Activity of Novel Antibiotic Lock Solutions in a Model Against Isolates of Catheter-Related Bloodstream Infections

Abstract

Catheter-related bloodstream infections (CRBSIs) are a primary concern in patients with indwelling central venous catheters (CVCs). Instillation of an antibiotic lock solution may serve as an adjunctive therapy.
To evaluate the efficacy of novel antibiotic-anticoagulant lock solutions using an in vitro model of CVC infection.
The following lock solutions were evaluated: daptomycin 1 mg/mL (reconstituted with lactated Ringer's [LR]) plus heparin 5000 units/mL, tigecycline 0.5 mg/mL plus ethylenediaminetetraacetate (EDTA) 30 mg/mL, gentamicin 5 mg/mL plus EDTA 30 mg/mL, cefazolin 5 mg/mL plus heparin 5000 units/mL, and phosphate-buffered NaCl 0.9% as the control solution. Analysis was performed on Hickman catheter segments inoculated with the following organisms: methicillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, and Pseudomonas aeruginosa. The catheters were incubated in the candidate lock solutions for 0, 2, 4, and 24 hours. Student's t-tests were conducted to evaluate reduction in log(10) colony-forming units/milliliter (cfu/mL) of individual lock solutions compared with the control solution. For each organism, analysis of variance and Student's t-tests were performed to determine whether differences existed among the lock solutions.
Gentamicin plus EDTA (G+EDTA) and tigecycline plus EDTA (Ti+EDTA) resulted in significant reductions (p < 0.05) of log(10) cfu/mL at 24 hours for all organisms tested. Daptomycin, reconstituted in LR, plus heparin (D+LR+H) demonstrated potent activity against all staphylococcal species (p < 0.05). With respect to MRSA, G+EDTA displayed significantly better activity than Ti+EDTA and cefazolin plus heparin (p < 0.05), but there was no significant difference compared with D+LR+H. No antagonism was noted with the addition of anticoagulants to the solutions.
Gentamicin, tigecycline, and daptomycin in combination with anticoagulants as lock solutions displayed potent activity against common pathogens responsible for CRBSIs. Each of these lock solutions deserves strong consideration for study in a clinical trial. Further data on compatibility and stability of these solutions are needed before routine clinical use can be recommended.

Full text

210 nThe Annals of Pharmacotherapy n2009 February, Volume 43 www.theannals.com
Catheter-related bloodstream infec-
tions (CRBSIs) occur in 5– 40% of
patients with indwelling catheters, with
estimates of over 200,000 cases occur-
ring annually in the US and attributable
mortality as high as 10%.1-4Gram-posi-
tive organisms, predominantly Staphylo-
coccus aureus and coagulase-negative
staphylococci, account for nearly 70% of
CRBSIs. Candida spp., predominantly
albicans,account for another 10–15% of
CRBSIs. Gram-negative organisms, in-
cluding Pseudomonas aeruginosa,may
cause severe, difficult-to-treat infections.
CRBSIs are increasingly detrimental to
the healthcare system, with estimated
costs of $10,000–$28,690 for each epi-
sode and an average increase in hospital-
ization of 6.5 days for critically ill pa-
tients.4Generally, catheter removal is a
component of definitive treatment for
CRBSIs; however, removal of the cathe-
ter is not always a viable option. Instilla-
tion of an antibiotic lock solution into the
catheter lumen may be used as an ad-
junct to systemic therapy. The Infectious
Diseases Society of America includes
antibiotic locksolutions in its most recent
guidelines as an option in both prevention
and treatment of CRBSIs.6,7 The potential
for use of antibiotic lock solutions may
Activity of Novel Antibiotic Lock Solutions in a Model Against
Isolates of Catheter-Related Bloodstream Infections
PBrandon Bookstaver, John C Williamson, Brian Keith Tucker, Issam I Raad, and Robert J Sherertz
Infectious Diseases
Author information provided at the end of the
text.
BACKGROUND:Catheter-related bloodstream infections (CRBSIs) are a primary
concern in patients with indwelling central venous catheters (CVCs). Instillation of
an antibiotic lock solution may serve as an adjunctive therapy.
OBJECTIVE:To evaluate the efficacy of novel antibiotic–anticoagulant lock
solutions using an in vitro model of CVC infection.
METHODS:The following lock solutions were evaluated: daptomycin 1 mg/mL
(reconstituted with lactated Ringer’s [LR]) plus heparin 5000 units/mL, tigecycline
0.5 mg/mL plus ethylenediaminetetraacetate (EDTA) 30 mg/mL, gentamicin 5
mg/mL plus EDTA30 mg/mL, cefazolin 5 mg/mL plus heparin 5000 units/mL, and
phosphate-buffered NaCl 0.9% as the control solution. Analysis was performed on
Hickman catheter segments inoculated with the following organisms: methicillin-
sensitive
Staphylococcus aureus
,methicillin-resistant
S. aureus
(MRSA),
Staphylococcus epidermidis
,and
Pseudomonas aeruginosa
.The catheters were
incubated in the candidate lock solutions for 0, 2, 4, and 24 hours. Student’s
t
-tests
were conducted to evaluate reduction in log10 colony-forming units/milliliter (cfu/mL)
of individual lock solutions compared with the control solution. For each organism,
analysis of variance and Student’s
t
-tests were performed to determine whether
differences existed among the lock solutions.
