A novel cell-associated protection assay demonstrates the ability of certain antibiotics to protect ocular surface cell lines from subsequent clinical Staphylococcus aureus challenge.
ABSTRACT In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.
- SourceAvailable from: Eric G Romanowski[Show abstract] [Hide abstract]
ABSTRACT: Ocular infections are a leading cause of vision loss. It has been previously suggested that predatory prokaryotes might be used as live antibiotics to control infections. In this study, Pseudomonas aeruginosa and Serratia marcescens ocular isolates were exposed to the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. All tested S. marcescens isolates were susceptible to predation by B. bacteriovorus strains 109J and HD100. Seven of the 10 P. aeruginosa isolates were susceptible to predation by B. bacteriovorus 109J with 80% being attacked by M. aeruginosavorus. All of the 19 tested isolates were found to be sensitive to at least one predator. To further investigate the effect of the predators on eukaryotic cells, human corneal-limbal epithelial (HCLE) cells were exposed to high concentrations of the predators. Cytotoxicity assays demonstrated that predatory bacteria do not damage ocular surface cells in vitro whereas the P. aeruginosa used as a positive control was highly toxic. Furthermore, no increase in the production of the proinflammatory cytokines IL-8 and TNF-alpha was measured in HCLE cells after exposure to the predators. Finally, injection of high concentration of predatory bacteria into the hemocoel of Galleria mellonella, an established model system used to study microbial pathogenesis, did not result in any measurable negative effect to the host. Our results suggest that predatory bacteria could be considered in the near future as a safe topical bio-control agent to treat ocular infections.PLoS ONE 06/2013; 8(6):e66723. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process.Clinical ophthalmology (Auckland, N.Z.) 01/2013; 7:965-71.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2011, p. 3788–3794
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 55, No. 8
A Novel Cell-Associated Protection Assay Demonstrates the Ability of
Certain Antibiotics To Protect Ocular Surface Cell Lines from
Subsequent Clinical Staphylococcus aureus Challenge?†
J. B. Wingard,1E. G. Romanowski,1,2R. P. Kowalski,1,2F. S. Mah,1,2Y. Ling,1,3
R. A. Bilonick,1,3and R. M. Q. Shanks1,2*
UPMC Eye Center, Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania1; The Charles T. Campbell Laboratory, UPMC Eye Center,
Ophthalmology and Visual Sciences Research Center, Eye and Ear Institute, Department of Ophthalmology, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania2; and Graduate School of Public Health, Department of
Biostatistics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania3
Received 29 December 2010/Returned for modification 25 March 2011/Accepted 20 May 2011
In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to
provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests.
We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin
(ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells
from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and
then incubated in medium containing a single antibiotic (0 to 512 ?g/ml). After incubation, the cells were
challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epi-
thelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective
concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was deter-
mined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and
growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and
TET at >64 ?g/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection
at higher concentrations. TET toxicity was demonstrated at >64 ?g/ml, whereas AZM displayed toxicity to one
cell line at 512 ?g/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to
BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was
displayed in this cell association assay, providing data that may be considered in addition to traditional testing
when determining therapeutic dosing regimens.
Traditional antibiotic efficacy tests, such as MIC, evaluate
the interaction between the pharmacologic agent and the bac-
terial cells in culture (12). Although this interaction is highly
important and has guided clinical decision-making regarding
antibiotic choice, these tests fail to incorporate information
about the host tissue that may affect bacterial susceptibility to
clinical therapy. While this phenomenon may be important in
many tissue types, it is especially important for the eye, where
antimicrobials may be delivered topically but may not remain
at the site of infection long enough to provide adequate ther-
apy without very frequent dosing.
When antibiotics are applied directly to the ocular surface,
they may adhere to or become incorporated within epithelial
cells. Since the tear film is made briskly and quickly circulates
away from the eye via the nasolacrimal system (20), there is a
theoretical advantage to antibiotics that have a prolonged tis-
sue half-life due to tissue absorption. In the current study, we
evaluate the efficacy of various antibiotics to control clinical
ocular Staphylococcus aureus strains using a novel cell-associ-
ated assay. Specifically, we report the different extents to
which azithromycin (AZM), erythromycin (ERY), tetracycline
(TET), and bacitracin (BAC) protect Chang and human
corneal limbal epithelial (HCLE) cell lines against challenge
with S. aureus ocular isolates after all free drug had been
removed from the cell culture. Antibiotic toxicity was also
We chose S. aureus as a challenge in this assay because it is
a major pathogen, associated with a variety of ocular infec-
tions, including blepharitis, conjunctivitis, keratitis, and endo-
phthalmitis (1, 6, 7, 17, 19). Our approach was to evaluate
possible treatments for conjunctivitis and blepharoconjunctivi-
tis, and so the six strains chosen in this assay were isolated from
patients with these conditions. The antibiotics that we evalu-
ated include two readily available antibiotics that are marketed
as ophthalmic ointments (erythromycin and bacitracin), one
that has recently received Food and Drug Administration ap-
proval as an ophthalmic solution intended to treat bacterial
conjunctivitis caused by S. aureus and other bacteria (azithro-
mycin), and one that historically was a treatment for a number
of ocular surface infections (tetracycline). This study therefore
addresses the effects of a range of antibiotics from different
drug classes in the protection of multiple ocular surface cell
lines of likely clinical relevance, measuring the antibiotic’s in
* Corresponding author. Mailing address: Department of Ophthal-
mology, University of Pittsburgh, 203 Lothrop St., Pittsburgh, PA
15208. Phone: (412) 647-3537. Fax: (412) 647-5880. E-mail: shanksrm
† Supplemental material for this article may be found at http://aac
?Published ahead of print on 31 May 2011.
vitro ability to protect epithelial cells against a clinically rele-
vant infectious agent.
