Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Eukaryotic Cell
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Antimicrobial Agents and Chemotherapy
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Archive
    • Minireviews
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About AAC
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • AAC Podcast
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
Susceptibility

In Vitro Susceptibility Testing of Eravacycline Is Unaffected by Medium Age and Nonstandard Assay Parameters

Trudy H. Grossman, Chris M. Pillar, Daniel F. Sahm, Joyce A. Sutcliffe
Trudy H. Grossman
aTetraphase Pharmaceuticals, Watertown, Massachusetts, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Chris M. Pillar
bEurofins Medinet, Chantilly, Virginia, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel F. Sahm
bEurofins Medinet, Chantilly, Virginia, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joyce A. Sutcliffe
aTetraphase Pharmaceuticals, Watertown, Massachusetts, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/AAC.04727-14
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

ABSTRACT

Eravacycline is a fluorocycline antibiotic in phase 3 clinical development for complicated intra-abdominal and urinary tract infections. To support its clinical development, a study was conducted to evaluate the effects of various susceptibility test parameters on the MIC values for aerobic bacteria. The results showed that eravacycline appears to be largely unaffected by medium age, medium additives, and other nonstandard assay conditions.

TEXT

This study investigated whether the age of the cation-adjusted Mueller-Hinton broth (CA-MHB) and other testing parameters affected the activity of eravacycline, a new broad-spectrum fluorocycline antibiotic (1) (Fig. 1) and whether any special accommodations need to be made for routine susceptibility testing with eravacycline. The in vitro activities of other tetracyclines, tigecycline and omadacycline, are known to be affected by the age of the test medium (2, 3, 4), and as a result, the Clinical and Laboratory Standards Institute (CLSI) guidelines specify the use of freshly prepared medium or medium that has been frozen within less than 12 h after preparation for susceptibility testing with these antibiotics. In addition to medium age, the effects of nonstandard conditions (addition of serum or blood, Haemophilus test medium [HTM], altered pH, high/low inoculum, various cation concentrations, polysorbate addition, or incubation in 5% CO2) on the eravacycline activity profile were evaluated.

FIG 1
  • Open in new tab
  • Download powerpoint
FIG 1

Chemical structure of eravacycline.

Testing was done using standardized CLSI methodology (1, 5) with CLSI quality control (QC) organisms (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, and Haemophilus influenzae ATCC 49247) alongside 10 clinical isolates of each species from the Eurofins Medinet repository (Chantilly, VA). Concurrent inocula of test organisms were used during testing for the comparison of different test conditions. Assays with quality control organisms were run in triplicate. Any change in the MIC value beyond one doubling dilution of that observed under standard CLSI conditions was noted, and essential agreement was defined as MIC values at or within one doubling dilution of standard. For E. faecalis, hazy growth at concentrations below the MIC was observed for all tetracyclines, and MIC values were defined at the lowest concentration producing a clear well.

Eravacycline was synthesized at Tetraphase Pharmaceuticals (1). Tigecycline (Thermo Fisher, Cambridge, MA) and tetracycline (Sigma-Aldrich, St. Louis, MO) were used as comparators. Antibiotic panels contained either cation-adjusted Mueller-Hinton broth (CA-MHB) (Difco; BD, Franklin Lakes, NJ) reconstituted from powder on the day of testing (fresh CA-MHB) or CA-MHB aged on the benchtop at room temperature for 2 weeks. Also, a set of fresh CA-MHB panels were frozen at −70°C for 2 weeks prior to use. Testing by broth microdilution was compared to that by agar dilution in Mueller-Hinton agar (BBL and Difco; BD). Nonstandard assay parameters evaluated for broth microdilution testing included the following: Haemophilus test medium (Thermo Fisher, Cambridge, MA, or prepared in-house), added human serum (5% or 10%; Bioreclamation, Westbury, NY) or lysed horse blood (Innovative Research, Novi, MI), altered pH (6.0 and 8.0), additional calcium (25 mg/liter Ca2+), added polysorbate (0.002%), carbon dioxide (5%), and altered inoculum size (5 × 104 CFU/ml and 5 × 106 CFU/ml).

The overall results showed that eravacycline MIC values in aged medium were unchanged or within one doubling dilution of the MIC values in fresh medium for 92.3% of all tested clinical E. coli, S. aureus, and E. faecalis isolates and the QC strains (Table 1). By organism, essential agreement of the eravacycline MIC values between fresh versus aged medium was seen for 100%, 92.3%, and 84.6% of E. coli, S. aureus, and E. faecalis isolates, respectively. Significantly less agreement was observed for tigecycline at 76.9% and 30.8% for E. coli and E. faecalis, respectively; however, 100% agreement was observed for S. aureus. The tetracycline MIC values were in 100% agreement for all organisms. Two E. faecalis isolates showing uncharacteristically poor agreement for eravacycline (≥+3 log2 difference between fresh versus aged medium) were retested in new batches of aged and fresh MHB in duplicate. The retesting results showed that the original eravacycline values were likely aberrant, and the essential agreement for E. faecalis was changed to 100%, while the agreements for tetracycline (100%) and tigecycline (30.8%) remained unchanged (see Table 1, footnote b).

