Poster
E-777
Eravacycline (TP-434) is Active In Vitro Against Biofilms Formed by Uropathogenic Escherichia coli
W. O’BRIEN, J. SUTCLIFFE, T. GROSSMAN
52nd Annual ICAAC
9 - 12 September, 2012
San Francisco, CA
Tetraphase Pharmaceuticals, Inc., Watertown, MA,
Results
Revised Abstract
Background: Bacteria can persist as biofilms in chronic and device-related infections. Eravacycline (TP-434), a novel broad-spectrum
fluorocycline, was evaluated in vitro against uropathogenic Escherichia coli (EC200) biofilms.
Conclusion: This in vitro activity of TP-434, if confirmed in vivo, would be important in the clinical treatment of chronic biofilm infections.
1
4.00
2.00
1.00
0.50
0.25
0.13
0.06
0.03
0.00
Turbidity after
treatment:
Gentamicin
48 hr
No drug/No cells
No drug
100
Tetracycline
•
10
Turbidity after
treatment:
1
Gentamicin g/ml:
128.00 64.00 32.00 16.00
8.00
4.00
2.00
1.00
0.00
Biofilm after
treatment:
128 .00
64.00
32.00
Tetracycline was inactive
against biofilms formed by E.
coli EC200, consistent with
antibacterial activity a
requirement for anti-biofilm
activity
• The microtiter MIC for
tetracycline MIC is >32 g/ml
16.00
Turbidity after
treatment:
Summary
Biofilm after
treatment:
Biofilm after
treatment:
Compound
Microtiter
MIC
(µg/ml)
Planktonic
MIC
(µg/ml)
Biofilm
MIC
(µg/ml)
% CFU Remaining
at Biofilm MIC vs.
No Drug Control
Eravacycline
Gentamicin
Colistin
Meropenem
0.25
8
0.25
0.031
0.25
64
32
0.13
0.5
64
32
2
0.9
3.5
1.1
1.3
TP-434
Meropenem
1000
Methods
Microtiter Minimal Inhibitory Concentration (MIC) Assays. All MIC assays were performed as per CLSI guidelines [1] with the exception of the use of
TSB/YE medium (Tryptic Soy Broth (TSB), Bacto BD#211825; Yeast Extract (YE), Bacto BD#210929; concentration of YE in reconstituted TSB was (1%).
EC200 Biofilm Formation. A log phase starter culture of a tetracycline-resistant, uropathogenic E. coli strain EC200 (ATCC BAA-1161; tet(B), bla(TEM))
grown for 2 hrs was diluted to ~106 colony forming units (CFU) in TSB/YE and 500 µl of culture was added to 5 ml round bottom polystyrene tubes (BD Falcon
#352054) to form biofilms in stationary cultures incubated at 35 °C for 24 hrs. At 24 hrs, planktonic cells were aseptically aspirated and biofilms were fed with
either 600µl of fresh TSB/YE, or serially diluted antibiotics in 600 µl TSB/YE, and tubes incubated for an additional 24 hrs at 35 °C without shaking.
Biofilm Staining. After overnight incubation with drug, planktonic cells were aspirated and tubes were washed with 750 µl tap water, followed by aspiration
of the wash and the addition of 750 µl 0.1% crystal violet (Sigma #C3886-100G0; prepared in deionized water) to each tube. Biofilms were stained for several
minutes. Dye was aspirated and tubes were rinsed with 1 ml of tap water and dried at room temperature prior to photography. Results presented in this
posterare representative of at least two independent experiments.
Biofilm Release and CFU Quantification. After overnight incubation with drug, planktonic cells were aspirated, washed with 1 ml sterile 0.9% saline. The
wash was aspirated and replaced with 1 ml sterile saline plus 2 sterile 6 mm borosilicate glass beads (Kimax, VWR 89001-520) to each tube. Tubes were
sonicated in a Branson 5510 water bath at room temperature for one minute and then placed on ice. Sonicates were serially diluted and 0.1 ml of each
dilution was plated on tryptic soy agar plates and incubated at 35 °C for CFU quantification. Untreated and treated tubes were run in duplicate.
Definitions
Microtiter MIC: the lowest concentration of drug that inhibited turbidity in standard microtiter plate assays using TSB/YE medium inoculated with
resuspended CFU, as per CLSI guidelines.