In this study, we demonstrated that a novel in vitro assay,
which we termed the cell-associated protection assay (CAPA),
can be employed to measure the relative protective efficacy of
an antibiotic based on its ability to associate with human ocular
surface cell layers composed of epithelial cells. We demon-
strated that certain antibiotics associated so closely with ocular
surface cell lines that the epithelial cell layers were protected
from clinical S. aureus challenge even after all free drug was
vigorously washed away from the cell culture. This protection
was observed throughout a 24-h S. aureus challenge, and sev-
eral assays suggested that the majority of the bacteria had
actually been killed by the cell-associated drugs. CAPA may be
used to analyze the ability of antibiotics to continue providing
effective antibacterial control throughout the day, between eye
drop doses or other antibiotic applications, an analysis that
could help guide dosing interval recommendations.
MATERIALS AND METHODS
Cell lines and culture conditions. Chang conjunctival cells (clone 1-5c-4
[Wong-Kilbourne derivative of Chang conjunctiva]; ATCC CCL-20.2; American
Type Culture Collection [ATCC], Manassas, VA) were maintained in Gibco
medium 199 with 1% Penn-Strep, 10% fetal bovine serum (FBS), 5% sodium
bicarbonate, and 0.1% gentamicin. When cells were plated for these experi-
ments, outgrowth medium (OG) for Chang cells consisted of Gibco medium 199
with 10% FBS and 5% sodium bicarbonate but without antibiotics. The ATCC
reports that the Chang cell line is contaminated with HeLa cells. Here, we use
Chang cells as sample mammalian cells and draw no conclusions based upon
their debatable ocular source.
HCLE cells (4) were obtained from Jes Klarlund with permission from Ilene
Gipson. OG for HCLE cells consisted of keratinocyte-SFM (serum-free medium)
with L-glutamine, supplemented with 25 ?g/ml bovine pituitary extract (BPE), 0.2
ng/ml epidermal growth factor (EGF), and 1 mM CaCl2, without any antibiotics.
Bacterial strains and growth medium. Six clinical isolates of S. aureus were
recovered from patients presenting with bacterial conjunctivitis and blepharo-
conjunctivitis to the UPMC Eye Center, with microbiologic testing performed in
the Charles T. Campbell Ophthalmic Microbiology Laboratory, Pittsburgh, PA.
Isolates were retrieved from a frozen ?75°C retrospective clinical collection that
was deidentified and stored for antibiotic validations. Isolates were selected from
the collection to include four isolates that were macrolide (AZM, ERY) suscep-
tible and two that were macrolide resistant, based on Kirby-Bauer disk diffusion
testing. No other antibiotic susceptibilities were tested as part of the inclusion
criteria, although the MIC of all isolates was later determined using Etests for
each studied antibiotic (bioMe ´rieux, Inc., Durham, NC) (Table 1). Bacteria were
grown in tryptic soy broth (TSB) and maintained on tryptic soy agar (TSA)
containing 5% sheep’s blood (BD BBL Becton, Dickinson and Co., Sparks, MD).
Experimental drugs. AZM 1% solution was provided by Inspire Pharmaceu-
ticals (Durham, NC). TET was purchased from Sigma-Aldrich (St. Louis, MO),
and a 1% solution was prepared in 95% ethanol. Stocks of ERY (Sigma-Aldrich)
and BAC (Sigma-Aldrich) were created prior to each set of experiments and
stored as a 1,024-?g/ml solution.
In vitro CAPA. (i) Step 1. Flat-bottom 96-well tissue culture plates (Costar
3595; Corning Inc., Corning, NY) containing Chang or HCLE cells were grown
to confluence without antibiotics, as described above.
(ii) Step 2. The medium from each well was removed, and 200 ?l of OG
medium with the test antibiotics (0 to 512 ?g/ml) was added to each well
according to the key detailed in Fig. 1A. Each concentration was plated in
triplicate wells on each of the 8 plates used per experiment. Plates were incu-
bated at 37°C in 5% CO2.
(iii) Step 3. Plates containing the epithelial cell cultures were removed from
incubation after 24 h with the antibiotics, medium was removed, and all wells
were washed twice with 200 ?l phosphate-buffered saline (MP Biomedicals,
(iv) Step 4. Bacterial inocula were 1 ? 105CFU in 200 ?l of OG medium per
well (confirmed range by colony counts: 3.68 ? 104to 4.76 ? 105CFU/well). The
bacterial strains are listed in Table 1. Plates were incubated for 24 h at 37°C in
5% CO2. Two plates were not inoculated with bacteria, one as a mock infection
plate, and the other for toxicity assays noted below.