RESULTS:Gentamicin plus EDTA (G+EDTA) and tigecycline plus EDTA
(Ti+EDTA) resulted in significant reductions (p < 0.05) of log10 cfu/mL at 24 hours
for all organisms tested. Daptomycin, reconstituted in LR, plus heparin (D+LR+H)
demonstrated potent activity against all staphylococcal species (p < 0.05). With
respect to MRSA, G+EDTA displayed significantly better activity than Ti+EDTA
and cefazolin plus heparin (p < 0.05), but there was no significant difference
compared with D+LR+H. No antagonism was noted with the addition of
anticoagulants to the solutions.
CONCLUSIONS:Gentamicin, tigecycline, and daptomycin in combination with
anticoagulants as lock solutions displayed potent activity against common
pathogens responsible for CRBSIs. Each of these lock solutions deserves strong
consideration for study in a clinical trial. Further data on compatibility and stability
of these solutions are needed before routine clinical use can be recommended.
KEY WORDS:antibiotic lock, catheter-related bloodstream infection, central
venous catheter, daptomycin, gentamicin, tigecycline.
Ann Pharmacother
2009;43:210-9.
Published Online, 3 Feb 2009,
www.theannals.com
,DOI 10.1345/aph.1L145
by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from by guest on October 11, 2013aop.sagepub.comDownloaded from

expand with the recent announcement of the Centers for
Medicare and Medicaid Services’ decision to cease payment
for nosocomial-acquired infections, including CRBSIs.8
The majority of studies on this subject have investigated
the in vitro activity of various antimicrobial–anticoagulant
combinations. Additional clinical studies have focused pri-
marily on use of antibiotic lock solutions in a prophylactic
modality, demonstrating predominantly positive results.
Prophylactic use of vancomycin–heparin and gentamicin–
heparin lock solutions reduces catheter colonization and
the rate of CRBSI when compared with heparin alone.9-11 In
one randomized controlled study, gentamicin–heparin lock
solutions reduced the incidence of CRBSI from 3.12 to 0.76
per 1000 catheter days (p < 0.05).11 Chatzinikolaou et al.5
demonstrated the efficacy of a minocycline–ethylene-
diaminetetraacetate (M-EDTA) lock solution in reducing the
number of port infections in a series of pediatric cancer pa-
tients. Additional studies with M-EDTA have supported
these results.12,13 There is a paucity of data evaluating treat-
ment outcomes of antibiotic lock therapy. Rijinders et al.14
have published, to our knowledge, the only randomized con-
trolled trial evaluating treatment outcomes, demonstrating a
trend toward fewer treatment failures in the antibiotic lock
arm. Additionally,in a recent retrospective analysis, the end-
point of successful treatment was achieved in 84% versus
65% of the antibiotic lock and control groups, respectively.15
Overall, numerous antimicrobial–anticoagulant combi-
nations have been investigated for prevention and treatment
of CRBSI. Few studies, however,have included the newer
antibiotics daptomycin and tigecycline, and one prospective
study has investigated tigecycline in combination with an
anticoagulant in solution.16-18 There are additional limited
data on alternatives to heparin, such as EDTA. The expand-
ing use of central venous catheters (CVCs) combined with a
growing concern over drug-resistant pathogens warrants
study of further options with novel lock solutions in the
treatment of CRBSI. This study evaluated the efficacy of
daptomycin, tigecycline, gentamicin, and cefazolin in antibi-
otic–anticoagulant lock solutions using an in vitro model of
intravenous catheter infection.
Methods
The following antibiotics were evaluated as candidate
lock solutions: daptomycin 1 mg/mL, tigecycline 0.5
mg/mL, gentamicin 5 mg/mL, and cefazolin 5 mg/mL.
Phosphate-buffered NaCl 0.9% (saline; PBS) served as the
control solution. Concentrations of antibiotics were select-
ed to estimate 500–1000 times 90% minimum inhibitory
concentration (MIC90)values for typical staphylococcal
isolates. Antibiotics were reconstituted according to the
manufacturers’ recommended procedures using commer-
cially available product intended for intravenous adminis-
tration. The candidate antibiotics were tested with and
without anticoagulants to evaluate for potential variations
in antimicrobial activity; all solutions tested are listed in
Table 1. The following organisms were used in the model:
methicillin-sensitive S. aureus (MSSA), methicillin-resistant
S. aureus (MRSA), Staphylococcus epidermidis (CoNS),
and P. aeruginosa.The P1 MSSA strain, the slime-produc-
ing RP62A CoNS, and clinical isolates of MRSA and P.
