scholarly journals Occurrence of Escherichia coli in captive psittaciformes: antimicrobial susceptibility and virulence genes

2020 ◽  
Vol 21 ◽  
Author(s):  
Karine Louise Calaça ◽  
Renato Clini Cervi ◽  
Silvânia Andrade Reis ◽  
Iolanda Aparecida Nunes ◽  
Valéria de Sá Jayme ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Virulence Genes ◽  
Susceptibility Testing ◽  
Metropolitan Region ◽  
Serum Resistance ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
Hygienic Conditions

Abstract Captive Psittaciformes may harbor Gram-negative bacteria in their digestive tract, mainly due to poor hygienic conditions and confinement. The present study was carried out with the objective of isolating and identifying Escherichia coli in samples collected from Psittaciformes cages in 50 commercial establishments in the metropolitan region of Goiania, with subsequent antimicrobial susceptibility testing and detection of virulence genes. A total of 141 samples of excreta and swab samples from feeders and water bowls were collected, totaling 423 samples. Escherichia coli was isolated from 9.7% (41/423) samples: 12% (17/141) in excreta, 8.5% (12/141) in feed, and 8.5% (12 /141) in waterers. To determine the susceptibility profile of E. coli isolates, resistance to ciprofloxacin 4.9% (2/41), gentamicin 17.0% (7/41), doxycycline 34.1% (14/41), florfenicol 34.1% (14/41), trimethoprim 39.0% (16/41), tetracycline 41.5% (17/41), enrofloxacin 43.9% (18/41), amoxicillin 48.8% (20/41), neomycin 61.0% (25/41), and sulfonamide 90.2% (37/41) was determined. In 20 isolates, resistance was determined at 4 or more antimicrobials, seven of excreta (7/17), five of feed (5/12), and eight of waterers (8/12). One of the isolates from the waterers showed resistance to all antimicrobials. The iss gene was detected in three isolates, the tsh gene in three, the papC gene in two, traT and eae genes were not detected. In this study, it can be concluded that Psittaciformes commercialized as pet are carry E. coli isolates resistant to most commonly used antimicrobials, mainly sulfonamides and neomycin, besides having virulence and serum resistance genes, which highlights the possibility of the to cause disease in humans.

scholarly journals Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC β-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization

2019 ◽  
Vol 74 (12) ◽  
pp. 3481-3488 ◽  
Author(s):  
Jordy P M Coolen ◽  
Evert P M den Drijver ◽  
Jan A J W Kluytmans ◽  
Jaco J Verweij ◽  
Bram A Lamberts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Susceptibility Testing ◽  
Resistance Mechanism ◽  
Gram Negative Bacteria ◽  
Third Generation ◽  
Training Set ◽  
Gram Negative ◽  
E Coli ◽  
Validation Set ◽  
Mic Value

Abstract Objectives AmpC-β-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common β-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. Methods We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. Results In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79–0.94) on the training set, 0.79 (95% CI=0.64–0.89) on validation set 1 and 0.85 (95% CI=0.71–0.94) on validation set 2. Conclusions This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.

scholarly journals Colonization and Resistance Dynamics of Gram-Negative Bacteria in Patients during and after Hospitalization

2005 ◽  
Vol 49 (7) ◽  
pp. 2879-2886 ◽  
Author(s):  
P. Margreet G. Filius ◽  
Inge C. Gyssens ◽  
Irma M. Kershof ◽  
Patty J. E. Roovers ◽  
Alewijn Ott ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Susceptibility Testing ◽  
General Ward ◽  
Gram Negative Bacteria ◽  
Icu Patients ◽  
Gram Negative ◽  
E Coli ◽  
General Wards ◽  
Colonization Rates

