systems on the resistance to antipseudomonal antibiotics

    Resistance to β-lactams due to altered outer membrane permeability

    Many of the imipenem-resistant P. aeruginosa clinical isolates are characterized by a deficiency of OprD (referred to as D2 porins) (Pechere & Kohler, 1999). OprD proteins form specific channels promoting the entry of basic amino acids and carbapenems, but no other β-lactam antibiotics (Livermore, 2001). In comparison with imipenem, meropenem cell influx is less affected by OprD deficiency. While imipenem MICs of the oprD mutants are within the range 8–32 mg l−1, MICs for meropenem are 2–4 mg l−1 (Pechere & Kohler, 1999; Pai et al., 2001). Loss of OprD determines resistance to carbapenems only in cases of expressed chromosomal AmpC β-lactamase, and this demonstrates the close cooperation between these two mechanisms (Livermore, 1992). Selection of resistant P. aeruginosa strains during imipenem treatment is a much more frequent phenomenon than the rise of ceftazidime-, piperacillin- or ciprofloxacin-resistant mutants (Livermore, 2001).

    Resistance to β-lactams due to an altered target

    The rarest mechanism of resistance to β-lactams in P. aeruginosa involves modification of the target site – PBPs. Altered PBP-4s with low affinity were reported after imipenem treatment, as well as after administration of high doses of piperacillin in patients suffering from cystic fibrosis. There are reports of reduced susceptibility to β-lactams in P. aeruginosa strains with overproduction of PBP-3s (Pechere & Kohler, 1999).

    Mechanisms of resistance to aminoglycosides

    Several groups of aminoglycoside resistance mechanisms are known: enzyme modification (major), low outer membrane permeability, active efflux and, rarely, target modification (Vakulenko & Mobashery, 2003; Poole, 2005; Magnet & Blanchard, 2005).

    AMEs

    AMEs attach a phosphate, adenyl or acetyl radical to the antibiotic molecule, and thus they decrease the binding affinity of the modified antibiotics to the target in the bacterial cell (30S ribosomal subunit) (Llano-Sotelo et al., 2002). AMEs are plasmid encoded and are divided into three classes: aminoglycoside phosphoryltransferases (APHs), aminoglycoside adenylyltransferases (also known as nucleotidyltransferases) (AADs or ANTs) and aminoglycoside acetyltransferases (AACs) (Vakulenko & Mobashery, 2003). Most frequently P. aeruginosa expresses the following AMEs: AAC(6′)-II (determines resistance to gentamicin, tobramycin and netilmicin), AAC(3)-I (resistance to gentamicin), AAC(3)-II (resistance to gentamicin, tobramycin and netilmicin), AAC(6′)-I (resistance to tobramycin, netilmicin and amikacin) and ANT(2′)-I (resistance to gentamicin and tobramycin) (Miller et al., 1997).

    Impermeability

    Aminoglycoside resistance that is independent from AMEs is characterized by resistance to all aminoglycosides, and often associated with reduced aminoglycoside accumulation (Bryan et al., 1976). This resistance is attributed to a reduced uptake due to diminished outer membrane permeability and is typically referred to as impermeability resistance. Numerous studies have highlighted the significance of impermeability resistance in aminoglycoside-resistant clinical isolates, particularly in cystic fibrosis isolates in which it is often the most common aminoglycoside resistance mechanism (MacLeod et al., 2000).

    Active efflux

    Active aminoglycoside efflux is a relatively rare resistance mechanism that is due to MexXY proteins operating simultaneously with OprM (Masuda et al., 2000a; Vogne et al., 2004), as well as with some other outer membrane proteins – OpmB, OpmG, OpmI (Jo et al., 2003) – thus forming three-component active efflux systems.

    Target modification

    Methylation of 16S rRNA has recently emerged as a new mechanism of resistance against aminoglycosides among Gram-negative pathogens belonging to the family Enterobacteriaceae and glucose-nonfermentative microbes, including P. aeruginosa and Acinetobacter species (Doi & Arakawa, 2007). This event is mediated by a newly recognized group of 16S rRNA methylases, which share modest similarity to those produced by aminoglycoside-producing actinomycetes. The responsible genes are usually located on transposons within transferable plasmids, which provides them with the potential to spread horizontally, and may partially explain the worldwide distribution of this novel resistance mechanism.

    The first 16S rRNA methylase, called RmtA, was reported in an aminoglycoside-resistant P. aeruginosa clinical isolate from Japan, in 2003 (Yokoyama et al., 2003). The enzyme was found to confer a high-level resistance to all parenterally administered aminoglycosides, including amikacin, tobramycin, isepamicin, kanamycin, arbekacin and gentamicin (MICs >1024 mg l−1). The structural gene of RmtA was associated with a genetic element that resembled a mercury-resistance transposon Tn5041 on a transferable plasmid (Yamane et al., 2004). The mol% G+C of rmtA was 55 %, suggesting its origin from some mol% G+C rich microbes, including actinomycetes.

    RmtD, a novel 16S rRNA methylase, was identified in a panresistant P. aeruginosa strain isolated in 2005 from an in-patient in Brazil (Doi et al., 2006). The methylase accounts for a high-level resistance to all 4,6-disubstituted deoxystreptamine aminoglycosides, such as amikacin, tobramycin, and gentamicin. RmtD shares a moderate degree of identity with RmtA. β-Lactam resistance of the studied isolate is conferred by the production of the MBL SPM-1.