aeruginosa cultured from patients with CRBSI at the study
institution were used.19-22 MIC (mg/L) data were deter-
mined or previously known for the test organisms:
1. P1 MSSA: oxacillin ≤0.12, cefazolin ≤0.5, genta-
micin ≤0.12, minocycline ≤0.5, vancomycin ≤1, dap-
tomycin = 0.25, tigecycline ≤0.5;
2. RP62A CoNS: oxacillin and gentamicin reported as re-
sistant by Kirby-Bauer disk method, minocycline ≤0.5,
vancomycin ≤1, daptomycin = 0.5, tigecycline ≤0.5;
3. clinical isolate of MRSA: tigecycline reported as sus-
ceptible, oxacillin ≥2, gentamicin ≤1, tetracycline ≤4,
vancomycin ≤2, daptomycin = 0.5, tigecycline ≤0.5;
4. P. aeruginosa:minocycline = 4, gentamicin = 2, van-
comycin >16, tigecycline ≤4.
Prior studies have demonstrated physical and chemical
compatibility of daptomycin and heparin.17,23 Due to lack
of published compatibility data at the time of this study,
each antibiotic–anticoagulant combination was visually in-
spected for particulate matter prior to testing in the model.
Solutions were stored at room temperature (25 ˚C) and at
37 ˚Cfor 72 hours and were visually inspected every 24
hours for particulate matter and/or color change.
Toestablish catheter infection, 1-cm silicone Hickman
catheter segments (4 per condition) were incubated
overnight at 37 ˚Cin inoculated tryptic soy broth with each
organism. The catheters were washed in PBS and incubated
in 4-mL candidate lock solutions for 0, 2, 4, and 24 hours.
After the specified period, the segments were removed and
washed twice in PBS, sonicated for 3 minutes, and vortexed
for 10 seconds. The resulting solution was serial diluted and
The Annals of Pharmacotherapy n2009 February, Volume 43 n211
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Table 1. Antibiotic–Anticoagulant Lock Solutions
Daptomycin 1 mg/mL ± heparin 5000 units/mL, reconstituted in NS
Cubicin, Cubist Pharmaceuticals, Lexington, MA
Heparin 10,000 units/mL vials, Baxter Healthcare, Deerfield, IL
Daptomycin 1 mg/mL ±heparin 5000 units/mL, reconstituted in LR
Tigecycline 0.5 mg/mL ± EDTA 30 mg/mL
Tygacil, Wyeth Pharmaceuticals, Madison, NJ
Gentamicin 5 mg/mL ± EDTA 30 mg/mL
Gensia Laboratories, Irvine, CA
Cefazolin 5 mg/mL ± heparin 5000 units/mL
Abbott Pharmaceutical, Abbott Park, IL
Phosphate-buffered saline (control solution)
EDTA = ethylenediaminetetraacetate; LR = lactated Ringer’s solution;
NS = NaCl 0.9%.

surface plated on blood agar. After incubation for 24 hours,
colony-forming units were counted and converted to log10
colony-forming units/milliliter (cfu/mL). Each condition was
tested with fresh solutions in quadruplicate and a geometric
mean was determined. Previous experimentation with this
model has demonstrated good reproducibility.
Time-kill analyses are represented through graphs of the
mean values. Statistical analysis evaluated the reduction in
cfu/mL by individual lock solutions compared with the
control solution, PBS, at the 24-hour data point. This mea-
sure served as the primary endpoint. Student’s t-tests were
performed to evaluate results of individual lock solutions
compared with control solution. For each organism, analy-
sis of variance and Student’s t-test were performed to de-
termine whether differences existed among the lock solu-
tions tested. In comparison, a p value less than 0.05 was
determined to be significant.
Results
Performance of each antibiotic lock solution with re-
spect to the test organisms at the 24-hour time points is
summarized in Table 2. The time-kill analyses for each or-
ganism are depicted in Figures 1–5, with minimal devia-
tion in the results.
COMPATIBILITY
Upon admixture, precipitation was not visualized in any
of the lock solutions. None of the lock solutions containing
anticoagulants performed worse than the corresponding
solution without anticoagulant, suggesting no significant
antagonistic effects of the anticoagulant. As such, and be-
cause most authorities recommend using an anticoagulant,
the results of lock solutions without anticoagulant are not
presented here. This study was not designed to evaluate
compatibility or stability of the solutions; however, no vi-
sual precipitation was seen at 72 hours for the test solu-
tions at 25 ˚Cand 37˚C. A slight color change from yel-
low to brown was noted in several syringes containing
tigecycline plus EDTA; however, this did not occur until
after 48 hours at both of these temperatures.