ABSTRACT The colonization and resistance dynamics of aerobic gram-negative bacteria in the intestinal and oropharyngeal microfloras of patients admitted to intensive care units (ICU) and general wards were investigated during and after hospitalization. A total of 3,316 specimens were obtained from patients upon admission, once weekly during hospitalization, at discharge from the ICU, at discharge from the hospital, and 1 and 3 months after discharge from the hospital. Five colonies per specimen were selected for identification and susceptibility testing. In both patient populations, the gram-negative colonization rates in oropharyngeal specimens increased during hospitalization and did not decrease in the 3 months after discharge. In rectal specimens, colonization rates decreased during hospitalization and increased after discharge. There was a change in species distribution among the dominant microfloras during hospitalization. Klebsiella spp., Enterobacter spp., Serratia marcescens, and Pseudomonas aeruginosa were isolated more often, whereas the frequency of Escherichia coli declined. The percentage of ICU patients colonized with ampicillin- and/or cephalothin-resistant fecal E. coli was significantly increased at discharge from the hospital and did not change in the 3 months after discharge. The emergence of multidrug resistance was observed for E. coli during patient stays in the ICU. Resistance frequencies in E. coli significantly increased with the length of stay in the ICU. For the general ward population, no significant changes in resistance frequencies were found during hospitalization. From a population perspective, the risk of dissemination of resistant gram-negative bacteria into the community through hospitalized patients appears to be low for general ward patients but is noticeably higher among ICU patients.

scholarly journals Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase producing urinary isolates of Escherichia coli in outpatients

2013 ◽  
Vol 141 (11-12) ◽  
pp. 775-779 ◽  
Author(s):  
Tatjana Markovic ◽  
Ljiljana Jeinic ◽  
Aleksandra Smitran ◽  
Miroslav Petkovic
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Age Group ◽  
Gram Negative ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Esbl Producers ◽  
Urinary Isolates

Introduction. In Gram-negative bacteria, the production of beta-lactamases is the most important mechanism of resistance to beta-lactam antibiotics. In the Banja Luka region, there were no extensive researches on the prevalence and antimicrobial resistance of the extended-spectrum beta-lactamase (ESBL) producing Escherichia coli (E. coli) isolates. Objective. The aim of the present study was to determine the presence of ESBL producing E. coli isolates as the cause of the urinary tract infections in outpatients, the distribution of these ESBL isolates according to age and gender of patients and their susceptibility to antimicrobials. Methods. Urine specimens obtained from outpatients were cultured on chromogenic CPS-ID3 media. All plates showing significant (>105 cfu/ml) growth of E. coli in pure culture were further processed. Antimicrobial susceptibility testing was performed on VITEK TWO Compact using AST-GN27 cards for testing Gram negative bacteria and detection of ESBL producers. Results. Out of 2,195 isolates, 177 (8.1%) were ESBL producers. Ninety-two isolates were obtained from female patients (5% of E. coli isolated from women) and 85 isolates from male patients (23% of E. coli isolated from men). High percentage of ESBL isolates was detected in the infant age group under one year (36.7%) and in the age group over 60 years (28.8%). All ESBL isolates were susceptible to imipenem and resistant to ampicillin, piperacillin, cefazolin, cefotaxime, ceftazidime and cefepime. There was a significant resistance to amikacin (79.1%), gentamicin (76.8%), amoxicillin/clavulanate (54.8%) and trimethoprim/sulphamethoxazole (45.8%). Resistance to nutrofurantoin was 13.6%. Conclusion. This study has demonstrated the presence of ESBL producing E. coli urinary isolates in outpatients, and their extensive susceptibility to imipenem and nitrofurantoin.

Rapid susceptibility testing of multi-drug resistant Escherichia coli and Klebsiella by glucose metabolization monitoring

2019 ◽  
Vol 57 (8) ◽  
pp. 1271-1279 ◽  
Author(s):  
Maximilian Kittel ◽  
Peter Findeisen ◽  
Beniam Ghebremedhin ◽  
Thomas Miethke ◽  
Alexander Grundt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Therapy ◽  
Susceptibility Testing ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Gram Negative ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Tof Ms

Abstract Background The increasing number of multi-drug resistant (MDR) bacteria provides enormous challenges for choosing an appropriate antibiotic therapy in the early phase of sepsis. While bacterial identification has been greatly accelerated by the introduction of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), the antibiotic susceptibility testing (AST) remains time-consuming. Here, we present a rapid susceptibility testing method for testing Gram-negative bacteria, exemplarily validated for Escherichia coli and Klebsiella spp. Methods Gram-negative isolates (E. coli and Klebsiella spp.) were either taken as single colonies from agar plates (n=136) or directly extracted and identified from positive blood cultures (n=42) using MALDI-TOF MS. Bacteria were incubated in glucose-supplemented Luria broths (LBs) each containing one antibiotic (ceftazidime, piperacillin, imipenem and ciprofloxacin), routinely used to classify Gram-negative bacteria in Germany. To determine susceptibility the dynamics of glucose utilization in bacterial suspensions were quantitatively measured in the presence or absence of antibiotics designated liquid-AST (L-AST). Results The L-AST can be run on clinical-chemistry analyzers and integrated into laboratory routines. It yields critical resistance information within 90–150 min downstream of a MS-based identification. The results showed a high concordance with routine susceptibility testing, with less than 1% very major errors (VME) and 3.51% major errors (ME) for 178 assessed isolates. Analysis of turnaround time (TAT) for 42 clinical samples indicated that L-AST results could be obtained 34 h earlier than the routine results. Conclusions As exemplified for E. coli and Klebsiella spp., L-AST provides substantial acceleration of susceptibility testing following MALDI-TOF MS identification. The assay is a simple and low-cost method that can be integrated into clinical laboratory to allow for 24/7 AST. This approach could improve antibiotic therapy.

