LABORATORY SCIENCE

In vitro efficacy of a povidone–iodine 0.4%

and dexamethasone 0.1% suspension
against ocular pathogens
Jesse S. Pelletier, MD, Darlene Miller, DHSc M, Bo Liang, PhD, Joseph A. Capriotti, MD

PURPOSE: To assess the efficacy of a povidone–iodine 0.4%–dexamethasone 0.1% suspension

against bacterial, fungal, and Acanthamoeba clinical isolates.
SETTING: Bascom Palmer Eye Institute, McKnight Research Building, Miami, Florida, USA.
DESIGN: Experimental study.
METHODS: One hundred milliliters of 104 colony-forming units/mL of ocular isolates of methicillin-
resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Serratia marcescens, Candida
albicans, Fusarium solani, and Acanthamoeba castellanii were inoculated into 100 mL of a povidone–
iodine 0.4%–dexamethasone 0.1% suspension in a 96-well microtiter plate incubated at room
temperature. Organism viability was assessed at 15, 30, and 60 seconds by removing 10 mL
aliquots and streaking onto a 5.0% sheep blood agar plate (fungi and bacteria) and agar-agar
(Acanthamoeba) using a 0.001 calibrated loop. The plates were then incubated at 35 C and
monitored for up to 7 days. Isolates were inoculated into 200 mL of trypticase soy broth as
controls. The number of colonies was counted and compared with controls to determine the kill rate.
RESULTS: A 99.9% kill was observed for MRSA, P aeruginosa, S marcescens, and C albicans after
15 seconds of exposure and for F solani after 60 seconds. Acanthamoeba castellanii cyst viability
was not inhibited by exposure to the povidone–iodine and dexamethasone suspension. Organism
growth was achieved on all control broth.
CONCLUSIONS: Povidone–iodine 0.4%–dexamethasone 0.1% suspension killed all bacterial and
candida isolates within 15 seconds of exposure. It killed Fusarium isolates at 60 seconds but
was ineffective against Acanthamoeba cysts.
Financial Disclosure: Drs. Pelletier and Miller have no financial or proprietary interest in any
material or method mentioned. Additional disclosures are found in the footnotes.
J Cataract Refract Surg 2011; 37:763–766 Q 2011 ASCRS and ESCRS

Molecular iodine is a powerful oxidizing agent with
broad application as an antiseptic and disinfectant
agent. The medical use of elemental iodine has always
been complicated by poor aqueous solubility, limited
room-temperature stability, and variable tolerability
by mucosal surfaces.1 Lugol first described a method
to improve the aqueous solubility of iodine by using
mixed iodine (I2) and iodide (I-) systems that could
generate the in situ formation of soluble triiodide
(I3-).1 Since Lugol, attempts to incorporate elemental
iodine into useful medical formulations have histori-
cally used strategies that modify the solvent system,
the ion content, or both.2 Iodophors are a class of
molecules that are capable of complexing, solubilizing,
and releasing molecular iodine through a variety of
equilibrium reactions in a host of solvents.3 They im-
prove solubility, stability, and tolerability without sac-
rificing the chemical availability of molecular iodine.
The organic polymer povidone has been successfully
used for decades as an iodophor in human and veter-
inary medicine and has been routinely used before
ophthalmic surgery.4 Aqueous povidone–iodine
complexes have utility in skin disinfection, presurgical
preparation, and topical antisepsis.
Interest in povidone–iodine as a treatment for exter-
nal ophthalmic infections has led to the development
of a new combination formulation containing both po-
vidone–iodine and dexamethasone.5 This combination