DAPTOMYCIN PLUS HEPARIN RECONSTITUTED WITH NACL
0.9% OR LACTATED RINGER’S
Overall, daptomycin plus heparin reconstituted in NaCl
0.9% (D+NS+H) displayed poor ability to reduce cfu/mL
at 24 hours (Figure 1). There was no significant difference
between D+NS+H and PBS among any of the organisms
tested (p > 0.05). Daptomycin plus heparin in lactated
Ringer’s (LR; D+LR+H) was significantly better (p <
0.05) at reducing 24-hour cfu/mL counts when compared
with D+NS+H for all staphylococcal species tested. For
this reason, further results for daptomycin are henceforth
reported only for D+LR+H.
METHICILLIN-SUSCEPTIBLE
S. AUREUS
All antibiotic–anticoagulant lock solutions displayed
good activity against the P1 MSSA strain (Figure 2) and
were found to be significantly better than PBS at reducing
cfu/mL at 24 hours (p < 0.05). Interestingly,there was no
statistically significant difference in activity found among
the different antibiotic solutions. Tigecycline plus EDTA
(Ti+EDTA) and gentamicin plus EDTA (G+EDTA) dis-
played the greatest mean reduction in 24-hour log10
cfu/mL (5.02 ± 0 and 5.02 ± 0) compared with PBS (0.52
± 1.32). No growth was seen from any of the catheters fol-
lowing 24 hours of exposure to Ti+EDTA or G+EDTA. Of
note, G+EDTAwas the lone solution resulting in a statisti-
cally significant reduction in cfu/mL compared with PBS
prior to the 24-hour endpoint. At the 4-hour time point,
cfu/mL were reduced by an average of 3.98 log units with
G+EDTA, a significantly greater reduction compared with
all other solutions (p < 0.05). D+LR+H reduced 24-hour
cfu/mL by an average of 3.82 log units.
METHICILLIN-RESISTANT
S. AUREUS
D+LR+H, Ti+EDTA, and G+EDTAeach resulted in
significant reductions in 24-hour cfu/mL compared with
PBS (p < 0.05) against the clinical isolate of MRSA. No
significant difference was noted between the mean log10
cfu/mL reduction at 24 hours between cefazolin plus hep-
arin (C+H) and PBS (2.99 vs 2.09; p > 0.05). G+EDTA re-
sulted in the greatest reduction in log10 cfu/mL at 24 hours
(6.37). In addition, G+EDTA was significantly more active
than both Ti+EDTA and C+H (p < 0.0001). The reduction
212 nThe Annals of Pharmacotherapy n2009 February, Volume 43 www.theannals.com
PB Bookstaver et al.
Table 2. Mean Log Colony-Forming Unit
Reduction at 24 Hours
Lock
Solutions MRSA MSSA CoNS PSA
C+H 2.99 4.23a2.04 1.26
D+NS+H 1.68 1.33 2.16 0.52
D+LR+H 4.67a3.82a4.25a1.09
Ti+EDTA 3.97a5.02a5.87a4.41a
G+EDTA 6.37a5.02a6.03a5.13a
PBS 2.09 0.52 1.23 0.01
C=cefazolin 5 mg/mL; CoNS = coagulase-negative
Staphylococcus
epidermidis
;D=daptomycin 1 mg/mL; EDTA = ethylenediaminete-
traacetate 30 mg/mL; G = gentamicin 5 mg/mL; H = heparin 5000
units/mL; LR = lactated Ringer’s solution; MRSA = methicillin-resis-
tant
Staphylococcus aureus
;MSSA = methicillin-susceptible
S. au-
reus
;NS = NaCl 0.9%; PBS = phosphate-buffered saline; PSA =
Pseu-
domonas aeruginosa
; Ti=tigecycline 0.5 mg/mL.
ap<0.05 compared with PBS.

in log10 cfu/mL at 24 hours was greater for G+EDTA than
for D+LR+H (6.37 vs 4.67, respectively); however, this
difference was not found to be statistically significant (p =
0.063). There also was no significant difference in the activi-
ty of D+LR+H when compared with either Ti+EDTA or
C+H (3.97 and 2.99; p > 0.05). No significant differences in
cfu/mL reductions were seen among any of the lock solu-
tions prior to the 24-hour time point. Time-kill activity of all
lock solutions against MRSA is shown in Figure 3.
COAGULASE-NEGATIVE S. EPIDERMIDIS
D+LR+H, Ti+EDTA, and G+EDTA all exhibited ex-
cellent activity against the slime-producing CoNS strain,
Novel Antibiotic Lock Solutions in a Model Against Isolates of Catheter-Related Bloodstream Infections
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Figure 2. Methicillin-sensitive
Staphylococcus aureus
time-kill analysis. C = cefazolin; D = daptomycin; EDTA = ethylenediaminetetraacetate; G =
gentamicin; H = heparin; LR = lactated Ringer’s solution; PBS = phosphate-buffered saline; Ti = tigecycline.