(v) Step 5. Remaining medium was removed from all wells, and plates were
washed twice with water to remove unattached cells and debris. Epithelial cell
layers were then fixed and stained with a gentian violet solution (0.5% gentian
violet, 0.9% NaCl, 1.85% formaldehyde, and 50% ethanol) and allowed to dry.
Plates were scanned to provide qualitative data on the survival of intact epithelial
MCPC determination. To assign the minimum cell layer protective concen-
tration (MCPC) of an antibiotic, we observed scanned plate images for wells in
which gentian violet staining was apparent. With Chang cells, the cell layers
without antibiotic treatment were destroyed, and no staining was apparent, so
that any purple staining was counted as a positive. Two plates from independent
experiments were observed, each with three replicate wells for each bacterium-
antibiotic concentration combination. At least two of the three replicate wells in
one experiment had to agree in order to consider an antibiotic concentration as
having a protective effect, and if the MCPCs of the two experiments differed,
then the lower value was used. For the HCLE cells, the cell layers were often
more resistant to bacterial challenge so that there was more background gentian
violet staining, making identification of wells with increased protection more
difficult. For HCLE cells, three independent experimental plate scans were
observed. In this case, we recorded for the lowest concentration with a difference
in staining compared to the no-antibiotic treatment. As with the Chang cell
MCPC prediction, at least two wells at a concentration were required for MCPC
estimation, and the lowest value was taken.
Quantification of stained cell layers and microscopy. Glacial acetic acid (200
?l per well of 30% [vol/vol]; Fisher Scientific) was then added to each well to
solubilize the gentian violet. After gentle vortexing, 20 ?l from each well was
transferred to corresponding wells in new plates that contained 180 ?l of 95%
ethanol. A single well in each plate was filled with 200 ?l of 95% ethanol and
used as a blank, and plates were read for absorbance at 590 nm on the Synergy
2 microplate reader (BioTek, Winooski, VT).
In addition to gentian violet staining, cell layers were assessed by microscopy,
and representative photomicrographs were obtained. Phase-contrast images
were obtained using a Nikon Eclipse TE2000-U microscope equipped with a
CoolSnap HQ charge-coupled device camera, and images were acquired using
TABLE 1. Bacterial strains used in this study and their MICs and MCPCs
Antibiotic MICb?MCPCc? (?g/ml)
32 (R) ?512/256?
24 (R) ?256/?512?
48 (R) ??512/?512?
?256 (R) ??512/?512?
48 (R) ??512/?512?
?256 (R) ??512/?512?
64 (R) ??512/?512?
64 (R) ??512/?512?
aAll strains are conjunctivitis or blepharoconjunctivitis clinical isolates.
bBacterial strains with MICs (?g/ml) for each antibiotic by Etest. Resistance is based on the CLSI (Clinical and Laboratory Standards Institute) systemic breakpoints
for each antibiotic. (R) indicates resistance to the antibiotic by MIC; where this is not listed, the strain was susceptible to the given antibiotic by MIC.
cBacterial strains with MCPCs (?g/ml), indicating the minimum concentration of antibiotic for which cell layer protection was evident in the CAPA. The left number
is for Chang cells, and the right number is for HCLE cells.
VOL. 55, 2011 CELL-ASSOCIATED ANTIBIOTIC SUSCEPTIBILITY ASSAY3789
Metamorph software. Photomicrographs were taken of plates of cells challenged
using bacterial strain B1370 before we determined that it was BAC resistant, as
evaluated by MIC determination (Table 1); micrographs were not taken of plates
challenged with other bacterial strains.
Mathematical modeling. Mathematical modeling was used to analyze gentian
violet staining data. Models were fitted for the logarithm of output (staining)
against the 4th root of the antibiotic dose. Various kinds of nonlinear regression
models (including sigmoid and bell-shaped curves) and linear regression models
(including linear, quadratic, or cubic curves) were fitted. Statistically significant
dose-dependent relationships were determined based on the statistical signifi-
cance of the coefficients estimated.
When a statistically significant dose-dependent relationship was found, a 50%
effectiveness dose was calculated, i.e., the theoretical antibiotic dose at which a
half maximal gentian violet staining was attained. To determine this metric, the
output values of the lower and upper asymptotes were determined within the
dose range of 0 to 512 ?g/ml (4th root of dose 0 to 4.76 ?g/ml), and the halfway
point on the output axis (epithelial cell integrity) was determined, such that the
corresponding antibiotic dose(s) on the dose axis could be calculated. All statis-
tical computations and graphics were produced using the R statistical program-
ming language and environment (14).
