ACQUIRED ANTIBIOTIC RESISTANCE

In addition to its remarkable intrinsic resistance, P. aeruginosa shows an extraordinary capacity for further developing resistance to all available antibiotics. In general, bacteria can increase its intrinsic resistance by either acquiring horizontal resistance determinants and/or through the selection of certain chromosomal mutations that alter their expression and/or function.

1.8.1. Transferable resistance determinants in CF isolates

The CF airway hosts a complex microbiome [Lim YW et al, 2014] where genetic exchange could theoretically occur effectively, thus theoretically contributing to the emergence of antibiotic resistance. Most mobile antibiotic resistance genes are encoded on plasmids and transposons, but a recent study have also suggested that phages may also play an important role in the CF setting as the CF virome encodes more antimicrobial resistance sequences than the non-CF virome [Fancello L et al, 2011].

Among the transferable resistance determinants, extended spectrum b-lactamases and carbapenemeses are widely distributed worldwide but, with some exceptions, horizontal gene transfer of resistance determinants seems not to be frequent in P. aeruginosa [Oliver A et al, 2015], especially among CF isolates. Although biofilms are known to provide cell-to-cell contact and stabilise mating pair formation, biofilms theirselves appear to limit the horizontal plasmid spread through a combination of physicochemical and biological factors inherent to the spatial structure and heterogeneity of these structures [Stalder T & Top E, 2016].

However, it should be mentioned that although rare, in late years some authors have reported several cases of CF patients infections with P. aeruginosa isolates producing ESBL and/or carbapenemases, including IMP and VIM metallo- β-lactamases [Agarwal G et al, 2005; Cardoso O et al, 2008; Pollini S et al, 2011].

1.8.2. Mutation-driven resistance

In comparison with P. aeruginosa isolates causing acute infections, mutation-driven resistance has been shown to be the major contributor to antimicrobial resistance development in CF P. aeruginosa isolates [Ferroni A et al, 2009], development which is indeed catalyzed by the unusual high prevalence of mutators in the CF airways.

All antibiotics compounds are prone to being compromised by acquiring mutations that eventually lead to alter the expression of chromosomally-encoded resistance mechanisms or that modify the function of its encoded-protein. In P. aeruginosa, major mutational resistance mechanisms include overexpression of the chromosomal AmpC cephalosporinase, efflux pumps overexpression, porin loss or altered antibiotic targets (Table 1.3.).

Table 1.3. Mutation-driven resistance mechanisms in P. aeruginosa.

Mutation Resistance mechanisms / Altered target Antibiotics affected^a

gyrA, gyrB DNA gyrase FQ

parC, parE DNA topoisomerase IV FQ

pmrAB LPS (lipid A) CO

mexR MexAB–OprM hyperproduction FQ, TZ, PM, PPT, MP

nalC MexAB-OprM hyperproduction FQ, TZ, PM, PPT, MP

nalD MexAB-OprM hyperproduction FQ, TZ, PM, PPT, MP

nfxB MexCD-OprJ Hyperproduction FQ, PM

mexS MexEF-OprN hyperproduction FQ

OprD downregulation IP, MP

mexT MexEF-OprN hyperproduction FQ

OprD downregulation IP, MP

mvaT MexEF-OprN hyperproduction FQ

mexZ MexXY –OprM hyperproduction FQ, AMG, PM

PA5471.1 MexXY –OprM hyperproduction FQ, AMG, PM

amgS MexXY –OprM hyperproduction FQ, AMG, PM

oprD OprD porin inactivation IP, MP

ampC AmpC structural modification PPT, TZ, PM, IP, MP

ampD AmpC hyperproduction TZ, PM, PPT

ampDh2 AmpC hyperproduction TZ, PM, PPT

ampDh3 AmpC hyperproduction TZ, PM, PPT

ampR AmpC hyperproduction TZ, PM, PPT

dacB AmpC hyperproduction TZ, PM, PPT

glpT Transporter protein GlpT FO

rpoB RNA polymerase β-chain RIF

β-lactam resistance mechanisms. Development of resistance to antipseudomonal penicillins (ticarcillin and piperacillin), cephalosporins (ceftazidime and cefepime) and monobactams (aztreonam) is the selection of mutations within PGN-recycling genes (ampD, dacB, ampR) that eventually leads to the constitutive overexpression of the chromosomal cephalosporinase AmpC [Cabot G et al, 2011; Juan C et al, 2005; Moyà B et al, 2009].

Besides ampC overexpression, recent studies have revealed that β-lactam resistance development, including novel β-lactam-β-lactamase inhibitor combinations such as ceftolozane/tazobactam, may also result from mutations leading to the structural modification of AmpC [Cabot G et al, 2014; Lahiri SD et al, 2014].

Beyond the chromosomal cephalosporinase AmpC, another contributing factor to β-lactam resistance is MexAB-OprM overexpression. This efflux system displays the broadest substrate profile (Table 1.1. and 1.3.) and its mutational overexpression determines reduced susceptibility to all β-lactams with the single exception of imipenem. MexAB-OprM-overproducing mutants can be readily generated in vitro in the presence of antibiotic by the selection of any mutational event leading to the inactivation or impairment of the mexR, nalC or nalD regulator genes. As well, these mutants have been shown to be very prevalent among multiresistant non-CF strains, and, of note, rates of MexAB-OprM overproducers of near 50% have been recorded in subpopulations of isolates exhibiting a reduced susceptibility to ticarcillin (≥32 μg/ml) [Li XZ et al, 2015].

