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1.
Artigo em Inglês | MEDLINE | ID: mdl-31430372

RESUMO

OBJECTIVES: To evaluate the correlation of O-antigen serotypes with resistance profiles and high-risk clones in a Spanish nationwide survey. METHODS: Up to 30 consecutive healthcare-associated Pseudomonas aeruginosa isolates were collected during October 2017 from each of 51 hospitals (covering all Spanish regions) with a total of 1445 isolates studied. MICs of 13 antipseudomonal agents and MDR/XDR profiles had been previously determined, as well as whole-genome sequences of 185 representative XDR isolates. O-antigen serotypes (O1-O16) were determined by agglutination using serotype-specific antisera (BioRad). The Pseudomonas aeruginosa serotyper (PAst) program was used for in silico serotyping. RESULTS: The most frequent serotypes were O6 (17.8%), O1 (15.4%) and O11 (13.3%). In contrast, the most frequent serotype among XDR isolates (17.3%) was O4 (34.1%), distantly followed by O11 (15.9%). Within serotypes, XDR phenotypes were more frequent for O12 (60.0%) and O4 (57.3%). The most frequent clone among the XDR isolates was ST175 (40.9%), followed by CC235 (10.7%), ST308 (5.2%) and CC111 (3.6%). Up to 81.6% of XDR ST175 isolates typed O4, whereas 18.4% were non-typeable. O4 genotype was detected in all sequenced (n = 55) ST175 isolates. On the other hand, CC235 and ST308 were associated with O11, whereas CC111 was linked to serotype O12. CONCLUSIONS: O4 serotype is linked to the MDR/XDR profile of widespread ST175 (typically only susceptible to colistin, amikacin and the novel combinations ceftolozane/tazobactam and ceftazidime/avibactam) and therefore, after local validation, its detection in the microbiology laboratory might be useful for guiding semi-empirical antipseudomonal therapies and infection control measures in Spanish hospitals.

2.
J Glob Antimicrob Resist ; 18: 37-44, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31154007

RESUMO

BACKGROUND: Extensively drug-resistant (XDR) Pseudomonas aeruginosa (P. aeruginosa) and particularly P. aeruginosa high-risk clones, are of growing concern because treatment options are limited. For years, colistin monotherapy has been the only available treatment, but is well known that is not an optimal treatment. A combination of colistin with another antibiotic could be a possible therapeutic option. OBJECTIVES: This study aimed to investigate effective antibiotic combinations against 20 XDR P. aeruginosa isolates obtained in a Spanish multicentre study (2015). METHODS: Forty-five checkerboards with six antipseudomonal antibiotics (amikacin, aztreonam, ceftazidime, meropenem, colistin, and ceftolozane/tazobactam) were performed to determine whether combinations were synergic or additive by fractional inhibitory concentration indices. On average, 15 different regimens were evaluated in duplicate against the three most prevalent high-risk clones (ST175, ST235, ST111) by time-kill analyses over 24h. The combination showing synergism in the three high-risk clones was validated in all studied XDR isolates. RESULTS: In time-kill curves, the untreated control failed, as did each study regimen when administered alone. Two combinations were synergistic in the three high-risk clones that were initially studied: amikacin plus ceftazidime and colistin plus meropenem, with the second being the most effective combination. The efficacy of colistin plus meropenem was then tested in all 20 isolates. A synergistic bacterial density reduction for the duration of the study occurred in 80% of the entire XDR collection. CONCLUSIONS: These data suggest that colistin plus meropenem may be a useful combination for the treatment of infections due to XDR P. aeruginosa, including high-risk clones, which warrants evaluation in a clinical trial.