Culture turbidity and bacterial growth. After overnight culture incubation,
culture turbidity was assessed as an indicator of bacterial growth by reading
absorbance at 600 nm by using a Synergy 2 microplate reader, using wells with
OG medium as a blank. To determine whether there was viable S. aureus
remaining in the inoculated wells, a 48-pronged, alcohol- and flame-sterilized
plate replicator (Dan-Kar, Woburn, MA) was used to transfer ?2 ?l of each
culture well to blood agar plates. These blood agar plates were incubated at 37°C
overnight and subsequently graded for degree of S. aureus growth (0, no growth;
1, 1 to 5 colonies; 2, 6 or more colonies; 3, confluent growth). Wells that showed
obvious contamination with microbes other than S. aureus were excluded from
further analysis, as were wells displaying visible turbidity prior to bacterial inoc-
ulation. Control experiments indicated that epithelial cell layers were necessary
for the observed antibacterial activity.
Antibiotic toxicity assays. For antibiotic toxicity plates, after the overnight
epithelial cell incubation with antibiotics, 200 ?l of OG was added instead of a
bacterial culture. These plates were incubated for 20 to 24 h at 37°C in 5% CO2
and then assessed for cytotoxicity using alamarBlue (Invitrogen, Camarillo, CA).
Healthy cells are able to reduce alamarBlue, shifting supernatant color from blue
to pink and increasing its fluorescence over 100-fold. First, to serve as a negative
control for reducing power, 2 ?l of Promega lysis solution (Madison, WI) was
added to 3 wells to lyse cell layers. After 15 min, medium was removed from all
wells, and 200 ?l of a 4% alamarBlue solution in clear OG (without phenol red)
was added to each well. The plate was returned to the incubator for 2 h at 37°C
in 5% CO2, and then fluorescence was measured using the Synergy 2 microplate
reader (excitation filter, 500/27; emission filter, 620/40).
In vitro epithelial assay to measure the cell-associated effi-
cacy of antibiotics. A new in vitro method was devised to
measure the contribution of an antibiotic’s ability to interact
with epithelial cells and protection of the cell layer from in-
fectious challenge. The key component to the CAPA is that
after the cell layer is incubated with antibiotics, the layer is
vigorously washed and challenged by pathogenic bacteria in
antibiotic-free medium. Therefore, any protective effect occurs
from antibiotics that associate with the epithelial cell surface or
enter the cells. We tested this model using four antibiotics, six
ocular clinical isolates of S. aureus, and two mammalian cell
lines. We report the results of the above experiments for one
representative bacterial strain, B1412 (Fig. 1 and 2). Strain
B1412 was chosen as the representative strain because it was
susceptible to all studied antibiotics by MIC (Table 1). Results
for the additional tested strains are shown in Fig. S1 and
Tables S1 and S2 in the supplemental material.
Epithelial cell layer protection as a function of pretreatment
with antibiotics. Visual inspection of gentian violet staining of
the Chang and HCLE cells shows that without antibiotic pre-
treatment, mammalian cell layers were destroyed by the bac-
terial challenge (Fig. 1B and C). AZM and ERY show some
degree of protection throughout the tested range when cell
layers were challenged with macrolide-susceptible S. aureus
strains: TET shows protection in the middle of the tested
range, with apparent toxicity at higher doses, and BAC shows
no obvious protective efficacy at any tested concentration with
either mammalian cell type (Fig. 1B and C). Results similar to
those using the Chang cell line were measured with the A549
lung carcinoma cell line using AZM and ERY and a subset of
the S. aureus strains (data not shown).
FIG. 1. Different antibiotics and different ocular cell lines exhibit
contrastive staining zones in the cell-associated protection assay.
(A) Assay plate legend showing the concentration of antibiotics, with
which each well was incubated before free antibiotic was washed away
and before challenge with S. aureus. TET (0 ?g/ml) was supplemented
with ethanol to a concentration equal to the ethanol concentration in
512 ?g/ml TET. AZM, azithromycin; ERY, erythromycin; TET, tet-
racycline; BAC, bacitracin. (B and C) Gentian violet staining of a
representative experiment using Chang (B) and HCLE (C) cells after
pretreatment with study antibiotics and subsequent inoculation with S.
aureus strain B1412. Dark-purple-stained wells are where pretreat-
ment of the monolayer with the indicated antibiotic concentration was
sufficient to afford protection to the monolayer from S. aureus infec-
tion. Clear wells did not have sufficient cell-associated antibiotic to
protect the wells from bacteria, and the mammalian monolayer was
obliterated, or antibiotic toxicity destroyed the cell layers. The MCPCs
for Chang cells for S. aureus strain B1412 were 32 ?g/ml for AZM, 64
?g/ml for ERY, 32 ?g/ml for TET, and ?512 ?g/ml for BAC. The
MCPCs for HCLE cells for S. aureus strain B1412 were 8 ?g/ml for
AZM, 8 ?g/ml for ERY, 8 ?g/ml for TET, and ?512 ?g/ml for BAC.
3790WINGARD ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Microscopic analysis of protected and challenged epithelial
cells supports that the gentian violet staining corresponds to
intact monolayers of epithelial cells and that biofilms evident in
low-antibiotic wells do not contribute to the A590measure-
ments (data not shown), as there was negligible staining in the
wells with 0 ?g/ml antibiotic (Fig. 1). This is consistent with
previous reports showing that S. aureus requires specific me-
dium conditions to form wash-resistant biofilms capable of
being stained with gentian violet (16).