Figure 1. Comparison of daptomycin plus heparin reconstituted in NS versus LR time-kill analysis. CoNS = coagulase-negative
Staphylococcus epi-
dermidis
;D=daptomycin; H = heparin; LR = lactated Ringer’s solution; MRSA = methicillin-resistant
Staphylococcus aureus
;MSSA = methicillin-sus-
ceptible
S. aureus
;NS = NaCl 0.9%.

resulting in significant reductions in bacterial counts at
24hours when compared with PBS (p < 0.05) (Figure 4).
C+H was found to be no better than PBS (p > 0.05) and
inferior in activity to all other lock solutions (p < 0.05).
G+EDTA again demonstrated the greatest reduction in
log10 cfu/mL at 24 hours and was found to be significantly
more active than D+LR+H (p < 0.05). There was no sig-
nificant difference observed in the cfu/mL reduction be-
tween G+EDTA and Ti+EDTA (6.03 vs 5.87; p > 0.05).
Comparison of Ti+EDTA and D+LR+H demonstrated sig-
nificantly better activity with Ti+EDTA (p = 0.041). When
data points prior to 24 hours are compared, G+EDTA
demonstrated a reduction in cfu/mL at 4 hours of 4.40 log
units, statistically superior in activity to all other lock solu-
tions (p < 0.001). No other significant differences prior to
the 24-hour time point were noted.
214 nThe Annals of Pharmacotherapy n2009 February, Volume 43 www.theannals.com
PB Bookstaver et al.
Figure 4.
Staphylococcus epidermidis
time-kill analysis. C = cefazolin; D = daptomycin; EDTA = ethylenediaminetetraacetate; G = gentamicin; H =
heparin; LR = lactated Ringer’s solution; PBS = phosphate-buffered saline; Ti = tigecycline.
Figure 3. Methicillin-resistant
Staphylococcus aureus
time-kill analysis. C = cefazolin; D = daptomycin; EDTA = ethylenediaminetetraacetate; G = gen-
tamicin; H = heparin; LR = lactated Ringer’s solution; PBS = phosphate-buffered saline; Ti = tigecycline.

P
.AERUGINOSA
Only G+EDTA and Ti+EDTA displayed effective activ-
ity against the clinical isolate of P. aeruginosa.There was
no significant difference in mean log10cfu/mL reductions
between G+EDTA and Ti+EDTA (5.13 vs 4.41, respec-
tively; p = 0.158); however, both were significantly
superior in activity compared with D+LR+H and C+H (p <
0.0001). Reductions of 3.33 and 3.29 log units for Ti+EDTA
and G+EDTA, respectively, at the 2-hour time point were
significantly greater than were reductions for PBS (p <
0.05). Significant reductions in cfu/mL continued at the 4-
and 24-hour time points. The time-kill analysis in Figure 5
demonstrates the overall activity of each lock solution.
Discussion
The lock solutions of daptomycin reconstituted with
LR+H, Ti+EDTA, and G+EDTA represent novel combi-
nations of antibiotics and anticoagulants. The use of C+H
in both a prevention and treatment modality for CRBSI
has been noted; however, specific clinical outcomes of ce-
fazolin as a single antimicrobial agent in solution have not
been reported.12,24,25 In our study, cefazolin exhibited activi-
ty against only the P1 MSSA strain. The intrinsic resis-
tance of many organisms responsible for CRBSI and the
results of the present study do not support the use of C+H
as an option for adjunctive treatment for CRBSI in the ab-
sence of proven antibiotic susceptibility.Compatibility and
stability data for C+H solutions are well documented.24,26
Daptomycin activity is dependent on the presence of un-
bound calcium ions. Unlike NaCl 0.9%, LR provides ap-
proximately 3 mEq/L of calcium in the form of calcium
chloride, dihydrate, optimizing antibacterial activity of
daptomycin.27,28 In our study, daptomycin 1 mg/mL recon-
stituted in NS+H was no better than PBS (p > 0.05) against
all organisms tested. Daptomycin 1 mg/mL reconstituted
with LR+H showed good activity against all staphylococ-
cal species tested. These results support those in a recent
report by Laplante et al.17 that demonstrated good in vitro
activity of daptomycin 5 mg/mL against biofilm-produc-
ing S. aureus.In a separate report, daptomycin 2 mg/mL
demonstrated significant reductions in cfu/mL in MRSA
biofilm after only 24 hours of exposure.17
In our study, as expected, daptomycin did not result in
any appreciable activity against the P. aeruginosa isolate;
however, testing was necessary to maintain laboratory con-
sistency and timing of the experimentation.
The potent activity of daptomycin against common
gram-positive CRBSI pathogens and results of the present
study and others support further investigation of dapto-
mycin reconstituted in LR in a clinical trial. Lack of gram-
negative activity may be a limiting factor.Another limiting
factor is that the activity of daptomycin against staphylo-
cocci embedded in biofilm is dependent upon calcium-en-
riched media such as LR. Calcium has been shown to pro-
mote microbial adherence of bacterial organisms through
the enhancement of slime production and biofilm forma-
tion, while calcium chelators such as EDTAwere shown to
disrupt biofilm.29-33 Because EDTA(as a calcium chelator)
Novel Antibiotic Lock Solutions in a Model Against Isolates of Catheter-Related Bloodstream Infections
The Annals of Pharmacotherapy n2009 February, Volume 43 n215
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Figure 5.
