No abstract

bWe compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ⌬mutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64؋ MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64؋ MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 g/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 g/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillintazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.

Modification of intrinsic (AmpC) and horizontally acquired β-lactamases appears to be the main mechanism leading to ceftolozane/tazobactam resistance in MDR P. aeruginosa.

T he increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate treatments and is therefore associated with significant morbidity and mortality (1-3). This growing threat results from the interplay of the extraordinary capacity of this pathogen for developing resistance to nearly all available antibiotics by the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (metallo-␤-lactamases [MBLs]) or extended-spectrum ␤-lactamases (ESBLs), frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (4). Over the last decade, multiple reports have warned about the epidemic dissemination of XDR/ MDR strains in multiple hospitals (5-10). Even more concerning are recent reports which have provided evidence of the existence of MDR/XDR clones of P. aeruginosa disseminated in multiple institutions worldwide, denominated epidemic highrisk clones (11). Among them, are the most widespread (12-18). Moreover, in a recent multicenter study of P. aeruginosa bloodstream infections, we showed that all XDR isolates (10.5% of all isolates) and some MDR isolates belonged to the epidemic high-risk clones, which were not detected among susceptible isolates (19); the specific genetic resistance markers of these clones, which may include multiple combinations of chromosomal mutations and/or horizontally acquired resistance elements, were also described in detail for the first time (19).The global success of bacterial pathogens is expected to be determined by a complex interplay between pathogenicity, epidemicity, and antibiotic resistance (20). The fitness cost of antibiotic resistance mechanisms (21), the existence of regulatory networks interconnecting resistance and virulence (22,23), and natural genetic engineering linking antibiotic resistance determinants and clonal success through genetic capitalism (24) are thought to be the main elements of this intricate equation (25). While the impacts on virulence and fitness of several individual antibiotic resistance mechanisms of P. aeruginosa have been evaluated (26-28), the specific adaptive traits that may explain the global success of epidemic high-risk clones remains unexplored. Since understanding the reasons for the success of these clones could be crucial for designing specific treatment and infection control strategies (29), the objective of this work was to determine the potential underlying biological parameters. For this purpose, using a large collection of well-characterized strains with different resistance profiles from a Spanish multicenter study of bloodstream infections (19,30) and control

Although the prevalence of MBL producers is increasing, mutation-driven resistance is still more frequent in Spain. Imipenem resistance was driven by OprD inactivation, while additional AmpC and particularly efflux pump hyperproduction had a lower impact on the activity of doripenem compared with meropenem.

A prospective, descriptive observational study of consecutive patients treated with ceftolozane/tazobactam in the reference hospital of the Balearic Islands (Spain), between May 2016 and September 2017, was performed. Demographic, clinical, and microbiological variables were recorded. The later included resistance profile, molecular typing, and whole genome sequencing of isolates showing resistance development. Fifty-eight patients were treated with ceftolozane/tazobactam. Thirty-five (60.3%) showed respiratory tract infections, 21 (36.2%) received monotherapy, and 37 (63.8%) combined therapy for ≥ 72 h, mainly with colistin (45.9%). In 46.6% of the patients, a dose of 1/0.5 g/8 h was used, whereas 2/1 g/8 h was used in 41.4%. In 56 of the cases (96.6%), the initial Pseudomonas aeruginosa isolates recovered showed a multidrug resistant (MDR) phenotype, and 50 of them (86.2%) additionally met the extensively drug resistant (XDR) criteria and were only susceptible colistin and/or aminoglycosides (mostly amikacin). The epidemic high-risk clone ST175 was detected in 50% of the patients. Clinical cure was documented in 37 patients (63.8%) and resistance development in 8 (13.8%). Clinical failure was associated with disease severity (SOFA), ventilator-dependent respiratory failure, XDR profile, high-risk clone ST175, negative control culture, and resistance development. In 6 of the 8 cases, resistance development was caused by structural mutations in AmpC, including some mutations described for the first time in vivo, whereas in the other 2, by mutations in OXA-10 leading to the extended spectrum OXA-14. Although further clinical experience is still needed, our results suggest that ceftolozane/tazobactam is an attractive option for the treatment of MDR/XDR P. aeruginosa infections.