Whereas Chang cells were highly susceptible to challenge by
all of the S. aureus strains used, HCLE cells not exposed to
antibiotics showed a range of damage by the different bacterial
strains; this difference can be clearly seen by the basal levels of
staining in the zero-antibiotic treatment groups when gentian
violet staining was quantified, described below (Fig. 2; see also
Fig. S1 in the supplemental material). Nevertheless, the range
in which pretreatment with antibiotics confers protection to
the cell layer against bacterial challenge can clearly be deter-
To characterize the extent of cell layer protection, we estab-
lished a metric that we titled the minimum cell layer protective
concentration (MCPC). The MCPC represents the minimum
concentration of a given antibiotic that protected the cell layer
from destruction by S. aureus challenge in the CAPA assay
(Table 1). It was noted that AZM exhibited the same or lower
MCPC values than ERY (Table 1), despite the fact that the
strains had lower MIC values to ERY (Table 1). All bacterial
strains had low MCPCs with TET, although these were some-
what difficult to determine with HCLE cells, as the staining
differences were subtle. BAC up to 512 ?g/ml produced no
measure of protection, so a value of ?512 ?g/ml was desig-
Quantification and modeling of gentian violet staining.
Gentian violet was solubilized and measured to quantify the
observed cell layer protection effect, both to validate the
MCPC values and to further characterize the ability of antibi-
otics to associate with and protect mammalian cells. The com-
bined solubilized gentian violet data from replicate experi-
ments shows that Chang cell layer integrity is enhanced by
AZM (32 to 512 ?g/ml), ERY (64 to 512 ?g/ml), and TET (32
to 128 ?g/ml) when susceptible bacteria were used (Fig. 2A;
see also Fig. S1 in the supplemental material). There is a
drop-off in staining from TET between 64 ?g/ml and 128 ?g/ml
with all S. aureus strains, suggesting that TET begins to show
toxicity at ?64 ?g/ml. BAC shows little difference from un-
treated controls at any concentration even though strain B1412
is susceptible to BAC (Fig. 2A; see also Fig. S1 in the supple-
The combined solubilized gentian violet data suggest that
HCLE cell layer integrity is enhanced by pretreatment with
AZM (16 to 128 ?g/ml) and ERY (32 to 512 ?g/ml) and to a
minor extent by TET at 32 ?g/ml (Fig. 2B). BAC shows little
or no protection at any concentration. The five additional
tested bacterial strains showed comparable results, with similar
concentrations of antibiotics providing epithelial cell layer pro-
tection when the strain was susceptible to that antibiotic by
MIC (see Fig. S1 in the supplemental material).
Mathematical modeling was applied to the gentian violet
spectrophotometric data so that 50% effectiveness doses could
be determined from antibiotic-bacterium strain combinations
where protection was observed. This 50% effectiveness dose
corresponds to the theoretical antibiotic concentration that
should yield 50% maximal gentian violet staining in that ex-
periment (see Table S1 in the supplemental material). The
results of this modeling predict that for strain B1412 in the
Chang cell line, the 50% dose was lowest for AZM (see Table
S1). For the other macrolide-susceptible bacterial strains, TET
and AZM have lower 50% predicted doses than ERY with
respect to protecting the Chang cell line. With HCLE cells,
AZM had lower 50% protection doses than ERY in all mac-
rolide-susceptible bacterial strains. TET provided protection
only against B1412 (and not other bacterial strains), although
it did so efficiently, exhibiting the lowest 50% effectiveness
dose for this strain (see Table S1). The MCPC values (Table 1)
and the 50% effectiveness doses (see Table S1) exhibit a sim-
ilar pattern in all cases except when tetracycline was used on
Culture turbidity and bacterial cell growth. The simplest
model for the mechanism of protection of epithelial layers
from S. aureus infection in this cell protection assay is that
epithelial cells secrete antibiotics into the medium at sufficient
concentrations to prevent proliferation of S. aureus to numbers
toxic to the epithelial cells. If this model is true, then there
should be fewer bacteria in wells that exhibit protection. Cul-
ture turbidity provided a measurement of bacterial density
within the supernatant (Fig. 3A), and growth on blood agar
FIG. 2. Quantitative analysis of gentian violet staining for Chang and HCLE cells challenged with S. aureus strain B1412. Gentian violet stain
was solubilized and measured at A590. (A) CAPA with Chang cells. The experiment was performed twice on different days (n ? 6 total wells per
condition), and the average is shown. (B) CAPA with HCLE cells. The experiment was performed three times on different days (n ? 9 total wells
per condition), and the average is shown. Error bars, standard errors of the means.
VOL. 55, 2011CELL-ASSOCIATED ANTIBIOTIC SUSCEPTIBILITY ASSAY3791
plates from well aliquots provided confirmation that this as-
sessment correlated with bacterial survival (Fig. 3B). Figure 3A
shows a representative culture turbidity assay performed with
HCLE cell lines after inoculation with strain B1412. In both
Chang and HCLE cell lines, AZM, ERY, and TET show an
inverse relationship between antibiotic concentration and
culture turbidity. These results are correlated with bacterial
growth on blood agar plates. Figure 3B shows 1-day growth
from one replicate of the HCLE experiment with strain B1412.