Pseudomonas aeruginosa
time-kill analysis. C = cefazolin; D = daptomycin; EDTA = ethylenediaminetetraacetate; G = gentamicin; H = hep-
arin; LR = lactated Ringer’ssolution; PBS = phosphate-buffered saline; Ti = tigecycline.

cannot be used with daptomycin, heparin is the only anti-
coagulant that can be used concurrently with this antibiot-
ic. Independently, heparin has been shown to enhance
biofilm formation.34,35 Despite this phenomenon of biofilm
enhancement, the addition of calcium to the solution is
critical for daptomycin activity and so should always be
added when daptomycin is used as a lock solution.
To our knowledge, this is the first study evaluating the
activity of tigecycline in combination with EDTA in a lock
solution. Previous studies have demonstrated in vitro and in
vivo activity against gram-positive pathogens, including
biofilm-producing MRSA isolates.16 EDTA was chosen as
the anticoagulant based on previous data from its use in
combination with minocycline. Minocycline plus EDTA in
alock solution demonstrated excellent in vitro activity and
resulted in significant reductions in the incidence of CRBSI
and catheter colonization when compared with standard
heparin or saline locks.5,13,19,36-41 In our study, the addition
of EDTA to tigecycline greatly enhanced the activity of
tigecycline alone, resulting in improved reductions of
cfu/mL for the majority of organisms tested at 24 hours
(data not shown). Ti+EDTA demonstrated significant re-
ductions of cfu/mL at 24 hours against all organisms test-
ed. Although our P.aeruginosa isolate was susceptible to
tigecycline, typical MIC90 values of tigecycline are greater
than 16 mg/mL, perhaps resulting in difficulty in eradicat-
ing the organism. Given the broad spectrum of activity for
tigecycline and potent activity demonstrated in the present
study,the combination of tigecycline and EDTA deserves
further investigation as an antibiotic lock solution.
G+H has been one of the most extensively studied and
used antibiotic lock solutions in clinical practice. However,
there are reports of incompatibility and potential inactiva-
tion of heparin in solution with gentamicin.25,42,43 The addi-
tion of EDTA to gentamicin has not been previously inves-
tigated, although another calcium chelator,citrate, has been
used in combination with gentamicin.40,44 In our study,
G+EDTA demonstrated the most potent activity at 24
hours of exposure against all organisms tested. Each or-
ganism, except the RP62A CoNS strain, displayed in vitro
susceptibility to gentamicin. G+EDTAwas the only solu-
tion to result in a significant reduction of cfu/mL at the 4-
hour time point (p < 0.05) for all organisms tested, except
MRSA. However, with regard to MRSA, G+EDTA was
superior to Ti+EDTA, C+H, and PBS (p < 0.0001) and re-
sulted in a 1.7 log unit further reduction in cfu/mL com-
pared with D+LR+H (p = 0.063) at the 24-hour time point.
G+EDTA at the 24-hour time point resulted in a reduction
in cfu/mL to the lower limit of detectability (1.59 log units)
of our model for all organisms tested. The potent activity
demonstrated by G+EDTA and previous successes of gen-
tamicin plus citrate firmly support the investigation of this
solution in a clinical trial.
Clinical use of antibiotic lock solutions in a treatment
modality for CRBSI is directed at specific patient popula-
tions; the CVC serves as the lifeline for many of these pa-
tients. In vitro models like the one presented here offer ef-
ficacy data to support use of specific antibiotics in a lock
solution as an adjunct to systemic therapy. Gentamicin,
tigecycline, and daptomycin all demonstrated potent activ-
ity against common pathogens associated with CRBSI.
Further compatibility and safety data are required before
these agents should be used routinely in clinical practice.
Antibiotic lock solutions do have a place in therapy for
specific patients, and that role may be expanding with the
new payment decisions from Centers for Medicare and
Medicaid Services. Use of antibiotic lock solutions may
lead to catheter flushing or leakage of solution from the
catheter lumen, exposing patients to low systemic concen-
trations of antibiotics. Thus, the potential for development
of resistance exists. Removal of the infected CVC, when
feasible, is the standard of care.
There are several limitations to our study.The exclusion
of drugs such as vancomycin and ethanol, which are com-
monly used in antimicrobial lock solutions, limits our abil-
ity to make further conclusions regarding their compara-
tive efficacy. Vancomycin and ethanol have been exam-
ined in previous studies utilizing this model and, as
concluded from other in vitro models, vancomycin appears
to have limited activity against organisms within a
biofilm.17Exclusion of a gentamicin plus heparin solution
is also a limitation. Unpublished results of gentamicin plus
heparin in this model demonstrate potent activity of this
combination. However, compatibility of gentamicin plus
heparin is variable, which is why the present data with
gentamicin plus EDTAare useful. The lower limit of de-
tectability of this model (1.59 cfu/mL) does not allow for
the extent of efficacy to be fully expressed when specific
agents are compared. Our model examined activity for
only 24 hours; thus, we cannot conclude whether rebound
growth would occur with continued use of these agents.
