scholarly journals MexY-Promoted Aminoglycoside Resistance in Pseudomonas aeruginosa: Involvement of a Putative Proximal Binding Pocket in Aminoglycoside Recognition

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Calvin Ho-Fung Lau ◽  
Daniel Hughes ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Drug Binding ◽  
Dimensional Structure ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Export Pathway ◽  
The Impact

ABSTRACTThe resistance-nodulation-division (RND) family multidrug efflux system MexXY-OprM is a major determinant of aminoglycoside resistance inPseudomonas aeruginosa, although the details of aminoglycoside recognition and export by MexY, the substrate-binding RND component of this efflux system, have not been elucidated. To identify regions/residues of MexY important for aminoglycoside resistance, plasmid-bornemexYwas mutagenized and mutations that impaired MexY-promoted aminoglycoside (streptomycin) resistance were identified in a ΔmexYstrain ofP. aeruginosa. Sixty-one streptomycin-sensitivemexYmutants were recovered; among these, 7 unique mutations that yielded wild-type levels of MexY expression were identified. These mutations compromised resistance to additional aminoglycosides and to other antimicrobials and occurred in both the transmembrane and periplasmic regions of the protein. Mapping of the mutated residues onto a 3-dimensional structure of MexY modeled onEscherichia coliAcrB revealed that these tended to occur in regions implicated in general pump operation (transmembrane domain) and MexY trimer assembly (docking domain) and, thus, did not provide insights into aminoglycoside recognition. A region corresponding to a proximal binding pocket connected to a periplasm-linked cleft, part of a drug export pathway of AcrB, was identified in MexY and proposed to play a role in aminoglycoside recognition. To test this, selected residues (K79, D133, and Y613) within this pocket were mutagenized and the impact on aminoglycoside resistance was assessed. Mutations of D133 and Y613 compromised aminoglycoside resistance, while, surprisingly, the K79 mutation enhanced aminoglycoside resistance, confirming a role for this putative proximal binding pocket in aminoglycoside recognition and export.IMPORTANCEBacterial RND pumps do not typically accommodate highly hydrophilic agents such as aminoglycosides, and it is unclear how those, such as MexY, which accommodate these unique substrates, do so. The results presented here indicate that aminoglycosides are likely not captured and exported by this RND pump component in a unique manner but rather utilize a previously defined export pathway that involves a proximal drug-binding pocket that is also implicated in the export of nonaminoglycosides. The observation, too, that a mutation in this pocket enhances MexY-mediated aminoglycoside resistance (K79A), an indication that it is not optimally designed to accommodate these agents, lends further support to earlier proposals that antimicrobials are not the intended pump substrates.

scholarly journals Expression of the MexXY Aminoglycoside Efflux Pump and Presence of an Aminoglycoside-Modifying Enzyme in Clinical Pseudomonas aeruginosa Isolates Are Highly Correlated

2020 ◽  
Vol 65 (1) ◽  
pp. e01166-20
Author(s):  
Alexander Seupt ◽  
Monika Schniederjans ◽  
Jürgen Tomasch ◽  
Susanne Häussler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Main Driver ◽  
Gentamicin Resistance ◽  
Bacterial Cell Membrane ◽  
Highly Correlated ◽  
The Impact ◽  
Gene Expression Levels

ABSTRACTThe impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that, in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels; however, those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside-modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyltransferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME-harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.

scholarly journals Reduced Expression of therplU-rpmARibosomal Protein Operon inmexXY-Expressing Pan-Aminoglycoside-Resistant Mutants of Pseudomonas aeruginosa

2012 ◽  
Vol 56 (10) ◽  
pp. 5171-5179 ◽  
Author(s):  
Calvin Ho-Fung Lau ◽  
Sebastien Fraud ◽  
Marcus Jones ◽  
Scott N. Peterson ◽  
Keith Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Promoter Region ◽  
Ribosomal Proteins ◽  
Antimicrobial Agents ◽  
Resistant Mutant ◽  
Wild Type ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Content Type