scholarly journals Evaluation of the Clinical Systems for Polymyxin Susceptibility Testing of Clinical Gram-Negative Bacteria in China

2021 ◽  
Vol 11 ◽  
Author(s):  
Ying Zhu ◽  
Peiyao Jia ◽  
Menglan Zhou ◽  
Jingjia Zhang ◽  
Ge Zhang ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Polymyxin B ◽  
Antimicrobial Susceptibility Testing ◽  
Gram Negative Bacteria ◽  
Major Error ◽  
Gram Negative ◽  
E Coli ◽  
Vitek 2 ◽  
Clinical Systems

ObjectivesThe performance of mainstream commercial antimicrobial susceptibility testing systems on polymyxins has not been well evaluated in China. In this study, three antimicrobial susceptibility testing systems were evaluated for polymyxin B and colistin.MethodsThe MICs of 257 Gram-negative strains collected from clinical cases and livestock were determined and analyzed. Using Broth Microdilution as the gold standard, the performance of VITEK 2® COMPACT, PhoenixTM M50, and Bio-kont AST System were evaluated. Essential agreement (EA), category agreement (CA), very major error (VME), and major error (ME) were calculated for comparison. The results of mcr-1 positive strains were separately discussed.ResultsThe EA, CA, VME, and ME to polymyxin B for Bio-kont were 83.5, 95.6, 13.1, and 0.6%, respectively. The EAs, CAs, VMEs, and MEs to colistin were as follows: Bio-kont, 86.7%/96.5%/7.2%/1.7%; Vitek 2, 64.2%/86.8%/41.0%/0%, and Phoenix M50, 92.9%/92.9%/21.7%/0%. The performance of Bio-kont to polymyxin B and colistin for Pseudomonas spp. (EA, CA < 90%, VME > 1.5%, ME = 5.6%/10%) and Enterobacter spp. (EA, CA < 90%, VME > 1.5% and ME = 0%), Vitek to colistin for most genera, and Phoenix to colistin for Enterobacter spp. (EA, CA < 90%, VME > 1.5%, ME = 0%) were unsatisfactory compared with other genera. The performance of Bio-kont to polymyxins for Escherichia spp. and Phoenix to colistin for Citrobacter spp., Escherichia spp., and Klebsiella spp., which all met the CLSI standard, were satisfactory. When the susceptibility of mcr-1 positive E. coli was tested, Bio-kont and Phoenix M50 presented excellent performance with no category errors, while Vitek 2 performed a high VME (25.5%).ConclusionWith relatively more accurate results for polymyxin B and colistin and lower VME, Bio-kont has an advantage in polymyxin antimicrobial susceptibility testing, especially for Escherichia spp., Klebsiella spp., Citrobacter spp. and Acinetobacter spp.

scholarly journals Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tessa B. Moyer ◽  
Ashleigh L. Purvis ◽  
Andrew J. Wommack ◽  
Leslie M. Hicks
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
Gram Negative Bacteria ◽  
Amaranthus Tricolor ◽  
Core Motif ◽  
Gram Negative ◽  
E Coli ◽  
Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Antimicrobial Resistance in Campylobacter, Salmonella, and Escherichia coli Isolated from Retail Grain-Fed Veal Meat from Southern Ontario, Canada

2011 ◽  
Vol 74 (8) ◽  
pp. 1245-1251 ◽  
Author(s):  
ANGELA COOK ◽  
RICHARD J. REID-SMITH ◽  
REBECCA J. IRWIN ◽  
SCOTT A. McEWEN ◽  
VIRGINIA YOUNG ◽  
...  
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Tetracycline Resistance ◽  
Antimicrobial Susceptibility Testing ◽  
E Coli ◽  
Testing Resistance ◽  
Resistance Patterns

