scholarly journals Efflux-Related Resistance to Norfloxacin, Dyes, and Biocides in Bloodstream Isolates of Staphylococcus aureus

2007 ◽  
Vol 51 (9) ◽  
pp. 3235-3239 ◽  
Author(s):  
Carmen E. DeMarco ◽  
Laurel A. Cushing ◽  
Emmanuel Frempong-Manso ◽  
Susan M. Seo ◽  
Tinevimbo A. A. Jaravaza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Efflux Pump Inhibitor ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Environmental Surfaces ◽  
Genes Encoding ◽  
High Level

ABSTRACT Efflux is an important resistance mechanism in Staphylococcus aureus, but its frequency in patients with bacteremia is unknown. Nonreplicate bloodstream isolates were collected over an 8-month period, and MICs of four common efflux pump substrates, with and without the broad-spectrum efflux pump inhibitor reserpine, were determined (n = 232). A reserpine-associated fourfold decrease in MIC was considered indicative of efflux. Strains exhibiting efflux of at least two of the four substrates were identified (“effluxing strains” [n = 114]). For these strains, MICs with or without reserpine for an array of typical substrates and the expression of mepA, mdeA, norA, norB, norC, and qacA/B were determined using quantitative real-time reverse transcription-PCR (qRT-PCR). A fourfold or greater increase in gene expression was considered significant. The most commonly effluxed substrates were ethidium bromide and chlorhexidine (100 and 96% of effluxing strains, respectively). qRT-PCR identified strains overexpressing mepA (5 [4.4%]), mdeA (13 [11.4%]), norA (26 [22.8%]), norB (29 [25.4%]), and norC (19 [16.7%]); 23 strains overexpressed two or more genes. Mutations probably associated with increased gene expression included a MepR-inactivating substitution and norA promoter region insertions or deletions. Mutations possibly associated with increased expression of the other analyzed genes were also observed. Effluxing strains comprised 49% of all strains studied (114/232 strains), with nearly half of these overexpressing genes encoding MepA, MdeA, and/or NorABC (54/114 strains). Reduced susceptibility to biocides may contribute to persistence on environmental surfaces, and efflux of drugs such as fluoroquinolones may predispose strains to high-level target-based resistance.

scholarly journals Staphylococcus aureus Mutants Isolated via Exposure to Nonfluorinated Quinolones: Detection of Known and Unique Mutations

2001 ◽  
Vol 45 (12) ◽  
pp. 3422-3426 ◽  
Author(s):  
Siddhartha Roychoudhury ◽  
Tracy L. Twinem ◽  
Kelly M. Makin ◽  
Mark A. Nienaber ◽  
Chuiying Li ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Sequence Analysis ◽  
Efflux Pump ◽  
Serial Passage ◽  
Efflux Pump Inhibitor ◽  
In Vitro Development ◽  
Development Of Resistance ◽  
High Level ◽  
Vitro Development

ABSTRACT The in vitro development of resistance to the new nonfluorinated quinolones (NFQs; PGE 9262932, PGE 4175997, and PGE 9509924) was investigated in Staphylococcus aureus. At concentrations two times the MIC, step 1 mutants were isolated more frequently with ciprofloxacin and trovafloxacin (9.1 × 10−8 and 5.7 × 10−9, respectively) than with the NFQs, gatifloxacin, or clinafloxacin (<5.7 × 10−10). Step 2 and step 3 mutants were selected via exposure of a step 1 mutant (selected with trovafloxacin) to four times the MICs of trovafloxacin and PGE 9262932. The step 1 mutant contained the known Ser80-Phe mutation in GrlA, and the step 2 and step 3 mutants contained the known Ser80-Phe and Ser84-Leu mutations in GrlA and GyrA, respectively. Compared to ciprofloxacin, the NFQs were 8-fold more potent against the parent and 16- to 128-fold more potent against the step 3 mutants. Mutants with high-level NFQ resistance (MIC, 32 μg/ml) were isolated by the spiral plater-based serial passage technique. DNA sequence analysis of three such mutants revealed the following mutations: (i) Ser84-Leu in GyrA and Glu84-Lys and His103-Tyr in GrlA; (ii) Ser-84Leu in GyrA, Ser52-Arg in GrlA, and Glu472-Val in GrlB; and (iii) Ser84-Leu in GyrA, Glu477-Val in GyrB, and Glu84-Lys and His103-Tyr in GrlA. Addition of the efflux pump inhibitor reserpine (10 μg/ml) resulted in 4- to 16-fold increases in the potencies of the NFQs against these mutants, whereas it resulted in 2-fold increases in the potencies of the NFQs against the parent.