3.
J Antimicrob Chemother ; 74(7): 1825-1835, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30989186

RESUMO

OBJECTIVES: To undertake a Spanish nationwide survey on Pseudomonas aeruginosa molecular epidemiology and antimicrobial resistance. METHODS: Up to 30 consecutive healthcare-associated P. aeruginosa isolates collected in 2017 from each of 51 hospitals were studied. MICs of 13 antipseudomonal agents were determined by broth microdilution. Horizontally acquired ß-lactamases were detected by phenotypic methods and PCR. Clonal epidemiology was evaluated through PFGE and MLST; at least one XDR isolate from each clone and hospital (n = 185) was sequenced. RESULTS: The most active antipseudomonals against the 1445 isolates studied were colistin and ceftolozane/tazobactam (both 94.6% susceptible, MIC50/90 = 1/2 mg/L) followed by ceftazidime/avibactam (94.2% susceptible, MIC50/90 = 2/8 mg/L). Up to 252 (17.3%) of the isolates were XDR. Carbapenemases/ESBLs were detected in 3.1% of the isolates, including VIM, IMP, GES, PER and OXA enzymes. The most frequent clone among the XDR isolates was ST175 (40.9%), followed by CC235 (10.7%), ST308 (5.2%) and CC111 (4.0%). Carbapenemase production varied geographically and involved diverse clones, including 16.5% of ST175 XDR isolates. Additionally, 56% of the sequenced XDR isolates showed horizontally acquired aminoglycoside-modifying enzymes, which correlated with tobramycin resistance. Two XDR isolates produced QnrVC1, but fluoroquinolone resistance was mostly caused by QRDR mutations. Beyond frequent mutations (>60%) in OprD and AmpC regulators, four isolates showed AmpC mutations associated with resistance to ceftolozane/tazobactam and ceftazidime/avibactam. CONCLUSIONS: ST175 is the most frequent XDR high-risk clone in Spanish hospitals, but this nationwide survey also indicates a complex scenario in which major differences in local epidemiology, including carbapenemase production, need to be acknowledged in order to guide antimicrobial therapy.

4.
Sci Rep ; 9(1): 3575, 2019 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837659

RESUMO

In the current scenario of high antibiotic resistance, the search for therapeutic options against Pseudomonas aeruginosa must be approached from different perspectives: cell-wall biology as source of bacterial weak points and our immune system as source of weapons. Our recent study suggests that once the permeability barrier has been overcome, the activity of our cell-wall-targeting immune proteins is notably enhanced, more in mutants with impaired peptidoglycan recycling. The present work aims at analyzing the activity of these proteins [lysozyme and Peptidoglycan-Recognition-Proteins (PGLYRPs)], alone or with a permeabilizer (subinhibitory colistin) in clinical strains, along with other features related to the cell-wall. We compared the most relevant and complementary scenarios: acute (bacteremia) and chronic infections [early/late isolates from lungs of cystic fibrosis (CF) patients]. Although a low activity of lysozyme/PGLYRPs per se (except punctual highly susceptible strains) was found, the colistin addition significantly increased their activity regardless of the strains' colistin resistance levels. Our results show increased susceptibility in late CF isolates, suggesting that CF adaptation renders P. aeruginosa more vulnerable to proteins targeting the cell-wall. Thus, our work suggests that attacking some P. aeruginosa cell-wall biology-related elements to increase the activity of our innate weapons could be a promising therapeutic strategy.

5.
Artigo em Inglês | MEDLINE | ID: mdl-30745381

RESUMO

Hypermutable Pseudomonas aeruginosa isolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutable P. aeruginosa isolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59 P. aeruginosa clinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterized P. aeruginosa strains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations in mutL or, less commonly, in mutS Hypermutable isolates also contained a range of mutations that are likely associated with adaptation of P. aeruginosa to the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutable P. aeruginosa strains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance.

6.
Eur J Clin Microbiol Infect Dis ; 37(11): 2191-2200, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30141088

RESUMO

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.

7.
J Antimicrob Chemother ; 73(10): 2770-2776, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30052973

RESUMO

Background: The appearance and dissemination of MDR among pathogenic bacteria has forced the search for new antimicrobials. Bacteriocins have been proposed as potential alternatives for the treatment of infections due to multiresistant strains. Objectives: To analyse the activity of R-pyocins against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis and other sources and evaluate them as a potential adjuvant or alternative to the current antibiotic treatment. Methods: The activity of R-pyocins against 150 strains of P. aeruginosa isolated from patients with cystic fibrosis or bacteraemia was studied through spot assay. Interactions between R-pyocins and antipseudomonal agents were quantitatively studied by the chequerboard method. Results: The proportion of P. aeruginosa isolates susceptible to R-pyocins was found to be higher in cystic fibrosis isolates compared with bacteraemia isolates (79.41% versus 50%). Moreover, no interactions were found between common antipseudomonal agents and R-pyocin susceptibility, except for the ST175 high-risk clone. Conclusions: Our results highlight the possibility of using R-pyocins as therapeutic agents, alone or as adjuvants, against P. aeruginosa in cystic fibrosis.

8.
Front Microbiol ; 9: 685, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29681898

RESUMO

One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including ß-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

9.
Artigo em Inglês | MEDLINE | ID: mdl-29530842

RESUMO

The aim of this study was to investigate the efficacy of ceftolozane-tazobactam in combination with meropenem against an extensively drug-resistant (XDR) Pseudomonas aeruginosa high-risk clone, sequence type 175, isolated in a Spanish university hospital. A 14-day hollow-fiber infection model was used to simulate clinical exposure of the two drug regimens alone and in combination, and serial samples were collected to determine drug concentrations and CFU counts. The untreated control failed, as did each study regimen when administered alone. However, when ceftolozane-tazobactam was administered in combination with meropenem, there was a >4-log10 CFU/ml bacterial density reduction and suppression of resistance for the duration of the study. These data suggest that ceftolozane-tazobactam plus meropenem may be a useful combination for treating XDR P. aeruginosa.