Q Published by Elsevier Inc. on behalf of ASCRS and ESCRS 0886-3350/$ - see front matter 763
doi:10.1016/j.jcrs.2010.11.028
764 LABORATORY SCIENCE: EFFICACY OF POVIDONE–IODINE 0.4% AND DEXAMETHASONE 0.1% SUSPENSION
suspension was developed to eliminate the organisms
responsible for infection while simultaneously reduc-
ing the inflammatory damage that often accompanies
ocular surface infection. Successful use has been re-
ported in human adenoviral conjunctivitis.6 Although
adenoviruses are often the cause of prolonged ocular
morbidity and outbreaks of infectious conjunctivitis,
other, more virulent, organisms may rapidly destroy
ocular tissue. Some of these microbes are highly
resistant bacteria with multiple virulence factors. For
example, methicillin-resistant Staphylococcus aureus
(MRSA) ocular infections are currently reported in
community and hospital settings with greater fre-
quency than in previous years.7,A Infectious manifes-
tations can include conjunctivitis, aggressive
keratitis, and endophthalmitis. Pseudomonas aeruginosa,
particularly prominent in contact lens–associated
keratitis, can also cause a rapidly destructive endoph-
thalmitis and uses a variety of virulence factors to
inflict damage. Serratia marcescens is another gram-
negative rod with the propensity for the development
of contact lens and post-refractive surgery keratitis.8,9
Filamentous fungi and yeasts are known to cause
progressive recalcitrant ocular infections for which
current pharmacologic therapeutics often remain sub-
optimal. Finally, Acanthamoeba species, with the poten-
tial of cystic encapsulation, are among the most
challenging microbes to eradicate.
Although several concentrations of povidone–
iodine alone have shown in vitro efficacy against
viruses, bacteria, fungi, and protozoans, this is the first
reported in vitro study of dilute (0.4%) povidone–
iodine in a single formulation with suspended aque-
ous dexamethasone.1 The purpose of this study
was to assess in vitro activity against selected ocular
pathogens with time-kill assessments performed out
to 60 seconds.
Submitted: August 26, 2010.
Final revision submitted: November 4, 2010.
Accepted: November 7, 2010.
From the Ocean Ophthalmology Group (Pelletier, Capriotti), North
Miami Beach, and Bascom Palmer Eye Institute (Pelletier, Miller),
Miami, Florida; CLS Pharmaceuticals, Inc. (Liang, Capriotti),
New York, New York, USA.
Additional financial disclosures: Drs. Liang and Capriotti have a
proprietary interest in the antiinfective product.
Funded by a research grant from Foresight Biotherapeutics,
New York, New York, USA.
Corresponding author: Jesse S. Pelletier, MD, 1400 Northeast
Miami Gardens Drive, Suite 203, North Miami Beach, Florida
33179, USA. E-mail: jessepelletier@yahoo.com.
J CATARACT REFRACT SURG - VOL 37, APRIL 2011
MATERIALS AND METHODS
Isolates of MRSA, P aeruginosa, S marcescens, Candida albicans,
Fusarium solani, and Acanthamoeba castellanii were obtained
from the human ocular isolates collection at Bascom Palmer
Eye Institute, McKnight Research Center, Miami, Florida.
The proprietary formulation of povidone–iodine 0.4% and
dexamethasone 0.1% used in this study was obtained from
Leiter’s Pharmacy, San Jose, California.
All bacterial and fungal isolates were grown on blood agar
plates. Acanthamoeba isolates were grown on agar-agar me-
dia. After incubation at 35 C for 24 hours, bacterial colonies
were suspended in saline and the optical densities of bacte-
rial suspensions were appropriately adjusted. One hundred
milliliters of a 104 colony-forming units (CFU)/mL suspen-
sion was inoculated into a 100 mL povidone–iodine 0.4%–
dexamethasone 0.1% suspension in a 96-well microtiter plate
and incubated at room temperature. Organism viability was
checked at 15, 30, and 60 seconds by removing 10 mL aliquots
from the microtiter plate, neutralizing the suspension, and
streaking with a 0.001 calibrated loop onto 5% blood agar
plates for bacteria and fungi and agar-agar plates for Acan-
thamoeba. These inoculated plates were then incubated at
35 C and monitored for growth up to 7 days. For controls,
100 mL of 104 CFU/mL of each isolate was inoculated in tryp-
ticase soy broth and observed for growth at 35 C. Kill rate
was determined by counting the number of colonies and
comparing this with that of controls.
RESULTS
One clinical isolate of each organism was used for each
experiment. After 15 seconds of exposure to the povi-
done–iodine 0.4%–dexamethasone 0.1% suspension,
99.9% kill of MRSA, P aeruginosa, S marcescens, and
C albicans was noted, as evidenced by lack of growth
on appropriate media for 7 days. Fusarium solani also
had 99.9% kill after exposure to the povidone–iodine
0.4%–dexamethasone 0.1% suspension for 60 seconds.
Acanthamoeba castellanii cysts were not inhibited after
exposure to the suspension for 60 seconds, as evidenced
by the presence of trophozoites by light microscopy. Kill
rate determinations for A castellanii were not performed.
Organism growth was noted in all trypticase soy broth
controls. Table 1 shows all results.
DISCUSSION
Recent interest in the development of antiseptics as
polymicrobial therapeutic agents alone and in combi-
nation with existing drugs has led to new topical treat-
ments used in current human trials for a variety of
indications.10–13 Important for these proposed agents,
particularly when incorporating antimicrobials into
combination antiinfective therapies, is the demonstra-
tion of efficacy against clinical isolates. An ideal anti-
infectious agent would show biocidal efficacy against
a variety of pathogen classes, including gram-
positive and gram-negative bacteria, yeasts, fungi,
protozoans, and viruses, and render them unable to
mount resistance over time. Povidone–iodine is
 LABORATORY SCIENCE: EFFICACY OF POVIDONE–IODINE 0.4% AND DEXAMETHASONE 0.1% SUSPENSION 765

Table 1. Effect of exposure time to test suspension on organism survival.