The use of EDTAas an anticoagulant may be a limitation
for institutional use, as its availability is somewhat limited
and it is costly. The lack of compatibility and stability data is
also a limitation, making it difficult for adoption of EDTA
into clinical practice. Investigators from this team are cur-
rently working on such studies. Application to clinical prac-
tice may be further limited by the continuous exposure of
catheters to antibiotic lock solutions in this model. Often in
clinical practice, the CVC will need to be accessed multiple
times daily, not allowing for a continuous dwell time.
Webelieve that Ti+EDTA and G+EDTA deserve strong
consideration for study in a clinical trial. Further study of
D+H should be targeted at gram-positive pathogens re-
sponsible for CRBSI. Additional compatibility and stabili-
ty information is also needed to support use of these candi-
date lock solutions.
216 nThe Annals of Pharmacotherapy n2009 February, Volume 43 www.theannals.com
PB Bookstaver et al.

PBrandon Bookstaver PharmD BCPS AAHIVE, Clinical Assis-
tant Professor, College of Pharmacy, University of South Carolina
Campus, Columbia, SC
John C Williamson PharmD BCPS AAHIVE, Clinical Coordina-
tor, Infectious Diseases, Department of Pharmacy, Wake Forest Uni-
versity Baptist Medical Center, Winston-Salem, NC
Brian Keith Tucker BS, Laboratory Technician, Department of In-
fectious Diseases, School of Medicine, Wake Forest University
Issam I Raad MD, Professor and Chair, Department of Infectious
Diseases, Infection Control & Employee Health, The University of
Texas MD Anderson Cancer Center, Houston, TX
Robert J Sherertz MD, Professor of Medicine and Hospital Epi-
demiologist, Section on Infectious Diseases, School of Medicine,
Wake Forest University
Reprints: Dr. Bookstaver, South Carolina College of Pharmacy,
University of South Carolina Campus, 715 Sumter St., CLS-312C,
Columbia, SC 29208, fax 803/777-2820, bookstaver@sccp.sc.edu
This study was funded in part by Cubist Pharmaceuticals.
Drs. Raad and Sherertz are co-patent holders of M-EDTA pharma-
ceutical preparations and uses thereof (US Patent 5,362,754. Novem-
ber 1994). It is currently not licensed or in production.
Findings of this study were presented in poster format (abstract 214)
at the 17th Scientific Meeting of the Society for Healthcare Epi-
demiology of America, Baltimore, MD, April 14–17, 2007.
Wethank Beth Williams PharmD BCPS, Director,Department of Pharmacy, Wake
Forest University Baptist Medical Center, Winston-Salem, NC, for assistance in pro-
viding laboratory supplies.
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Actividad de Nuevas Soluciones Antibióticas de Sellado en un
Modelo de Muestras Aisladas de Infecciones Relacionadas con
Catéteres Intravasculares
PB Bookstaver, JC Williamson, BK Tucker, II Raad, y RJ Sherertz
Ann Pharmacother 2009;43:210-9.
EXTRACTO
TRASFONDO: Las infecciones relacionadas con catéteres intravasculares
(CRBSI) son motivo de preocupación en pacientes con catéteres venosos
centrales (CVC) permanentes. La instilación de una solución antibiótica
de sellado puede servir como terapia adyuvante.
OBJETIVO: Evaluar la eficacia de nuevas soluciones antibióticas-antico-
agulantes de sellado mediante un modelo in vitro de infección de
CVC.
MÉTODOS: Se evaluaron las siguientes soluciones de sellado: daptomicina
1mg/mL (reconstituida con ringer lactato [RL]) más heparina 5000
unidades/mL, tigeciclina 0.5 mg/mL más ácido etileno-diamino-tetra-
acético (EDTA) 30 mg/mL, gentamicina 5 mg/mL más EDTA30 mg/mL,
cefazolina 5 mg/ml más heparina 5000 unidades/mL, y solución salina
tamponada con fosfato como solución control. El análisis se realizó en
segmentos de catéter Hickman inoculados con los siguientes organismos:
Staphylococcus aureus sensible a meticilina y resistente a la meticilina
(MRSA), Staphylococcus epidermidis, y Pseudomonas aeruginosa.Las
soluciones se incubaron en las soluciones de sellado candidatas durante
períodos de 0, 2, 4, y 24 horas. Se realizó el test de tde Student para
evaluar la reducción en log10 de unidades formadoras de colonias (cfu/mL)
de las soluciones de sellado por separado en comparación con la solución
control. Para cada organismo, se efectuaron los análisis de varianza y los
test de tpara determinar si había diferencias entre las soluciones de
sellado.