10.
Artigo em Inglês | MEDLINE | ID: mdl-29437613

RESUMO

Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments.

13.
Artigo em Inglês | MEDLINE | ID: mdl-28923877

RESUMO

The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large (n = 140) collection of well-characterized P. aeruginosa isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a Caenorhabditis elegans infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the C. elegans model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its exoU+ type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our C. elegans model. Other markers, such as motility or pigment production, were not essential for virulence in the C. elegans model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the C. elegans model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between P. aeruginosa resistance profiles, high-risk clones, and virulence.


Assuntos
Proteínas de Bactérias/genética , Caenorhabditis elegans/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Pseudomonas aeruginosa/genética , Sistemas de Secreção Tipo III/genética , Animais , Antibacterianos/farmacologia , Bacteriemia/microbiologia , Bacteriemia/patologia , Proteínas de Bactérias/metabolismo , Células Clonais , Infecção Hospitalar/microbiologia , Infecção Hospitalar/patologia , Modelos Animais de Doenças , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Sistemas de Secreção Tipo III/metabolismo , Virulência
14.
Artigo em Inglês | MEDLINE | ID: mdl-28874376

RESUMO

This study assessed the molecular epidemiology, resistance mechanisms, and susceptibility profiles of a collection of 150 extensively drug-resistant (XDR) Pseudomonas aeruginosa clinical isolates obtained from a 2015 Spanish multicenter study, with a particular focus on resistome analysis in relation to ceftolozane-tazobactam susceptibility. Broth microdilution MICs revealed that nearly all (>95%) of the isolates were nonsusceptible to piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, and ciprofloxacin. Most of them were also resistant to tobramycin (77%), whereas nonsusceptibility rates were lower for ceftolozane-tazobactam (31%), amikacin (7%), and colistin (2%). Pulsed-field gel electrophoresis-multilocus sequence typing (PFGE-MLST) analysis revealed that nearly all of the isolates belonged to previously described high-risk clones. Sequence type 175 (ST175) was detected in all 9 participating hospitals and accounted for 68% (n = 101) of the XDR isolates, distantly followed by ST244 (n = 16), ST253 (n = 12), ST235 (n = 8), and ST111 (n = 2), which were detected only in 1 to 2 hospitals. Through phenotypic and molecular methods, the presence of horizontally acquired carbapenemases was detected in 21% of the isolates, mostly VIM (17%) and GES enzymes (4%). At least two representative isolates from each clone and hospital (n = 44) were fully sequenced on an Illumina MiSeq. Classical mutational mechanisms, such as those leading to the overexpression of the ß-lactamase AmpC or efflux pumps, OprD inactivation, and/or quinolone resistance-determining regions (QRDR) mutations, were confirmed in most isolates and correlated well with the resistance phenotypes in the absence of horizontally acquired determinants. Ceftolozane-tazobactam resistance was not detected in carbapenemase-negative isolates, in agreement with sequencing data showing the absence of ampC mutations. The unique set of mutations responsible for the XDR phenotype of ST175 clone documented 7 years earlier were found to be conserved, denoting the long-term persistence of this specific XDR lineage in Spanish hospitals. Finally, other potentially relevant mutations were evidenced, including those in penicillin-binding protein 3 (PBP3), which is involved in ß-lactam (including ceftolozane-tazobactam) resistance, and FusA1, which is linked to aminoglycoside resistance.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Cefalosporinas/farmacologia , Fluoroquinolonas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Ácido Penicilânico/análogos & derivados , Ácido Penicilânico/farmacologia , Polimixinas/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Espanha/epidemiologia , Tazobactam , Resistência beta-Lactâmica/genética , beta-Lactamases/genética
15.
Int J Antimicrob Agents ; 50(3): 334-341, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28735882

RESUMO

The first Spanish multi-centre study on the microbiology of cystic fibrosis (CF) was conducted from 2013 to 2014. The study involved 24 CF units from 17 hospitals, and recruited 341 patients. The aim of this study was to characterise Pseudomonas aeruginosa isolates, 79 of which were recovered from 75 (22%) patients. The study determined the population structure, antibiotic susceptibility profile and genetic background of the strains. Fifty-five percent of the isolates were multi-drug-resistant, and 16% were extensively-drug-resistant. Defective mutS and mutL genes were observed in mutator isolates (15.2%). Considerable genetic diversity was observed by pulsed-field gel electrophoresis (70 patterns) and multi-locus sequence typing (72 sequence types). International epidemic clones were not detected. Fifty-one new and 14 previously described array tube (AT) genotypes were detected by AT technology. This study found a genetically unrelated and highly diverse CF P. aeruginosa population in Spain, not represented by the epidemic clones widely distributed across Europe, with multiple combinations of virulence factors and high antimicrobial resistance rates (except for colistin).