Bacterial growth from three experiments was graded (Fig. 3C),
and AZM and ERY show less growth at ?128 ?g/ml, and TET
shows less growth at 256 to 512 ?g/ml. BAC had no effect on
bacterial growth to the sensitivity of either assay. Combined
turbidity and blood agar data are correlative to the epithelial
protection assay results above, and Chang cells show similar
results (data not shown).
Toxicity assays. In addition to the toxicity results implied
above, two dedicated toxicity assays were performed during
each run of the experiments without bacterial challenge. First,
mock experiment plates (no S. aureus challenge) were stained
with gentian violet as described above (Fig. 1B and C). Visual
inspection of these gentian violet-stained cells showed clear
cytotoxicity from TET only, an effect seen with both Chang and
HCLE cell lines (data not shown).
Solubilized gentian violet testing for mock-infected Chang
cells showed TET toxicity at ?64 ?g/ml, but no other antibiotic
tested showed staining at any concentration that was clearly
different from that of untreated cells (Fig. 4A). HCLE cell
testing showed a moderate reduction in staining when treated
with AZM at high concentrations, ERY at 8 to 16 ?g/ml, and
a stronger effect from TET at concentrations of 64 to 512
?g/ml (Fig. 4B).
Whereas gentian violet staining can be used to assess cell
layer integrity, it does not necessarily correlate to cell layer
viability. To more directly assess cell layer viability, the
alamarBlue vital stain was used (Fig. 4C and D). alamarBlue
testing confirmed the results found with gentian violet staining,
showing the toxicities of TET at similar concentrations be-
tween the two assays. With both Chang (Fig. 4C) and HCLE
(Fig. 4D) cells, the only antibiotic that conferred a dose-de-
pendent loss of cell viability was TET. The toxicity of TET to
Chang cells reduced viability staining to levels similar to those
of the negative control (lysis), in which the cell layer had been
killed using detergent (Fig. 4C).
The primary endpoint of infectious disease therapy is pres-
ervation of the host tissue, with microbial killing as a means to
that goal. Our study and new model system underscore impor-
tant differences in the ability of antibiotics to protect cells of
the ocular surface. Although the S. aureus strain B1412 was
susceptible to every study antibiotic, some antibiotics showed
the in vitro ability to associate with epithelial cells, thereby
prolonging their effective period of administration and provid-
ing superior protection against subsequent microbial challenge
in this assay.
We conclude that the protection from microbial challenge is
due to the antimicrobial effects of the antibiotics, as opposed to
an unknown mammalian cell response to the antibiotic that
leads to protection, for example, the upregulation of ?-defen-
sin production. This conclusion is based upon the observation
that mammalian cell lines were not protected against bacterial
strains that were antibiotic resistant. For example, when AZM-
resistant bacterial strain B1382 (MIC ? 64 ?g/ml) was used, no
tested concentration of AZM was able to protect Chang or
HCLE cells from the bacteria (Fig. 1 and 2; see also Fig. S1 in
the supplemental material).
The tendency of certain antibiotics, including AZM, ERY,
TET, and BAC, to associate with human cells has been studied.
Macrolide antibiotics, such as AZM and ERY, have been doc-
umented to reach high concentrations in bronchoalveolar cells,
lung tissue, leukocytes, lung epithelial cells, and alveolar mac-
FIG. 3. Analysis of bacterial growth and viability. (A) Turbidity of
bacteria and HCLE cultures 24 h after inoculation with S. aureus strain
B1412. Epithelial cells had been pretreated with the antibiotics listed
at the displayed concentrations in micrograms per milliliter. Similar
results were found using Chang cells. Error bars indicate one standard
deviation. (B) Growth of S. aureus strain B1412 after plate replication
from an inoculated HCLE culture that had been pretreated with the
study antibiotics according to the legend for Fig. 1A. [Ab], antibiotic
concentration. (C) Median graded growth of B1412 from blood agar
plates as shown in panel B. 0, no growth; 1, 1 to 5 CFU; 2, 6? CFU,
but not confluent; 3, confluent. Similar results were measured with
Chang cells. The experiment was repeated 3 times on different days;
n ? 9 for each data point.
3792WINGARD ET AL.ANTIMICROB. AGENTS CHEMOTHER.
rophages (8, 13, 21, 22). The ability of these drugs to penetrate
cells may be important in fighting obligate intracellular
pathogens (2). AZM was found to have a particularly long
tissue half-life, estimated at greater than 2 days in mamma-
lian phagocytic cells, and AZM accumulation was more than
20-fold higher than ERY accumulation in human polymorpho-
nuclear neutrophils (9). With respect to the eye, a recent study
described AZM reaching high concentrations in the human
conjunctiva and persisting in the cornea for at least 7 days after
treatment, with a half-life of 65.7 h (18). Another study showed
that ERY was taken up by rabbit primary corneal epithelial
cells, and its efflux was inhibited by steroids (10).
Tetracycline and related antibiotics were found to be con-
centrated severalfold by alveolar macrophages but to a lesser
extent than ERY, whereas other drugs, such as penicillin G,
cefamandole, and gentamicin exhibited very little uptake (8).