Likewise, the mutational overexpression of MexXY or MexCD-OprJ can also confer resistance to cefepime. MexCD-OprJ overexpression, which is more frequent among P.

aeruginosa isolates recovered from CRI not only confers increased cefepime resistance but have also been shown to determine hypersusceptibility to most β-lactams and aminoglycosides [Mulet X et al, 2011].

Finally, screenings of transposon mutant libraries have shown that inactivation of galU, a gene which code for an enzyme involved in the LPS core, increases ceftazidime and meropenem minimum inhibitory concentrations (MICs) [Dötsch A et al, 2009; Álvarez-Ortega C et al, 2010].

Carbapenem resistance mechanisms. Mutational inactivation of the porin OprD, together with the inducible expression of AmpC, confers resistance to imipenem and reduced susceptibility to meropenem [Livermore DM et al, 1992]. Indeed, the prevalence of imipenem resistant isolates frequently exceeds 20%, and nearly all them are OprD deficient [Cabot G et al, 2011; Riera E et al, 2011]. As well, MexAB-OprM mutational overexpression determines reduced susceptibility to meropenem and its overexpression plus OprD inactivation is one of the most relevant causes of clinical resistance to this carbapenem [Riera E et al, 2011].

Finally, although less frequent, mutation-driven resistance to carbapenems can also result from MexEF-OprN overexpression, as mutations within mexT,mexS and/or the ParRS two-component system not only lead to MexEF-OprN overexpression but also to OprD downregulation, which in turns determine a reduced susceptibility to carbapenems [Köhler T et al, 1999; Li XZ et al, 2015].

Aminoglycoside resistance mechanisms. Resistance to this antibiotic class has been clasically linked to the mutational overexpression of MexXY efflux pump, being its overexpression very frequent among clinical isolates and mainly caused by mexZ, amgS, or parRS mutations [Guénard S et al, 2014]. However, recent studies have revealed that the aminoglycoside mutational resistome extends far beyond MexXY overexpression and

several novel resistance determinants have been described; moreover accumulation of mutations within these genes can eventually lead to high-level antibiotic resistance [El’Garch F et al, 2007; Schurek KN et al, 2008].

Fluoroquinolone resistance mechanisms. Fluoroquinolone resistance in P. aeruginosa frequently results from gain-of-function mutations in topoisomerases, including DNA gyrases (GyrA/GyrB) and type IV topoisomerases (ParC/ParE) [Bruchmann S et al, 2013].

Besides, overexpression of all 4 major efflux-pumps systems also contributes to fluoroquinolone resistance (Table 1.1. and 1.3.). The overexpression of MexAB-OprM and MexXY-OprM is globally more frequent among clinical strains but its contribution to clinical fluoroquinolone resistance is likely more modest [Bruchmann S et al, 2013]. On the other hand, mutational overexpression of MexEF-OprN or MexCD-OprJ efflux pump is associated with high-level fluoroquinolone resistance, and although their prevalence is considered low except in the CF-CRI setting, recent data show that it might be higher than expected [Richardot C et al, 2015].

Polymyxin resistance mechanisms. The prevalence of polymyxin (polymyxin B and colistin) resistance is still very low (<5%) among P. aeruginosa isolates. Resistance to polymyxins most frequently results from the modification of the LPS caused by the addition of a 4-amino-4-deoxy-l-arabinose moiety in the lipid A structure [Olaitan AO et al, 2014] and the underlying mutations are frequently tracked to the PmrAB or PhoPQ two-component regulators, which in turns lead to the activation of the arnBCADTEF operon [Barrow K &

Kwon DH, 2009]. More recent studies have also revealed that mutations within the two-component regulator ParRS, in addition to conferring colistin resistance due to the activation of the arnBCADTEF operon, lead to a MDR profile caused by the overprexpression of MexXY, MexEF and the repression of OprD [Muller C et al, 2011]. Finally, two additional two-component regulators, ColRS and CprRS, have also been shown to play a role in polymyxin resistance [Gutu AD et al, 2013]. Moreover, recent in vitro evolution assays have revealed, through WGS, the implication of additional mutations in high level colistin resistance, facilitated by the emergence of mutator (mutS deficient) phenotypes [Döβelmann B et al, 2017]. Particularly noteworthy among them are those occurring in LptD (essentaial OMP involved in LPS transport), LpxC (UDP-3-O-[hydroxymyristoyl]-N-acetylglucosamine deacetylase involved in lipid A biosynthesis) or MigA (α-1,6-rhamnosyltransferase, involved in the synthesis of the LPS core region [Döβelmann B et al, 2017].

P. aeruginosa possesses a complex and large genome, thus, and given the current gaps and the crutial role of mutatrion-driven resistance mechanisms for acquiring antibiotic resistance, one of the aims of this work was to decipher the P. aeruginosa mutational resistome.