The Expression of Efflux Pump Genes in Methicillin-Resistant Staphylococcus aureus (MRSA) Strains Isolated from Blood Cultures in Iran

2020 ◽  
Vol 15 (5) ◽  
Author(s):  
Arezoo Bostanmaneshrad ◽  
Jamileh Norouzi ◽  
Gita Eslami ◽  
Ali Hashemi
Keyword(s):  
Staphylococcus Aureus ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Expression Level ◽  
Resistance Pattern ◽  
Dilution Method ◽  
University Hospitals ◽  
Efflux Pump Inhibitor ◽  
Disk Diffusion Test ◽  
Methicillin Resistant

Background: Efflux pump is a significant resistance mechanism in Staphylococcus aureus. A total of 100 patients with bacteremia from Shahid Beheshti University Hospitals of Tehran in Iran were tested for the expression of efflux pump genes, contributing to S. aureus antimicrobial resistance. Objectives: this study was conducted to identify resistance pattern, and to evaluate the inhibitory effect of efflux pump, MIC of ciprofloxacin, and expression levels of norA, norB, and norC efflux pump genes in the presence of an efflux pump inhibitor against MDR S. aureus. Methods: A total of 100 MRSA isolates were investigated in different hospitals of Shahid Beheshti University of Medical Sciences from April 2017-2018. Owing to new consensus guidelines from the Clinical and Laboratory Standards Institute (CLSI), both the Kirby-Bauer disk diffusion test and micro-dilution method were used to evaluate antimicrobial susceptibility. Efflux pump activity using carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was identified as a chemical efflux pump inhibitor. E-test was used to determine Vancomycin-resistant antibiotic. Broth micro-dilution method for S. aureus isolates resistant to ciprofloxacin has been developed for minimum inhibitory concentration (MIC) of ciprofloxacin and CCCP and their composition. Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) was used to investigate the expression level of norA, norB, and norC efflux pump genes. Results: A total of 38 of 45 MRSA isolates (84.4%) showed resistance to ciprofloxacin. Moreover, 100% of isolates had the norA and norB genes. Further, 95% of S. aureus isolates had the norC gene. According to this study, ciprofloxacin MIC has decreased by CCCP compared to ciprofloxacin. There was an increase in the expression level of norA, norB, and norC efflux pump genes in methicillin-resistant and ciprofloxacin-resistant S. aureus strains based on RT- PCR. In this study, four different spA types were obtained as the most prevalent type of spA by t037and t790 (23.3%) and t030 (14.1%) and t044 (12.2%). Conclusions: This study indicates that the prevalence of ciprofloxacin-resistant S. aureus strains has a rising trend among MRSA clinical isolates. The ability of S. aureus isolates to be converted into drug-resistant strains using efflux pump mechanism has become a widespread concern.

scholarly journals Activity of imipenem/relebactam on Klebsiella pneumoniae with different mechanisms of imipenem non-susceptibility

2021 ◽  
Author(s):  
Mervat El-Sayed Mashaly ◽  
Ghada El-Saeed Mashaly
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Efflux Pump ◽  
Molecular Profile ◽  
Efflux Pump Inhibitor ◽  
Extended Spectrum Beta Lactamase ◽  
Reverse Transcription Pcr ◽  
Treat Ment ◽  
Encoding Genes

Background and Objectives: Imipenem/relebactam (IMP/R) is a newly FDA approved β-lactam/β-lactamase inhibitor combination. Relebactam ability to restore IMP activity could differ according to the cause of imipenem non-susceptibility. Therefore, we investigated the in-vitro activity of IMP/R against Klebsiella pneumoniae with different mechanisms of imi- penem non-susceptibility. Materials and Methods: Imipenem-nonsusceptible (IMP-NS) K. pneumoniae isolates were collected and characterized for β-lactamase encoding genes by multiplex PCR. For IMP-NS carbapenemase-negative isolates, study of Ompk35 & Ompk36 gene expression was performed by reverse transcription-PCR while efflux pump activity was studied by minimum inhibitory concentration (MIC) reduction assay using efflux pump inhibitor. Susceptibility testing of K. pneumoniae to IMP and IMP/R were achieved by broth microdilution (BMD) method. Results: During the study period, 140 isolates of IMP-NS K. pneumoniae were collected. BMD method showed that relebac- tam restored IMP susceptibility in 100%, 60% and 49% of isolates that only harbor AmpC, extended spectrum beta lactamase (ESBL) and carbapenemases, respectively. IMP/R was most potent against all bla KPC and 50% of bla _producing isolates. No demonstrable activity of IMP/R against K. pneumoniae harboring metallo-β-lactamases (MBLs). Out of 18 isolates with IMP non-suceptibility due to porins loss with overproduction of ESBL and/or AmpC, 14 (77.7%) isolates were IMP/R sus- ceptible. IMP/R showed no activity against isolates with only efflux pump hyperactivity. Conclusion: Relebactam could restore IPM activity in KPC or AmpC-producing IMP/NS K. pneumoniae but with no ac- tivity against MBL- producing isolates. Relebactam activity against isolates harbouring-bla OXA-48 or with altered Ompk35 & Ompk36 gene expression and efflux pump hyperactivity need further studies. Therefore, using IMP/R antibiotic in the treat- ment of infections caused by IMP/NS K. pneumoniae should be based on its molecular profile of IMP resistance to optimize the utility of IMP/R.