Tetracycline was also reported to accumulate in lung epithelial
cells, red blood cells, and neutrophils, with neutrophil uptake
hypothesized to account for TET’s immune modulatory effects
Very few studies have looked at the uptake of bacitracin by
cells. In one study, the uptake of fluorescently labeled bacitra-
cin in Swiss albino mouse dermal fibroblasts was found to be
negligible without the use of ethosomes or liposomes as drug
delivery mechanisms (5). Bacitracin was also stated to have no
ability to penetrate through the cornea to the anterior cham-
ber, unlike vancomycin, chloramphenicol, fluoroquinolones,
macrolides, and aminoglycoside antibiotics, suggesting that it
cannot pass through cell layers (15). Together, these previous
antibiotic accumulation studies correlate with our findings that
macrolide and tetracycline antibiotics exhibit cellular protec-
tion and cellular accumulation, whereas bacitracin has previ-
ously shown no accumulation and, here, no protection.
The effect of cellular antibiotic uptake in an in vitro system
analyzing antibiotic efficacy has not been previously described
in the literature. It is striking that BAC, a commonly pre-
scribed antibiotic, shows scant if any ability to protect the
tested epithelial cells against bacterial challenge in this pre-
treatment assay, whereas AZM and ERY show a remarkable
ability to protect the cell lines even at very low pretreatment
concentrations which are far below the toxicity threshold.
These data suggest that AZM and ERY may be superior to
BAC for the treatment of ocular surface S. aureus infections
and that these drugs may also be superior to TET, since TET
was toxic within or very near to the therapeutic concentration
range for both cell lines and all bacterial strains tested. A study
using human gingival epithelial cells showed a similar trend,
with TET showing higher levels of cytotoxicity than both AZM
and ERY (11). Cell association could not overcome resistance
demonstrated by MIC, but MIC testing would have predicted
success with BAC treatment, a result called into question by
the cell association assay.
We describe a new metric for antibiotic characterization, the
MCPC. This value can be used in conjunction with MIC values
when determining antibiotic choice and dosing patterns. The
assay is reasonably inexpensive, easy to perform, and flexible.
FIG. 4. Toxicity controls show tetracycline toxicity. (A and B) Gentian violet from toxicity plates was solubilized and measured spectropho-
tometrically. Reduced staining indicates loss of cell layer integrity. (C and D) alamarBlue vital staining where reduced fluorescence compared to
the mock group (0 antibiotic) indicates loss of epithelial layer viability. Lysis indicates minimum fluorescence from a detergent-destroyed
monolayer. (A and C) Chang cells; each data point represents the average of results from two separate experiments performed on different days,
with measurements from at least 5 wells. (B and D) HCLE cells; each data point represents the average of results from three separate experiments
performed on different days, with measurements from at least 7 wells. Error bars, standard errors of the means.
VOL. 55, 2011 CELL-ASSOCIATED ANTIBIOTIC SUSCEPTIBILITY ASSAY3793
Our results suggest that while the CAPA can be performed
effectively with a wide range of mammalian cell lines, to em-
ploy the simple MCPC method, some care must be taken in
choosing a cell line appropriate for the assay, ensuring that the
bacterial challenge strain is sufficiently pathogenic to eliminate
background staining. Preliminary experiments with several cell
lines and with other antibiotics have shown that other antibi-
otic classes are capable of providing epithelial cell protection
against bacterial challenge. The incubation time for antibiotic
loading can be altered to further test the time it takes for a
certain antibiotic to associate, and other alterations of the
assay to test antibiotic disassociation from tissue can easily be
adapted from the primary assay. The benefit of the MCPC
value is that it provides a metric for antibiotic association to
tissue without the use of more expensive approaches, such as
mass spectroscopy or high-performance liquid chromatogra-
phy. One limitation to the MCPC value is that it does not
indicate antibiotic toxicity to a particular cell line; however,
toxicity can be observed when a protection zone fades at high
concentrations, as exhibited by TET in Fig. 1B.
The utility of this antibiotic testing approach will be in fur-
ther testing of various antibiotics to correlate with clinically
significant preventive and therapeutic efficacy against ocular or
other infections. Pretreatment effects as demonstrated in this
assay may be a reasonable paradigm to evaluate the utility of
an antibiotic in the environment of the ocular surface. An
active tear film turnover and drainage through the nasolacri-
mal system should quickly eliminate free drug from the envi-
ronment of the ocular surface. The treatment interval would
have to be very frequent to overcome this loss, unless epithelial
cells or other surface reservoirs can hold on to the antibiotic
and then continue to elute the drug. Our results demonstrate
that it is possible to evaluate antibiotic association with epi-
thelial cells in a clinically appropriate way, although the clinical
relevance remains to be demonstrated. The data presented in
this study directly pertain to in vitro antibiotic association with
particular cell types, and it may not be possible to generalize
these results or to assume an equivalent in vivo effect. How-
ever, it must be stated that the data presented here study only
antibiotic association in vitro using particular cell types and
may not represent the in vivo effectiveness of particular anti-
biotics. Furthermore, inflamed tissue may exhibit differential
interactions with antibiotics, and this will be the subject of
subsequent studies using this model.