Organisms Recovered per 0.1 mL After Contact Time (n)

Organism 15 Seconds 30 Seconds 45 Seconds 60 Seconds

MRSA 0 0 0 0
Pseudomonas aeruginosa 0 0 0 0
Serratia marcescens 0 0 0 0
Candida albicans 0 0 0 0
Fusarium solani 104 104 102 0
Acanthamoeba castellanii NA NA NA NA

NA Z not applicable

a promising compound that has historically shown
efficacy against practically all pathogens and has
been safely used for decades in ophthalmology.14
Improvements in understanding the relationship
between povidone–iodine aqueous concentration
and efficacy have led us to favor lower povidone–
iodine concentrations over the common presurgical
strengths of 5.0% to 10.0% in certain situations.15
Earlier in vitro work with dilute povidone–iodine
showed its paradoxical increase in bactericidal activity
against strains of S aureus, Mycobacterium chelonae,
Klebsiella pneumoniae, Streptococcus mitis, and Pseudo-
monas cepacia.15 In another in vitro study that used
hospital-acquired clinical isolates,16 dilute povidone–
iodine more effectively eradicated multiple serotypes
of staphylococci than higher povidone–iodine
concentrations.
The current study was designed to determine
whether adding dexamethasone 0.1% would render
the suspension less effective against pathogenic
microbes in vitro. The selected ocular pathogens in
this study are virulent and frequently implicated in
broad-spectrum ocular morbidity. Likewise, the infec-
tious settings may be diverse, ranging from postoper-
ative cataract and refractive procedures to contact lens
and trauma. None of these pathogens is typically part
of the normal ocular flora; however, all except Acantha-
moeba have been cultured from noninflamed eyes in
some populations.17,18 In these experiments, rapid kill-
ing was shown for all tested bacteria and fungi, most
of which were incapacitated after only 15 seconds of
exposure. Acanthamoeba castellanii viability was not in-
hibited after contact with the suspension for 60 sec-
onds. This is not unexpected given the difficulty of
Acanthamoeba eradication due to trophozoite encyst-
ment. Povidone–iodine 10.0% has been shown to be ef-
fective against Acanthamoeba cysts and trophozoites in
vitro.19 However, in a study by Gatti et al.,19 exposure
of cysts and trophozoites to povidone–iodine was for
48 hours. It is therefore plausible that exposure of
A castellanii to the challenge suspension for longer
than 60 seconds may have inhibited growth on the
agar-agar media.
The current study was performed on 1 clinical iso-
late for each test organism. The absence of experi-
ments on multiple serotypes for each microbe may
limit the interpretation of these results, although povi-
done–iodine is unlikely to have a serotype-dependent
effect based on its mechanism of action. Failure to
retest quantitatively in cases in which there was
incomplete killing after 15 seconds may also limit
the interpretation of the current results. It is under-
stood that in vitro studies do not always correlate
with in vivo outcomes. In vivo performance may
rely on factors such as concentration at the target tis-
sue and host interactions that could not be accounted
for in this study.
In conclusion, the combination povidone–iodine
0.4%–dexamethasone 0.1% suspension rapidly killed
common ocular pathogens, including gram-positive
bacteria, gram-negative bacteria, MRSA, and fungi. It
had no effect on Acanthamoeba cysts at the contact
times tested. Further investigation of the suspension
as a topical agent for surface infections appears to be
warranted.
REFERENCES
1. Gottardi W. Iodine and iodine compounds. In: Block SS, ed,
Disinfection, Sterilization, and Preservation, 5th ed. Philadel-
phia, PA, Lippincott Williams & Wilkins, 2001; 159–184
2. Shelanski HA, Shelnski MV. PVP iodine: history, toxicity and
therapeutic uses. J Intern College Surg 1956; 26:727–734
3. McDonnell G, Russell AD. Antiseptics and disinfectants: activity,
action, and resistance. Clin Microb Rev 1999; 12:147–179. erra-
tum 2001; 14:227. Available at: http://www.ncbi.nlm.nih.gov/
pmc/articles/PMC88911/pdf/cm000147.pdf. erratum available
at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC88971/. Ac-
cessed December 12, 2010
4. Chase RC, Ellis PP. Iodophors and skin asepsis: iodophors as
skin antiseptics before ophthalmic surgery. Ann Ophthalmol
1970; 12:312–317