scholarly journals Effect of Vitamin K3 Inhibiting the Function of NorA Efflux Pump and Its Gene Expression on Staphylococcus aureus

Membranes ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 130 ◽  
Author(s):  
Saulo R. Tintino ◽  
Veruska C. A. de Souza ◽  
Julia M. A. da Silva ◽  
Cícera Datiane de M. Oliveira-Tintino ◽  
Pedro S. Pereira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Structural Changes ◽  
Efflux Pump ◽  
Direct Interaction ◽  
Vitamin K3 ◽  
Efflux Pump Inhibitor ◽  
Microdilution Method ◽  
Dual Mechanism ◽  
Hospital Acquired

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

scholarly journals Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Saeed Sharifi ◽  
Bita Bakhshi ◽  
Shahin Najar-peerayeh
Keyword(s):  
Gene Expression ◽  
Point Mutation ◽  
Campylobacter Jejuni ◽  
Significant Contribution ◽  
Efflux Pump ◽  
Campylobacter Coli ◽  
Rapd Pcr ◽  
Resistant Strains ◽  
High Level ◽  
Level Resistance

Abstract Background Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. Methods A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. Results Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. Conclusion Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

scholarly journals Triclosan resistance in clinical isolates of Acinetobacter baumannii

2009 ◽  
Vol 58 (8) ◽  
pp. 1086-1091 ◽  
Author(s):  
Yagang Chen ◽  
Borui Pi ◽  
Hua Zhou ◽  
Yunsong Yu ◽  
Lanjuan Li
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Resistance Mechanism ◽  
Dilution Method ◽  
Agar Dilution Method ◽  
Active Efflux ◽  
Low Level ◽  
High Level ◽  
Resistance Rates ◽  
Triclosan Resistance

The susceptibility to triclosan of 732 clinical Acinetobacter baumannii isolates obtained from 25 hospitals in 16 cities in China from December 2004 to December 2005 was screened by using an agar dilution method. Triclosan MICs ranged between 0.015 and 16 mg l−1, and the MIC90 was 0.5 mg l−1, lower than the actual in-use concentration of triclosan. Twenty triclosan-resistant isolates (MICs ≥1 mg l−1) were characterized by antibiotic susceptibility, clonal relatedness, fabI mutation, fabI expression, and efflux pump phenotype and expression to elucidate the resistance mechanism of A. baumannii to triclosan. The resistance rates of triclosan-resistant isolates to imipenem, levofloxacin, amikacin and tetracycline were higher than those of triclosan-sensitive isolates. Triclosan resistance was artificially classified as low level (MICs 1–2 mg l−1) or high level (MICs ≥4 mg l−1). High-level triclosan resistance could be explained by a Gly95Ser mutation of FabI, whilst wild-type fabI was observed to be overexpressed in low-level resistant isolates. Active efflux did not appear to be a major reason for acquired triclosan resistance, but acquisition of resistance appeared to be dependent on a background of intrinsic triclosan efflux.

scholarly journals A Combination Fluorescence Assay Demonstrates Increased Efflux Pump Activity as a Resistance Mechanism in Azole-Resistant Vaginal Candida albicans Isolates

2016 ◽  
Vol 60 (10) ◽  
pp. 5858-5866 ◽  
Author(s):  
Somanon Bhattacharya ◽  
Jack D. Sobel ◽  
Theodore C. White
Keyword(s):  
Candida Albicans ◽  
Efflux Pump ◽  
Pathogenic Fungus ◽  
Resistance Mechanism ◽  
Efflux Pumps ◽  
Resistant Isolate ◽  
Vaginal Infections ◽  
Content Type ◽  
Qrt Pcr ◽  
Pump Activity