105
–
106
Planktonic MIC : the lowest concentration of drug that inhibited turbidity in compound-treated tubes inoculated with a 24 hour established biofilm.
Biofilm MIC: the lowest compound concentration that produced the maximum reduction in crystal violet staining in compound-treated tubes inoculated with a
24 hour established biofilm.
1Clinical
10
and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. CLSI document M07-A9 . Clinical and
Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2012.
% Growth (CFUs) of Untreated Control Biofilm
•
100
1000
Untreated Controls
24 hr
Pre-drug biofilm inoculum
Tetracycline µg/ml:
% Growth (CFUs) of Untreated Control Biofilm
•
1000
TP-434 g/ml:
Background
TP-434 is a broad spectrum fluorocycline antibiotic with potent activity against ESβLproducing Enterobacteriaceae (Fyfe et al., Poster No. P1149, 21st ECCMID/27th ICC;
May 7-10, 2011).
Bacterial biofilms formed on biological surfaces and medically implanted devices are
often difficult to treat due to their relative insensitivity to antibiotics vs. planktonically
growing cells.
The ability of TP-434, and comparators, to clear an established E. coli biofilm from a
polystyrene surface was tested in an in vitro assay system.
Eravacycline (TP-434)
% Growth (CFUs) of Untreated Control Biofilm
Results: The biofilm MIC of TP-434 (0.5 µg/ml) was similar to the planktonic MIC (0.25 µg/ml), showing that TP-434 was active against
both planktonic cells and cells growing in a biofilm. Gentamicin, colistin and meropenem also inhibited biofilm cells, but a higher
concentrations than TP-434, see Summary Table.
Results
Activity Against Biofilms Established for 24 Hours by Uropathogenic E. coli
% Growth (CFUs) of Untreated Control Biofilm
Methods: The minimal inhibitory concentration (MIC) of compounds was determined according to CLSI guidelines with the exception that
tryptic soy broth/yeast extract (TSB/YE) medium was used. A starter culture of EC200 grown for 2 hrs was diluted to ~106 colony forming
units (CFU) in TSB/YE and 500 µl of culture was added to 5 ml polystyrene tubes to form biofilms at 35°C for 24 hrs. At 24 hrs, planktonic
cells were aspirated and biofilms were fed with either 600 µl of fresh TSB/YE, or serially diluted antibiotics in TSB/YE, and incubated for an
additional 24 hrs at 35°C. The “planktonic MIC” was the lowest concentration that inhibited turbidity in compound-treated biofilm tubes.
For staining biofilms, planktonic cells were aspirated, tubes were rinsed with water and stained with 0.1% crystal violet (CV). The “biofilm
MIC” was the lowest compound concentration that produced the maximum reduction in CV staining. For biofilm quantification, planktonic
cells were aspirated, tubes washed with saline, and cells were released from biofilms by sonication. Cells were plated for CFUs. The %
CFU reduction at the biofilm MIC, versus untreated controls, was calculated.
•
Contact:
Leland Webster
Tetraphase Pharmaceuticals
lwebster@tphase.com
100
10
Meropenem g/ml:
1
4.00 2.00 1.00 0.50 0.25 0.13 0.06 0.03 0.02 0.01 0.00
1000
Colistin
100
Conclusions
10
1
Colistin g/ml: 128.00 64.00 32.00 16.00 8.00
Turbidity after
treatment:
Turbidity after
treatment:
Biofilm after
treatment:
Biofilm after
treatment:
4.00
2.00
1.00
0.00
• Eravacycline disrupted established biofilm growth of tetracyclineresistant, ESβL-producing uropathogenic E. coli strain EC200 at a
concentration (0.5 g/ml) within two-fold of its microtiter MIC (0.25
g/ml).
• Gram-negative-active antibiotics gentamicin, colistin, and
meropenem disrupted biofilm growth at concentrations
significantly higher than their microtiter MICs (8-, 128-, and 64fold, respectively).
• The planktonic MIC of eravacycline was equivalent to its microtiter
MIC in TSB/YE broth whereas the planktonic MICs of gentamicin,
colistin, and meropenem were 8-, 128-, and 4-fold higher than
their respective microtiter MICs.
• Eravacycline shows promising activity in vitro against E. coli
biofilms, and if confirmed in vivo, this activity would be
advantageous for the treatment of chronic biofilm infections, such
as complicated urinary tract infections.