J CATARACT REFRACT SURG - VOL 37, APRIL 2011

766 LABORATORY SCIENCE: EFFICACY OF POVIDONE–IODINE 0.4% AND DEXAMETHASONE 0.1% SUSPENSION
5. Isenberg SJ, Apt L. The ocular application of povidone-iodine.
Community Eye Health 2003; 16:30–31. Available at: http://
www.ncbi.nlm.nih.gov/pmc/articles/PMC1705857/pdf/jceh_16_
46_030.pdf. Accessed December 12, 2010
6. Pelletier JS, Stewart K, Trattler W, Ritterband DC, Braverman S,
Samson CM, Liang B, Capriotti JA. A combination povidone-
iodine 0.4%/dexamethasone 0.1% ophthalmic suspension in the
treatment of adenoviral conjunctivitis. Adv Ther 2009; 26:776–783
7. Major JC Jr, Engelbert M, Flynn HW Jr, Miller D, Smiddy WE,
Davis JL. Staphylococcus aureus endophthalmitis: antibiotic
susceptibilities, methicillin resistance, and clinical outcomes.
Am J Ophthalmol 2010; 149:278–283
8. Parment PA. The role of Serratia marcescens in soft contact lens
associated ocular infections; a review. Acta Ophthalmol Scand
1997; 75:67–71. Available at: http://onlinelibrary.wiley.com/doi/
10.1111/j.1600-0420.1997.tb00253.x/pdf. Accessed December
12, 2010
9. Mun ~oz G, Alio JL, Pe
 rez-Santonja J-J, Artola A, Abad JL. Ulcer-
ative keratitis caused by Serratia marcescens after laser in situ
keratomileusis. J Cataract Refract Surg 2004; 30:507–512
10. Neher A, Nagl M, Appenroth E, Gsto €ttner M, Wischatta M,
Reisigl F, Schindler M, Ulmer H, Stephan K. Acute otitis externa:
efficacy and tolerability of N-chlorotaurine, a novel endogenous
antiseptic agent. Laryngoscope 2004; 114:850–854. Available
at: http://www.pathogenics.com/PDF/Otitis-Laryngoscope0504.pdf
Accessed December 12, 2010
11. Breithaupt H. The new antibiotics. Nat Biotechnol 1999;
17:1165–1169
12. Isenberg SJ, Apt L, Valenton M, Del Signore M, Cubillan L,
Labrador MA, Chan P, Berman NG. A controlled trial of
povidone-iodine to treat infectious conjunctivitis in children.
Am J Ophthalmol 2002; 134:681–688
13. Nagl M, Nguyen VA, Gottardi W, Ulmer H, Ho €pfl R. Tolerability
and efficacy of N-chlorotaurine compared to chloramine T for
J CATARACT REFRACT SURG - VOL 37, APRIL 2011
treatment of chronic leg ulcers with purulent coating: a random-
ized phase II study. Br J Dermatol 2003; 149:590–597
14. Speaker MG, Menikoff JA. Prophylaxis of endophthalmitis with
topical povidone-iodine. Ophthalmology 1991; 98:1769–1765
15. Berkelman RL, Holland BW, Andersen RL. Increased bacteri-
cidal activity of dilute preparations of povidone-iodine solutions.
J Clin Microbiol 1982; 15:635–639. Available at: http://www.ncbi.
nlm.nih.gov/pmc/articles/PMC272159/pdf/jcm00153-0121.pdf.
Accessed December 12, 2010
16. Gocke DJ, Ponticas S, Pollack W. In vitro studies of the killing of
clinical isolates by povidone-iodine solutions. J Hosp Infect
1985; 6(suppl 1):59–66
17. Capriotti JA, Pelletier JS, Shah M, Caivano DM, Ritterband DC.
Normal ocular flora in healthy eyes from a rural population in
Sierra Leone. Int Ophthalmol 2009; 29:81–84
18. Ainley R, Smith B. Fungal flora of the conjunctival sac in healthy
and diseased eye. Br J Ophthalmol 1965; 49:505–515. Available
at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC506151/pdf/
brjopthal00382-0001.pdf. Accessed December 12, 2010
19. Gatti S, Cevini C, Bruno A, Penso G, Rama P, Scaglia M. In vitro
effectiveness of povidone-iodine on Acanthamoeba isolates
from human cornea. Antimicrob Agents Chemother 1998;
42:2330–2334. Available at: http://aac.asm.org/cgi/reprint/42/
9/2232. Accessed December 12, 2010
OTHER CITED MATERIAL
A. Miller D, Diaz MG, Perez EM, Newton J, Alfonso EC, “Preva-
lence of Community Acquired Methicillin Resistant Staphylo-
coccus Aureus Among Ocular MRSA Isolates,” poster
presented at the Association of Practitioners in Infection Con-
trol (APIC), Tampa, Florida, USA, June 2006. Abstract avail-
able in Am J Infect Control 2006; 34:E23–E24. Available at:
http://www.ajicjournal.org/article/S0196-6553(06)00703-6/abstract.
Accessed December 29, 2010