This study estimated the prevalence of Salmonella, Campylobacter, and Escherichia coli isolates in fresh retail grain-fed veal obtained in Ontario, Canada. The prevalence and antimicrobial resistance patterns were examined for points of public health significance. Veal samples (n = 528) were collected from February 2003 through May 2004. Twenty-one Salmonella isolates were recovered from 18 (4%) of 438 samples and underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 6 (29%) of 21 Salmonella isolates; 5 (24%) of 21 isolates were resistant to five or more antimicrobials. No resistance to antimicrobials of very high human health importance was observed. Ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance was found in 5 (3%) of 21 Salmonella isolates. Campylobacter isolates were recovered from 5 (1%) of 438 samples; 6 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was documented in 3 (50%) of 6 Campylobacter isolates. No Campylobacter isolates were resistant to five or more antimicrobials or category I antimicrobials. E. coli isolates were recovered from 387 (88%) of 438 samples; 1,258 isolates underwent antimicrobial susceptibility testing. Resistance to one or more antimicrobials was found in 678 (54%) of 1,258 E. coli isolates; 128 (10%) of 1,258 were resistant to five or more antimicrobials. Five (0.4%) and 7 (0.6%) of 1,258 E. coli isolates were resistant to ceftiofur and ceftriaxone, respectively, while 34 (3%) of 1,258 were resistant to nalidixic acid. Ciprofloxacin resistance was not detected. There were 101 different resistance patterns observed among E. coli isolates; resistance to tetracycline alone (12.7%, 161 of 1,258) was most frequently observed. This study provides baseline prevalence and antimicrobial resistance data and highlights potential public health concerns.

scholarly journals Detection of virulence genes and antimicrobial resistance profiles of Escherichia coli isolates from raw milk and artisanal cheese in Southern Brazil

2019 ◽  
Vol 40 (1) ◽  
pp. 163 ◽  
Author(s):  
Leandro Parussolo ◽  
Ricardo Antônio Pilegi Sfaciotte ◽  
Karine Andrezza Dalmina ◽  
Fernanda Danielle Melo ◽  
Ubirajara Maciel Costa ◽  
...  
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Antimicrobial Susceptibility ◽  
Virulence Genes ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Public Health Issue ◽  
Southern Brazil ◽  
E Coli

The serrano artisanal cheese is a typical product from South region of Brazil, which is produced by skilled cheesemakers using raw milk. The contamination of this food by Escherichia coli has a great impact on public health, since it could threat the consumers’ health. The study evaluated the presence of virulence genes, antimicrobial susceptibility profiles and bofilm-production ability of Escherichia coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil. A total of 117 isolates of E. coli were characterized by multiplex PCR to detect the following virulence genes: eae for enteropatogenic E. coli (EPEC), lt and st for enterotoxigenic E. coli (ETEC), stx for shiga toxin-producing E. coli (STEC), stx and eae for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E. coli (EIEC) and aggR for enteroaggregative E. coli (EAEC). In addition, antimicrobial susceptibility profile to 22 antimicrobial agents was also performed by disk diffusion method, and we searched for extended-spectrum beta-lactamases (ESBL) and/or carbapenemase- producing isolates. Isolates that were positive for ESBL and carbapenemase were further investigated for the presence of the genes: blaTEM, blaSHV, blaOXA, blaCTX-M, for ESBL and blaOXA-48 for carbapenemase. Further, isolates had their ability to form biofilms investigated by the red Congo agar method. Virulence genes of E. coli were identified in 21.37% of the tested isolates, which were classified as EPEC (the most prevalent pathotype) and ETEC or EAEC. Ten (8.55%) of the total studied E. coli isolates revealed a multidrug-resistant profile, since they were resistant to three or more antimicrobial classes; whereas four isolates (3.42%) were classified as ESBL-producers and showed the presence of blaTEM gene. None of the isolates exhibited carbapenemase activity nor did they carry carbapenemase genes. From the total of E. coli isolates, 79 (67.52%) were considered potential biofilm producers. These results address a serious public health issue, since artisanal cheeses pose a risk to consumers’ health, since may be sources of dissemination of diarrheogenic E. coli, that can cause from subclinical to severe and fatal infections in children and adults, and also emphasize the need to improve adaptations/adjustments in the manufacturing processes of these products.

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