ABSTRACTCandida albicansis a pathogenic fungus causing vulvovaginal candidiasis (VVC). Azole drugs, such as fluconazole, are the most common treatment for these infections. Recently, azole-resistant vaginalC. albicansisolates have been detected in patients with recurring and refractory vaginal infections. However, the mechanisms of resistance in vaginalC. albicansisolates have not been studied in detail. In oral and systemic resistant isolates, overexpression of the ABC transporters Cdr1p and Cdr2p and the major facilitator transporter Mdr1p is associated with resistance. Sixteen fluconazole-susceptible and 22 fluconazole-resistant vaginalC. albicansisolates were obtained, including six matched sets containing a susceptible and a resistant isolate, from individual patients. Using quantitative real-time reverse transcriptase PCR (qRT-PCR), 16 of 22 resistant isolates showed overexpression of at least one efflux pump gene, while only 1 of 16 susceptible isolates showed such overexpression. To evaluate the pump activity associated with overexpression, an assay that combined data from two separate fluorescent assays using rhodamine 6G and alanine β-naphthylamide was developed. The qRT-PCR results and activity assay results were in good agreement. This combination of two fluorescent assays can be used to study efflux pumps as resistance mechanisms in clinical isolates. These results demonstrate that efflux pumps are a significant resistance mechanism in vaginalC. albicansisolates.

scholarly journals Expression of the apoptosis-releated genes in patients with newly diagnosed chronic lymphocytic leukemia in clinical data context

2018 ◽  
Vol 17 (2) ◽  
pp. 41-46 ◽  
Author(s):  
S. G. Zakharov ◽  
A. K. Golenkov ◽  
A. V. Misyurin ◽  
E. V. Kataeva ◽  
A. A. Rudakova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Manifestations ◽  
Multivariate Regression Analysis ◽  
Lymphocytic Leukemia ◽  
Newly Diagnosed ◽  
Expression Of Genes ◽  
Gene Level ◽  
Genes Encoding ◽  
Group Ii ◽  
High Level

Introduction.The given data of fundamental studies of apoptosis processes in B-cell lymphocytic leukemia (B-CLL) testifies about the complexity and variety of mechanisms affecting the kinetics of normal cells and tumor lymphocytes in this disease. It is important to study the severity of clinical manifestations of the disease depending on the expression of the genes that modulate apoptosis.The purposeof the study is to compare the activity of genes encoding apoptosis modulators, the cell cycle and cancer-testicular PRAME protein with clinical manifestations of the disease in primary patients with B-CLL.Materials and methods.The level of expression of the proapoptotic genes FAS, TRAIL, TNFR2, DR4/5 and DR3, as well as the HSP27, XIAP genes, blocking apoptosis was determined in 23 patients with newly diagnosed chronic B-CLL. In addition, expression of genes TP53 and P21 and cancer-testis gene PRAME are tested.Results.According to the multivariate regression analysis, the FAS gene expression in the onset of the disease had the greatest impact on the clinical characteristics of the disease. In this connection, the patients were divided into groups with normal (group) and low gene level (group II). A low level of FAS expression (Me 387 %) was associated with stage II disease (p = 0.03), a large number of lympho cytes (p = 0.001), fewer erythrocytes (p = 0.08), and a lower level of TNFR2 gene expression (p = 0.08), high level of expression of XIAP, HSP27, P21. Overall, the anti-apoptotic potential in Group II patients was higher, which was accompanied by more pronounced clinical manifestations of the disease.Conclusions.The increased anti-apoptotic potential of tumor lymphocytes in newly diagnosed B-CLL is accompanied by a larger tumor mass and greater clinical and hematological manifestation of the disease.

Leveraging Automation toward Development of a High-Throughput Gene Expression Profiling Platform

2020 ◽  
pp. 247255522095659
Author(s):  
Jing Chen ◽  
Alan Futran ◽  
Austin Crithary ◽  
Sha Li ◽  
Alex Wolicki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Discovery ◽  
High Throughput ◽  
Screening Strategy ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Expression Platform ◽  
Program Support ◽  
Drug Discovery Program ◽  
One Step

We previously developed a panel of one-step real-time quantitative reverse transcription PCR (one-step qRT-PCR; hereafter referred to as qRT-PCR) assays to assess compound efficacy. However, these high-cost, conventional qRT-PCR manual assays are not amenable to high-throughput screen (HTS) analysis in a time-sensitive and complex drug discovery process. Here, we report the establishment of an automated gene expression platform using in-house lysis conditions that allows the study of various cell lines, including primary T cells. This process innovation provides the opportunity to perform genotypic profiling in both immunology and oncology therapeutic areas with quantitative studies as part of routine drug discovery program support. This newly instituted platform also enables a panel screening strategy to efficiently connect HTS, lead identification, and lead optimization in parallel.

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