Assuntos
Fibrose Cística/complicações , Farmacorresistência Bacteriana , Variação Genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Adolescente , Adulto , Criança , Pré-Escolar , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Proteínas MutL/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Espanha/epidemiologia , Virulência , Adulto Jovem
16.
Sci Rep ; 7(1): 5555, 2017 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-28717172

RESUMO

Emergence of epidemic clones and antibiotic resistance development compromises the management of Pseudomonas aeruginosa cystic fibrosis (CF) chronic respiratory infections. Whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, WGS mutator genotypes (mutome) and resistome of a widespread clone (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. The coexistence of two divergent CC274 clonal lineages was revealed, but without evident geographical barrier; phylogenetic reconstructions and mutational resistome demonstrated the interpatient transmission of mutators. The extraordinary capacity of P. aeruginosa to develop resistance was evidenced by the emergence of mutations in >100 genes related to antibiotic resistance during the evolution of CC274, catalyzed by mutator phenotypes. While the presence of classical mutational resistance mechanisms was confirmed and correlated with resistance phenotypes, results also showed a major role of unexpected mutations. Among them, PBP3 mutations, shaping up ß-lactam resistance, were noteworthy. A high selective pressure for mexZ mutations was evidenced, but we showed for the first time that high-level aminoglycoside resistance in CF is likely driven by mutations in fusA1/fusA2, coding for elongation factor G. Altogether, our results provide valuable information for understanding the evolution of the mutational resistome of CF P. aeruginosa.

17.
Antimicrob Agents Chemother ; 60(12): 7415-7423, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27736752

RESUMO

Whole-genome sequencing (WGS) was used for the characterization of the frequently extensively drug resistant (XDR) Pseudomonas aeruginosa sequence type 175 (ST175) high-risk clone. A total of 18 ST175 isolates recovered from 8 different Spanish hospitals were analyzed; 4 isolates from 4 different French hospitals were included for comparison. The typical resistance profile of ST175 included penicillins, cephalosporins, monobactams, carbapenems, aminoglycosides, and fluoroquinolones. In the phylogenetic analysis, the four French isolates clustered together with two isolates from one of the Spanish regions. Sequence variation was analyzed for 146 chromosomal genes related to antimicrobial resistance, and horizontally acquired genes were explored using online databases. The resistome of ST175 was determined mainly by mutational events; resistance traits common to all or nearly all of the strains included specific ampR mutations leading to ampC overexpression, specific mutations in oprD conferring carbapenem resistance, or a mexZ mutation leading to MexXY overexpression. All isolates additionally harbored an aadB gene conferring gentamicin and tobramycin resistance. Several other resistance traits were specific to certain geographic areas, such as a streptomycin resistance gene, aadA13, detected in all four isolates from France and in the two isolates from the Cantabria region and a glpT mutation conferring fosfomycin resistance, detected in all but these six isolates. Finally, several unique resistance mutations were detected in single isolates; particularly interesting were those in genes encoding penicillin-binding proteins (PBP1A, PBP3, and PBP4). Thus, these results provide information valuable for understanding the genetic basis of resistance and the dynamics of the dissemination and evolution of high-risk clones.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Genoma Bacteriano , Mutação , Filogenia , Pseudomonas aeruginosa/genética , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbapenêmicos/farmacologia , Cefalosporinas/farmacologia , Células Clonais , Fluoroquinolonas/farmacologia , França/epidemiologia , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Monobactamas/farmacologia , Penicilinas/farmacologia , Porinas/genética , Porinas/metabolismo , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/epidemiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/classificação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Espanha/epidemiologia , beta-Lactamases/genética , beta-Lactamases/metabolismo
18.
J Infect Dis ; 214(9): 1449-1455, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27543671