ABSTRACTPan-aminoglycoside-resistantPseudomonas aeruginosamutants expressing themexXYcomponents of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in themexZgene encoding a repressor of this efflux system and in themexXYpromoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068–1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of therplU-rpmAoperon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-typeP. aeruginosaPAO1. Moreover, correction of the mutation restoredrplU-rpmAexpression and, significantly, reversed the elevatedmexXYexpression and pan-aminoglycoside resistance of the mutant. ReducedrplU-rpmAexpression was also observed in a secondmexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in therplU-rpmApromoter region. Restoration ofrplU-rpmAexpression in the K2968 mutant following chromosomal integration of therplU-rpmAoperon derived from wild-typeP. aeruginosafailed, however, to reverse the elevatedmexXYexpression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impactingrplU-rpmAexpression in K2968 also impacts othermexXY-related genes. IncreasedmexXYexpression owing to reducedrplU-rpmAexpression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment ofmexXYand does so via PA5471, reminiscent ofmexXYinduction by ribosome-disrupting antimicrobial agents. Interestingly, reducedrplU-rpmAexpression was also observed in amexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals Changes in the Use of Broad-Spectrum Antibiotics after Cefepime Shortage: a Time Series Analysis

2011 ◽  
Vol 56 (2) ◽  
pp. 989-994 ◽  
Author(s):  
C. Plüss-Suard ◽  
A. Pannatier ◽  
C. Ruffieux ◽  
A. Kronenberg ◽  
K. Mühlemann ◽  
...  
Keyword(s):  
Time Series ◽  
Pseudomonas Aeruginosa ◽  
Broad Spectrum ◽  
Interrupted Time Series ◽  
Antibiotic Policy ◽  
Content Type ◽  
Broad Spectrum Antibiotics ◽  
Before And After ◽  
Alternative Antibiotic ◽  
The Impact

ABSTRACTThe original cefepime product was withdrawn from the Swiss market in January 2007 and replaced by a generic 10 months later. The goals of the study were to assess the impact of this cefepime shortage on the use and costs of alternative broad-spectrum antibiotics, on antibiotic policy, and on resistance ofPseudomonas aeruginosatoward carbapenems, ceftazidime, and piperacillin-tazobactam. A generalized regression-based interrupted time series model assessed how much the shortage changed the monthly use and costs of cefepime and of selected alternative broad-spectrum antibiotics (ceftazidime, imipenem-cilastatin, meropenem, piperacillin-tazobactam) in 15 Swiss acute care hospitals from January 2005 to December 2008. Resistance ofP. aeruginosawas compared before and after the cefepime shortage. There was a statistically significant increase in the consumption of piperacillin-tazobactam in hospitals with definitive interruption of cefepime supply and of meropenem in hospitals with transient interruption of cefepime supply. Consumption of each alternative antibiotic tended to increase during the cefepime shortage and to decrease when the cefepime generic was released. These shifts were associated with significantly higher overall costs. There was no significant change in hospitals with uninterrupted cefepime supply. The alternative antibiotics for which an increase in consumption showed the strongest association with a progression of resistance were the carbapenems. The use of alternative antibiotics after cefepime withdrawal was associated with a significant increase in piperacillin-tazobactam and meropenem use and in overall costs and with a decrease in susceptibility ofP. aeruginosain hospitals. This warrants caution with regard to shortages and withdrawals of antibiotics.

scholarly journals Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

2015 ◽  
Vol 59 (6) ◽  
pp. 3059-3065 ◽  
Author(s):  
C. Pitart ◽  
F. Marco ◽  
T. A. Keating ◽  
W. W. Nichols ◽  
J. Vila
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Mutant ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

Hotel customer segmentation according to eco-service quality perception: the case of Russian tourists