RESULTADOS: Gentamicina más EDTA (G+EDTA) y tigeciclina más EDTA
(Ti+EDTA) produjeron reducciones significativas (p < 0.05) de log10
cfu/mL a las 24 horas para todos los organismos analizados.
Daptomicina reconstituida con RL más heparina (D+RL+H) demostró
una potente actividad contra todas las especies estafilocócicas (p < 0.05).
Con respecto a MRSA, G+EDTA mostró una actividad significativa-
mente superior que Ti+EDTA y cefazolina más heparina (p < 0.05), pero
no hubo diferencias en comparación con D+RL+H (p = NS). No se
observó antagonismo con la adición de anticoagulantes a las soluciones.
CONCLUSIONES: Gentamicina, tigeciclina, y daptomicina en combinación
con anticoagulantes como soluciones de sellado muestran una potente
actividad contra los patógenos responsables de CRBSIs. Cada una de
estas soluciones de sellado merece que se estudie más profundamente en
un ensayo clínico. Se necesitan más datos sobre la compatibilidad y la
estabilidad de estas soluciones antes de poder recomendar su uso
clínico rutinario.
Traducido por Enrique Munoz Soler
Activité d’une Nouvelle Solution Antibiotique pour Instillation
Locale Dans un Modèle d’Infections Associées aux Cathéters
PB Bookstaver,JC Williamson, BK Tucker, II Raad, et RJ Sherertz
Ann Pharmacother 2009;43:210-9.
RÉSUMÉ
INTRODUCTION: Les infections associées au cathéter sont un problème
fréquent chez les patients ayant un cathéter veineux central permanent
(CVP). L’instillation d’une solution antibiotique à l’intérieur de la
lumière du cathéter peut agir comme thérapie adjuvante.
OBJECTIF: Évaluer l’efficacité d’une solution d’un nouvel antibiotique
associé à un anticoagulant dans un modèle in vitro d’infection de
CVP.
MÉTHODOLOGIE: Les solutions antibiotiques suivantes instillées
localement furent évaluées l’association daptomycine 1 mg/mL
(reconstituée avec du lactate ringer (LR) et héparine 5000 unités/mL)
(D+LR+H), l’association tigécycline 0.5 mg/mL et éthylènediamine-
tétraacétate (EDTA) 30 mg/mL (Ti+EDTA), l’association
gentamicine 5 mg/mL et EDTA30 mg/mL (G+EDTA), l’association
céfazoline 5 mg/mL et héparine 5000 unités/mL (C+H) et finalement,
une solution tamponnée par des phosphates fut utilisée comme
solution contrôle. L’analyse fut effectuée sur des sections de cathéter
Hickman inoculées avec les organismes suivants: Staphylococcus
aureus sensible à la méthicilline et résistant à la méthicilline (MRSA),
Staphylococcus epidermidis,et Pseudomonas aeruginosa.Les
cathéters furent incubés dans une des solutions antibiotiques pour une
période variant de 0, 2, 4, et 24 heures. Le test tde Student fut utilisé
pour évaluer la réduction de la colonisation (en log10 du nombre de
colonies) des différentes solutions d’instillation locale comparée à celle de
la solution contrôle. Pour chaque organisme, le test d’analyse de
variance et le test tde Student furent effectués pour déterminer la
présence de différences entre les diverses solutions d’instillation
locale.
RÉSULTATS: La G+EDTA et la Ti-EDTA ont démontré une réduction
significative (p < 0.05) du nombre de colonies/mL exprimé en log10 à24
heures pour tous les microorganismes testés. La combinaison D+LR+H
adémontré une activité puissante contre toutes les espèces de staphylo-
218 nThe Annals of Pharmacotherapy n2009 February, Volume 43 www.theannals.com
PB Bookstaver et al.

coques (p < 0.05). Pour le MRSA, la G+EDTA a démontré une activité
supérieure à celle de la Ti-EDTA et de la C+H (p < 0.05) mais aucune
différence:avec la D+LR+H (p = NS). Aucun antagonisme ne fut noté
avec l’addition d’anticoagulants à ces solutions antibiotiques.
CONCLUSIONS: L’instillation locale de solutions de gentamicine, de tigécy-
cline, et de daptomycine en combinaison avec un anticoagulant ont
démontré une activité puissante contre les pathogènes responsables des
infections associées au cathéter. Chacune de ces solutions d’instillation
devrait faire l’objet d’une étude clinique. De plus, des données de com-
patibilité et de stabilité seront nécessaires avant que leur utilisation de
routine ne soit recommandée.
Traduit par Marc M Perreault
Novel Antibiotic Lock Solutions in a Model Against Isolates of Catheter-Related Bloodstream Infections
The Annals of Pharmacotherapy n2009 February, Volume 43 n219
www.theannals.com
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