RESUMO

Surfactant protein A (SP-A) plays a critical role in the clearance of Pseudomonas aeruginosa from the lung. However, there is limited information about the interaction of this protein with P. aeruginosa isolates from individuals with cystic fibrosis (CF). We characterized the interplay between SP-A and a collection of isogenic sequential isolates from 7 patients with CF. We identified outer membrane protein OprH as a novel ligand for SP-A on P. aeruginosa The last-available (late) isolates from patients with CF bound significantly less SP-A than their respective first-available (early) isolates. This difference could be associated with a reduction in the expression of OprH. Binding of SP-A to OprH promoted phagocytic killing; thus, late CF isolates were at least 2-fold more resistant to SP-A-mediated killing by human macrophages than their respective early isolates. We postulate that the reduction of OprH expression is a previously unrecognized adaptation of P. aeruginosa to the lung of individuals with CF that facilitates the escape of the microorganism from SP-A-mediated phagocytic killing.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/microbiologia
19.
Antimicrob Agents Chemother ; 60(5): 2912-22, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26926631

RESUMO

Traditional therapeutic strategies to control chronic colonization in cystic fibrosis (CF) patients are based on the use of a single nebulized antibiotic. In this study, we evaluated the therapeutic efficacy and dynamics of antibiotic resistance in Pseudomonas aeruginosa biofilms under sequential therapy with inhaled aztreonam (ATM) and tobramycin (TOB). Laboratory strains PAO1, PAOMS (hypermutable), PAOMA (mucoid), and PAOMSA (mucoid and hypermutable) and two hypermutable CF strains, 146-HSE (Liverpool epidemic strain [LES-1]) and 1089-HSE (ST1089), were used. Biofilms were developed using the flow cell system. Mature biofilms were challenged with peak and 1/10-peak concentrations of ATM (700 mg/liter and 70 mg/liter), TOB (1,000 mg/liter and 100 mg/liter), and their alternations (ATM/TOB/ATM and TOB/ATM/TOB) for 2 (t = 2), 4 (t = 4), and 6 days (t = 6). The numbers of viable cells (CFU) and resistant mutants were determined. Biofilm structural dynamics were monitored by confocal laser scanning microscopy and processed with COMSTAT and IMARIS software programs. TOB monotherapy produced an intense decrease in CFU that was not always correlated with a reduction in biomass and/or a bactericidal effect on biofilms, particularly for the CF strains. The ATM monotherapy bactericidal effect was lower, but effects on biofilm biomass and/or structure, including intense filamentation, were documented. The alternation of TOB and ATM led to an enhancement of the antibiofilm activity against laboratory and CF strains compared to that with the individual regimens, potentiating the bactericidal effect and/or the reduction in biomass, particularly at peak concentrations. Resistant mutants were not documented in any of the regimens at the peak concentrations and only anecdotally at the 1/10-peak concentrations. These results support the clinical evaluation of sequential regimens with inhaled antibiotics in CF, as opposed to the current maintenance treatments with just one antibiotic in monotherapy.


Assuntos
Antibacterianos/farmacologia , Aztreonam/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/microbiologia
20.
Drug Resist Updat ; 21-22: 41-59, 2015 Jul-Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26304792

RESUMO

The increasing prevalence of chronic and hospital-acquired infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa strains is associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of this pathogen for developing resistance through chromosomal mutations and from the increasing prevalence of transferable resistance determinants, particularly those encoding carbapenemases or extended-spectrum ß-lactamases (ESBLs). P. aeruginosa has a nonclonal epidemic population structure, composed of a limited number of widespread clones which are selected from a background of a large quantity of rare and unrelated genotypes that are recombining at high frequency. Indeed, recent concerning reports have provided evidence of the existence of MDR/XDR global clones, denominated high-risk clones, disseminated in hospitals worldwide; ST235, ST111, and ST175 are likely those more widespread. Noteworthy, the vast majority of infections by MDR, and specially XDR, strains are produced by these and few other clones worldwide. Moreover, the association of high-risk clones, particularly ST235, with transferable resistance is overwhelming; nearly 100 different horizontally-acquired resistance elements and up to 39 different acquired ß-lactamases have been reported so far among ST235 isolates. Likewise, MDR internationally-disseminated epidemic strains, such as the Liverpool Epidemic Strain (LES, ST146), have been noted as well among cystic fibrosis patients. Here we review the population structure, epidemiology, antimicrobial resistance mechanisms and virulence of the P. aeruginosa high-risk clones. The phenotypic and genetic factors potentially driving the success of high-risk clones, the aspects related to their detection in the clinical microbiology laboratory and the implications for infection control and public health are also discussed.


Assuntos
Antibacterianos/farmacologia , Infecções por Pseudomonas/epidemiologia , Pseudomonas aeruginosa/isolamento & purificação , Células Clonais , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Genótipo , Humanos , Epidemiologia Molecular , Prevalência , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética
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