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Akin Aksu ◽  
Tahir Albayrak ◽  
Meltem Caber
Keyword(s):  
Service Quality ◽  
Customer Segmentation ◽  
Dimensional Structure ◽  
Research Approach ◽  
Theoretical Point ◽  
Regression Analyses ◽  
Content Type ◽  
Hotel Managers ◽  
The Impact ◽  
Hotel Customers

PurposeThis study aims to explore the components of eco-service quality at hotels and to cluster hotel customers based on their eco-service quality perceptions.Design/methodology/approachA quantitative research approach was adapted, and a survey study was performed on Russian tourists staying at the hotels located in Antalya, Turkey. Factor analysis results showed that the eco-service quality variable contains the dimensions of equipment, practice and staff and food. These factors were used to cluster hotel customers, and two groups were obtained as sensitive customers to eco-services and apathetic customers to eco-services. Cluster-based differences were identified by a series of cross-tabulations and regression analyses.FindingsSome socio-demographic and travel choice-related differences were obtained between the customer groups. The results of regression analyses showed that the most important determinant of sensitive customers' overall satisfaction was equipment, which was followed by staff and food and practice. The only significant determinant of apathetic customers' overall satisfaction was equipment.Practical implicationsHotel customers, who have different socio-demographic characteristics, are identified to have also distinct perceptions on the quality of eco-friendly equipment or services. Hence, hotel managers are suggested to develop proactive and value-generating environmentally friendly practices that appeal to different market segments. However, hotel managers should decide on prior areas and prefer low-cost options when “going green”, as some customer-groups do not notice such efforts.Originality/valueFrom the theoretical point of view, this study is original in showing the dimensional structure of the eco-service quality construct and the impact of each dimension on hotel customers' overall satisfaction. Both theoretically and practically, the findings offer valuable implications about the behavioural tendencies of Russian tourists towards eco-hotel practices.

scholarly journals Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives inEscherichia coliwith Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Sabine Schuster ◽  
Martina Vavra ◽  
Winfried V. Kern
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Drug Transport ◽  
Binding Pocket ◽  
Entrance Channel ◽  
Transport Pathways ◽  
Double Mutation ◽  
Content Type ◽  
Rnd Transporter ◽  
The Impact

ABSTRACTA major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives inEscherichia coliwas confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

scholarly journals Predictors of Mortality in Bloodstream Infections Caused by Pseudomonas aeruginosa and Impact of Antimicrobial Resistance and Bacterial Virulence

2019 ◽  
Vol 64 (2) ◽  
Author(s):  
Raúl Recio ◽  
Mikel Mancheño ◽  
Esther Viedma ◽  
Jennifer Villa ◽  
María Ángeles Orellana ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Resistance ◽  
Multivariate Logistic Regression Analysis ◽  
Empirical Therapy ◽  
Bloodstream Infections ◽  
Multivariate Logistic Regression ◽  
Related Factors ◽  
Content Type ◽  
Crude Mortality ◽  
The Impact