We thank Kathleen Yates, Nicholas Stella, Katherine O’Connor, Le
Zhang, and Kate Davoli for expert technical support, Kira Lathrop for
taking photomicrographs, and Ilene Gipson and Jes Klarlund for
HCLE cells and protocols.
This work was supported by Inspire Pharmaceuticals, NIH
AI085570, a Core Grant for Vision Research EY08098, and the Eye
and Ear Institute of Pittsburgh. R. M. Q. Shanks is supported by a
Research to Prevent Blindness Career Development Award.
1. Benz, M. S., I. U. Scott, H. W. Flynn, Jr., N. Unonius, and D. Miller. 2004.
Endophthalmitis isolates and antibiotic sensitivities: a 6-year review of cul-
ture-proven cases. Am. J. Ophthalmol. 137:38–42.
2. Bergogne-Berezin, E. 1995. Predicting the efficacy of antimicrobial agents in
respiratory infections—is tissue concentration a valid measure? J. Antimi-
crob. Chemother. 35:363–371.
3. Gabler, W. L. 1991. Fluxes and accumulation of tetracyclines by human
blood cells. Res. Commun. Chem. Pathol. Pharmacol. 72:39–51.
4. Gipson, I. K., et al. 2003. Mucin gene expression in immortalized human
corneal-limbal and conjunctival epithelial cell lines. Invest. Ophthalmol. Vis.
5. Godin, B., and E. Touitou. 2004. Mechanism of bacitracin permeation en-
hancement through the skin and cellular membranes from an ethosomal
carrier. J. Control Release 94:365–379.
6. Goldstein, M. H., R. P. Kowalski, and Y. J. Gordon. 1999. Emerging fluo-
roquinolone resistance in bacterial keratitis: a 5-year review. Ophthalmology
7. Green, M., A. Apel, and F. Stapleton. 2008. Risk factors and causative
organisms in microbial keratitis. Cornea 27:22–27.
8. Hand, W. L., R. W. Corwin, T. H. Steinberg, and G. D. Grossman. 1984.
Uptake of antibiotics by human alveolar macrophages. Am. Rev. Respir. Dis.
9. Hand, W. L., and D. L. Hand. 2001. Characteristics and mechanisms of
azithromycin accumulation and efflux in human polymorphonuclear leuko-
cytes. Int. J. Antimicrob. Agents 18:419–425.
10. Hariharan, S., S. Gunda, G. P. Mishra, D. Pal, and A. K. Mitra. 2009.
Enhanced corneal absorption of erythromycin by modulating P-glycoprotein
and MRP mediated efflux with corticosteroids. Pharm. Res. 26:1270–1282.
11. Inoue, K., S. Kumakura, M. Uchida, and T. Tsutsui. 2004. Effects of eight
antibacterial agents on cell survival and expression of epithelial-cell- or
cell-adhesion-related genes in human gingival epithelial cells. J. Periodontal
12. Kowalski, R. P., et al. 2005. An ophthalmologist’s guide to understanding
antibiotic susceptibility and minimum inhibitory concentration data. Oph-
13. Labro, M. T., C. Babin-Chevaye, and M. Mergey. 2005. Accumulation of
azithromycin and roxithromycin in tracheal epithelial fetal cell lines express-
ing wild type or mutated cystic fibrosis transmembrane conductance regula-
tor protein (CFTR). J. Chemother. 17:385–392.
14. R Development Core Team. 2010. R: a language and environment for sta-
tistical computing. R Development Core Team, Vienna, Austria.
15. Robert, P. Y., and J. P. Adenis. 2001. Comparative review of topical oph-
thalmic antibacterial preparations. Drugs 61:175–185.
16. Shanks, R. M., et al. 2005. Heparin stimulates Staphylococcus aureus biofilm
formation. Infect. Immun. 73:4596–4606.
17. Smolin, G., and M. Okumoto. 1977. Staphylococcal blepharitis. Arch. Oph-
18. Stewart, W. C., et al. 2010. Pharmacokinetics of azithromycin and moxifloxa-
cin in human conjunctiva and aqueous humor during and after the approved
dosing regimens. Am. J. Ophthalmol. 150:744–751.e2.
19. Tarabishy, A. B., G. S. Hall, G. W. Procop, and B. H. Jeng. 2006. Bacterial
culture isolates from hospitalized pediatric patients with conjunctivitis.
Am. J. Ophthalmol. 142:678–680.
20. Tiffany, J. M. 2008. The normal tear film. Dev. Ophthalmol. 41:1–20.
21. Venner, M., et al. 2010. Concentration of the macrolide antibiotic tulathro-
mycin in broncho-alveolar cells is influenced by comedication of rifampicin
in foals. Naunyn Schmiedebergs Arch. Pharmacol. 381:161–169.
22. Yamazaki, T., et al. 2008. The intracellular accumulation of phagocytic and
epithelial cells and the inhibitory effect on Chlamydophila (Chlamydia)
pneumoniae of telithromycin and comparator antimicrobials. J. Chemother.
3794 WINGARD ET AL.ANTIMICROB. AGENTS CHEMOTHER.