Azithromycin (AZM) has shown promising results in the treatment of Pseudomonas aeruginosa chronic lung infections such as those occurring in cystic fibrosis (CF) patients. We evaluated the effect of hypermutation and alginate hyperproduction on the bactericidal activity and resistance development to AZM in P. aeruginosa biofilms. Strains PAO1, its mucA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were used. Biofilms were incubated with several AZM concentrations for 1, 2, 4, or 7 days, and the numbers of viable cells were determined. During the first 2 days, AZM showed bactericidal activity for all the strains, but in extended AZM incubation for strain PAOMS and especially strain PAOMSA, a marked increased in the number of viable cells was observed, particularly at 4 g/ml. Biofilms formed by the lineages recovered from the 7-day experiments showed enhanced AZM resistance. Furthermore, most of the independent lineages studied, including those obtained from biofilms treated with AZM concentrations as low as 0.5 g/ml, showed MexCD-OprJ hyperexpression and mutations in nfxB. The role of nfxB mutation in AZM resistance was further confirmed through the characterization of nfxB and mexD knockout mutants. Results from this work show that, although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected and that, furthermore, they frequently show cross-resistance to other unrelated antipseudomonal agents such as ciprofloxacin or cefepime but hypersusceptibility to others such as imipenem or tobramycin. Therefore, these results should help guide the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.The establishment of Pseudomonas aeruginosa chronic respiratory infection is mediated by a complex adaptive process that includes physiological changes, mainly represented by the transition from a planktonic to a biofilm mode of growth (3) and by the selection of an important number of adaptive mutations required for long-term persistence (42). The biofilm mode of growth is one of the most important factors in the persistence of chronic lung infections due to its increased resistance to the host defense mechanisms, including mechanical clearance and clearance mediated by complement, antibodies, or phagocytes, and to its inherent resistance to antibiotics (14).A common feature of P. aeruginosa chronic lung infections, including those occurring in patients suffering from cystic fibrosis (CF), bronchiectasias, or chronic obstructive pulmonary disease, is the very high prevalence (30 to 60% of patients) of hypermutable (or mutator) strains deficient in the DNA mismatch repair system in contrast to what is observed in acute infections (Ͻ1%) (2,8,23,31,32). The presence of hypermutable strains has been found to be linked to the high antibiotic resistance rates of P. aeruginosa strains isolated from patients with chronic lung infections (23, 32). It has also been shown by in vitro and in...

Chronic respiratory infection by Pseudomonas aeruginosa is a major cause of mortality in cystic fibrosis (CF). We investigated the interplay between three key microbiological aspects of these infections: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of antibiotic resistance. For this purpose, 10 sequential isolates, covering up to an 8-year period, from each of 10 CF patients were studied. As anticipated, resistance significantly accumulated overtime, and occurred more frequently among mutator variants detected in 6 of the patients. Nevertheless, highest resistance was documented for the nonmutator CF epidemic strain LES-1 (ST-146) detected for the first time in Spain. A correlation between resistance profiles and resistance mechanisms evaluated [efflux pump (mexB, mexD, mexF, and mexY) and ampC overexpression and OprD production] was not always obvious and hypersusceptibility to certain antibiotics (such as aztreonam or meropenem) was frequently observed. The analysis of whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) revealed that a single genotype (clone FQSE-A) produced persistent infections in 4 of the patients. Multilocus Sequence typing (MLST) identified clone FQSE-A as the CF epidemic clone ST-274, but striking discrepancies between PFGE and MLST profiles were evidenced. While PFGE macrorestriction patterns remained stable, a new sequence type (ST-1089) was detected in two of the patients, differing from ST-274 by only two point mutations in two of the genes, each leading to a nonpreviously described allele. Moreover, detailed genetic analyses revealed that the new ST-1089 is a mutS deficient mutator lineage that evolved from the epidemic strain ST-274, acquired specific resistance mechanisms, and underwent further interpatient spread. Thus, presented results provide the first evidence of interpatient dissemination of mutator lineages and denote their potential for unexpected short-term sequence type evolution, illustrating the complexity of P. aeruginosa population biology in CF.