View this table:
  • View inline
  • View popup
TABLE 1

Comparison of MIC values for isolates of E. coli, S. aureus, and E. faecalis using fresh versus aged CA-MHB

For all tested clinical E. coli, S. aureus, and E. faecalis isolates and the QC strains, no effects on eravacycline microdilution MIC values were seen for fresh versus frozen medium. No effects on eravacycline MIC values were seen for the microdilution assays conducted with fresh broth versus those for agar dilution assays.

When the eravacycline MIC values were evaluated against the QC strains, 2- to 4-fold decreases were observed for S. pneumoniae at pH 6.0 and E. faecalis at pH 8.0. A 4-fold increase in the eravacycline and tigecycline MIC values was observed only for E. faecalis in 0.002% polysorbate and 5% and 10% human serum (eravacycline only). The variations in inoculum size, incubation in 5% CO2, and supplemental Ca2+ in test medium had no effect on the eravacycline MIC values. The tigecycline MIC values for S. aureus, E. faecalis, and S. pneumoniae were elevated in HTM by 8-, 32- and 8-fold, respectively, while the eravacycline MIC values were unchanged. Further, the tigecycline MIC values against H. influenzae ATCC 49247 were outside the CLSI QC range. These results were likely due to the use of HTM that was not freshly prepared for testing.

In conclusion, the in vitro antibacterial activity of eravacycline appears to be largely unaffected by medium age, medium additives, and other nonstandard assay conditions. In agreement with literature reports, the activity of tigecycline in this study was significantly affected by aged medium (3, 4).

FOOTNOTES

    • Received 5 November 2014.
    • Returned for modification 3 December 2014.
    • Accepted 11 January 2015.
    • Accepted manuscript posted online 20 January 2015.
  • Copyright © 2015, American Society for Microbiology. All Rights Reserved.

REFERENCES

  1. 1.↵
    1. Xiao X-Y,
    2. Hunt DK,
    3. Zhou J,
    4. Clark RB,
    5. Dunwoody N,
    6. Fyfe C,
    7. Grossman TH,
    8. O'Brien WJ,
    9. Plamondon L,
    10. Ronn M,
    11. Sun C,
    12. Zhang W-Y,
    13. Sutcliffe JA
    . 2012. Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: a potent, broad spectrum antibacterial agent. J Med Chem 55:597–605. doi:10.1021/jm201465w.
    OpenUrlCrossRefPubMed
  2. 2.↵
    Clinical Laboratory Standards Institute. 2012. Methods for dilution antimicrobial susceptibility for bacteria that grow aerobically; approved standard, 9th ed. CLSI document M07-A9E, vol 32, no. 2. Clinical Laboratory Standards Institute, Wayne, PA.
  3. 3.↵
    1. Petersen PJ,
    2. Bradford PA
    . 2005. Effect of medium age and supplementation with biocatalytic oxygen-reducing reagent oxyrase on in vitro activities of tigecycline against recent clinical isolates. Antimicrob Agents Chemother 49:3910–3918. doi:10.1128/AAC.49.9.3910-3918.2005.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Bradford PA,
    2. Petersen PJ,
    3. Young M,
    4. Jones CH,
    5. Tischler M,
    6. O'Connell J
    . 2005. Tigecycline MIC testing by broth dilution requires use of fresh medium or addition of biocatalytic oxygen-reducing reagent oxyrase to standardize the test method. Antimicrob Agents Chemother 49:3903–3909. doi:10.1128/AAC.49.9.3903-3909.2005.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Clinical Laboratory Standards Institute. 2014. Performance standards for antimicrobial susceptibility testing; 24th informational supplement. CLSI document M100-S24, vol. 34, no. 1. Clinical Laboratory Standards Institute, Wayne, PA.
PreviousNext
Back to top
Download PDF
Citation Tools
In Vitro Susceptibility Testing of Eravacycline Is Unaffected by Medium Age and Nonstandard Assay Parameters
Trudy H. Grossman, Chris M. Pillar, Daniel F. Sahm, Joyce A. Sutcliffe
Antimicrobial Agents and Chemotherapy Mar 2015, 59 (4) 2426-2427; DOI: 10.1128/AAC.04727-14

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Antimicrobial Agents and Chemotherapy article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
In Vitro Susceptibility Testing of Eravacycline Is Unaffected by Medium Age and Nonstandard Assay Parameters
(Your Name) has forwarded a page to you from Antimicrobial Agents and Chemotherapy
(Your Name) thought you would be interested in this article in Antimicrobial Agents and Chemotherapy.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
In Vitro Susceptibility Testing of Eravacycline Is Unaffected by Medium Age and Nonstandard Assay Parameters
Trudy H. Grossman, Chris M. Pillar, Daniel F. Sahm, Joyce A. Sutcliffe
Antimicrobial Agents and Chemotherapy Mar 2015, 59 (4) 2426-2427; DOI: 10.1128/AAC.04727-14
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • TEXT
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

Related Articles

Cited By...

About

  • About AAC
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • AAC Podcast
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #AACJournal

@ASMicrobiology

       

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0066-4804; Online ISSN: 1098-6596