ABSTRACT Whether multidrug resistance (MDR) is associated with mortality in patients with Pseudomonas aeruginosa bloodstream infections (BSI) remains controversial. Here, we explored the prognostic factors of P. aeruginosa BSI with emphasis on antimicrobial resistance and virulence. All P. aeruginosa BSI episodes in a 5-year period were retrospectively analyzed. The impact in early (5-day) and late (30-day) crude mortality of host, antibiotic treatment, and pathogen factors was assessed by multivariate logistic regression analysis. Of 243 episodes, 93 (38.3%) were caused by MDR-PA. Crude 5-day (20%) and 30-day (33%) mortality was more frequent in patients with MDR-PA (34.4% versus 11.3%, P < 0.001 and 52.7% versus 21.3%, P < 0.001, respectively). Early mortality was associated with neutropenia (adjusted odds ratio [aOR], 9.21; 95% confidence interval [CI], 3.40 to 24.9; P < 0.001), increased Pitt score (aOR, 2.42; 95% CI, 1.34 to 4.36; P = 0.003), respiratory source (aOR, 3.23; 95% CI,2.01 to 5.16; P < 0.001), inadequate empirical therapy (aOR, 4.57; 95% CI, 1.59 to 13.1; P = 0.005), shorter time to positivity of blood culture (aOR, 0.88; 95% CI, 0.80 to 0.97; P = 0.010), an exoU-positive genotype (aOR, 3.58; 95% CI, 1.31 to 9.79; P = 0.013), and the O11 serotype (aOR, 3.64; 95% CI, 1.20 to 11.1; P = 0.022). These risk factors were similarly identified for late mortality, along with an MDR phenotype (aOR, 2.18; 95% CI, 1.04 to 4.58; P = 0.040). Moreover, the O11 serotype (15.2%, 37/243) was common among MDR (78.4%, 29/37) and exoU-positive (89.2%, 33/37) strains. Besides relevant clinical variables and inadequate empirical therapy, pathogen-related factors such as an MDR phenotype, an exoU-positive genotype, and the O11 serotype adversely affect the outcome of P. aeruginosa BSI.

scholarly journals Light-Mediated Decreases in Cyclic di-GMP Levels Inhibit Structure Formation in Pseudomonas aeruginosa Biofilms

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Lisa Juliane Kahl ◽  
Alexa Price-Whelan ◽  
Lars E. P. Dietrich
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Infections ◽  
Prolonged Exposure ◽  
Light Exposure ◽  
Research Attention ◽  
Content Type ◽  
Dependent Inhibition ◽  
Matrix Production ◽  
Biofilm Development ◽  
The Impact

ABSTRACT Light is known to trigger regulatory responses in diverse organisms, including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial infections. We grew P. aeruginosa strain PA14 in a colony morphology assay and found that growth under prolonged exposure to low-intensity blue light inhibited biofilm matrix production and thereby the formation of vertical biofilm structures (i.e., “wrinkles”). Light-dependent inhibition of biofilm wrinkling was correlated with low levels of cyclic di-GMP (c-di-GMP), consistent with the role of this signal in stimulating matrix production. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified c-di-GMP phosphodiesterases that contribute to light-dependent inhibition of biofilm wrinkling. One of these, RmcA, was previously characterized by our group for its role in mediating the effect of redox-active P. aeruginosa metabolites called phenazines on biofilm wrinkle formation. Our results suggest that an RmcA sensory domain that is predicted to bind a flavin cofactor is involved in light-dependent inhibition of wrinkling. Together, these findings indicate that P. aeruginosa integrates information about light exposure and redox state in its regulation of biofilm development. IMPORTANCE Light exposure tunes circadian rhythms, which modulate the immune response and affect susceptibility to infection in plants and animals. Though molecular responses to light are defined for model plant and animal hosts, analogous pathways that function in bacterial pathogens are understudied. We examined the response to light exposure in biofilms (matrix-encased multicellular assemblages) of the nonphotosynthetic bacterium Pseudomonas aeruginosa. We found that light at intensities that are not harmful to human cells inhibited biofilm maturation via effects on cellular signals. Because biofilm formation is a critical factor in many types of P. aeruginosa infections, including burn wound infections that may be exposed to light, these effects could be relevant for pathogenicity.

scholarly journals Identification of Novel VEB β-Lactamase Enzymes and Their Impact on Avibactam Inhibition

2016 ◽  
Vol 60 (5) ◽  
pp. 3183-3186 ◽  
Author(s):  
Sushmita D. Lahiri ◽  
Richard A. Alm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Pocket ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Content Type ◽  
Class A ◽  
Extended Spectrum ◽  
Class C

ABSTRACTCeftazidime-avibactam has activity againstPseudomonas aeruginosaandEnterobacteriaceaeexpressing numerous class A and class C β-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A β-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB β-lactamases possessed an extended-spectrum β-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB β-lactamases.

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