    Non-enzymic mechanisms involved in the gradual development of aminoglycoside resistance

    Recently, El'Garch et al. (2007) examined the interplay and cumulative effects of different non-enzymic mechanisms engineered in a reference strain P. aeruginosa PAO1. Their data revealed that the accumulation of mutants leads to a gradual increase in the resistance to aminoglycosides, as seen in cystic fibrosis patients (Hurley et al., 1995; MacLeod et al., 2000). Four genes of P. aeruginosa (namely, galU, nuoG, mexZ and rplY) have been shown to be involved in the gradual increase in MICs of aminoglycosides (El'Garch et al., 2007).

    P. aeruginosa galU is required for synthesis of a complete LPS core and its inactivation results in the production of truncated (rough) LPS molecules lacking both A- and B-band polysaccharides in P. aeruginosa (Dean & Goldberg, 2002). Loss of the A- and B-band LPS was reported to impair the antibacterial activity of aminoglycosides by compromising their binding to the cell surface (Kadurugamuwa et al., 1993).

    Abolished nuoG expression promotes the disruption of the nuoABDEFGHIJKLMN operon that codes for proton-translocating type I NADH oxidoreductase (El'Garch et al., 2007). Inactivation of the enzymic complex that significantly contributes to proton electrochemical gradient impairs membrane energetics and thereby the uptake of aminoglycosides (Taber et al., 1987).

    Inactivation of the repressor gene mexZ results in increased expression of the mexY gene and constitutive overproduction of the multidrug transporter MexY (Vogne et al., 2004; El'Garch et al., 2007). Suppression of the gene rplY, which encodes for ribosomal protein L25, results in both moderate upregulation of the efflux system MexXY–OprM and hypersusceptibility to β-lactam antibiotics (El'Garch et al., 2007).

    Disruption of the described genes individually led to increased aminoglycoside resistance (in the region of twofold). Construction of double, triple and quadruple mutants demonstrated cumulative effects of the different mechanisms on aminoglycoside resistance, with MICs increasing from 16- to 64-fold in the quadruple mutant compared to the wild-type P. aeruginosa PAO1 strain. Altogether, these results illustrate how P. aeruginosa may gradually develop high resistance to these antibiotics via non-enzymic mechanisms, as seen in cystic fibrosis patients (El'Garch et al., 2007).

    Mechanisms of resistance to fluoroquinolones

    Two major mechanisms lead to fluoroquinolone resistance in P. aeruginosa: structural changes in target enzymes and active efflux (Hooper, 2001). Modification of the primary target for fluoroquinolones (DNA gyrase, also known as topoisomerase II) occurs by point mutations in gyrA/gyrB genes within the QRDR (quinolone-resistant-determinative region) motif, which is considered as the enzyme's active site. As a result of these mutations, the amino acid sequence of A and B subunits alters, which leads to synthesis of modified topoisomerase II with low binding affinity to quinolone molecules. Modifications of a secondary target (topoisomerase IV) occur as a result of point mutations in parC and parE genes encoding ParC and ParE enzyme subunits, respectively.

    As described above, four well known genetically different efflux systems were identified in P. aeruginosa: MexAB–OprM, MexCD–OprJ, MexEF–OprN and MexXY–OprM. While each pump has a preferential set of antimicrobial agent substrates, the fluoroquinolones are universal substrates for each of them (Masuda et al., 2000a). A new member of the tripartite multidrug efflux pumps, MexV (membrane fusion protein)–MexW (RND-type membrane protein)–OprM, was found in P. aeruginosa in 2003 (Li et al., 2003). It confers resistance to fluoroquinolones, tetracycline, chloramphenicol, erythromycin, ethidium bromide and acriflavine. High-level fluoroquinolone resistance in P. aeruginosa is attributable to the interplay of the efflux pump systems and mutations of the genes encoding DNA gyrase and topoisomerase IV (Nakajima et al., 2002; Wang et al., 2007).

    According to a number of studies, quinolones may select multidrug-resistant phenotypes in vitro, as well as in vivo (Masuda & Ohya, 1992; Kohler et al., 1997). The most common causes for their appearance are the following mutations: nalB, nfxB and nfxC leading, respectively, to overexpression of MexA–MexB–OprM, MexC–MexD–OprJ and MexE–MexF–OprN (Table 4⇑). The new fluoroquinolones select predominantly nfxB P. aeruginosa mutants, while older quinolones select for nfxC or nalB mutants (Kohler et al., 1997).

    Conclusion

    P. aeruginosa is a uniquely problematic nosocomial pathogen because of the following: the species' natural resistance to many drug classes; its ability to acquire resistance (via mutations) against all relevant treatments; its high resistance rates worldwide; and frequent implication in severe infections. Multidrug resistance (MDR) is common and increasing. A number of strains have now been identified that exhibit resistance to essentially all reliable antipseudomonal antibiotics. This problem grows with the incidence of integrons that carry gene cassettes encoding both carbapenemases and AACs.

    MDR in P. aeruginosa makes treatment of infections caused by this organism both difficult and expensive. Improved methods for antimicrobial susceptibility testing are needed, including detection of emerging strains producing ESBLs and MBLs. Clinical studies are needed to identify risk factors for MDR development, as well as to determine the most efficacious antimicrobial regimens and duration of therapy to maximize successful outcomes in the treatment of severe infections due to multiresistant P. aeruginosa. Prudent antimicrobial policies combined with good infection control practices worldwide could guarantee a limitation in the development and spread of resistance to β-lactams, aminoglycosides and fluoroquinolones, ensuring that these agents will maintain their place in the therapy of P. aeruginosa infections.

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