Prevention of Staphylococcus aureus Endophthalmitis

1
Prevention of Staphylococcus aureus
Endophthalmitis with Topical Gatifloxacin in a
Rabbit Prophylaxis Model
Luis E. Fernández de Castro, M.D., Helga P.
Sandoval, M.D., David T. Vroman, M.D., Kerry D.
Solomon, M.D.
Magill Research Center for Vision Correction,
Storm Eye Institute, Medical University of South
Carolina, 167 Ashley Avenue, Charleston, SC
29425, USA
Purpose (Click here to enlarge) Infectious
postoperative endophthalmitis is a rare but
catastrophic complication of intraocular
surgery.1 The incidence of postoperative
endophthalmitis after cataract surgery is
approximately 0.07. Early diagnosis is extremely
important, as delay of treatment can
substantially alter the visual prognosis. Despite
advancements in the medical treatment of
postoperative endophthalmitis, even with potent
intravitreal injection of antibiotics, the
prognosis of endophthalmitis remains poor.
Therefore, in recent years there has been an
increase in studies on the use of newly developed
broad-spectrum antibiotics by various means of
administration including the frequent and
extended administration of antimicrobial eye
drops. Although intravitreal injection of
antibiotics is the primary route of antimicrobial
management of endophthalmitis, topical
prophylactic antibiotic treatment may also play a
role. In fact, the ocular penetration of
topically administered antibiotics has been
improved with the newer fourth generation
fluoroquinolones.2 As a result, there have been
several studies evaluating the aqueous humor
concentration and vitreous humor concentration
after topical antibiotic administration.3,4 The
purpose of this study was to investigate the
prophylactic potential of the topically-applied
fourth generation fluoroquinolone, (0.3)
gatifloxacin, for the prevention of
endophthalmitis.
Rabbit Examination (Click here to
enlarge) Twenty-four hours after the anterior
chamber infection, rabbit eyes were examined and
graded by a masked examiner following the
criteria described by Kowalski and coauthors.5
The presence of clinical signs of endophthalmitis
(i.e., iritis, hypopyon, conjunctivitis, limbal
injection, blepharitis, anterior chamber cells,
anterior chamber flare, corneal infiltrates, red
reflex, and fibrin) were evaluated and scored
using a scale based on increasing severity (0,
0.5, 1, 2, and 3), and the eye was photographed.
At the end of the examination, all rabbits were
anesthetized, and under sterile conditions the
aqueous and vitreous material was aspirated using
a 27-Gauge needle on a tuberculin syringe, to
determine the number of viable bacteria present
using standard colony count dilution methods. The
animals were then euthanized as recommended by
the Panel on Euthanasia of the AVMA.
Prophylactic Study Protocol (Click here to
enlarge) The 40 rabbits were randomly placed into
two groups. Group A (20 rabbits) received one
drop of 0.3 gatifloxacin (commercially available
Zymar Allergan, Inc., Irvine, CA), and Group B
(20 rabbits) received one drop of balanced salt
solution (commercially available BSS Alcon
Laboratories, Inc., Fort Worth, TX) in their
right eye every 15 min (i.e., 0, 15, 30, and 45
minutes post-infection) respectively. Immediately
after the final pre-treatment drop was placed,
the anterior chamber of each rabbit was injected
with an inoculum of 0.025 mL containing 5 x 105
cfu of S. aureus. Following the staphylococcal
injection, the treatment regimen continued Group
A (1 drop of 0.3 gatifloxacin) or Group B (1
drop of BSS), each received immediately
post-infection, and at 6, 12, 18, and 24
hours. Twenty-four hours after the staphylococcal
injection, rabbit examination was performed as
previously mentioned. The animals were then
euthanized. Statistical Analysis (Click here to
enlarge) Statistical analysis was performed using
the Wilcoxon Rank-sum test, Fisher and Chi square
with SAS System 9.1 for Windows (SAS Institute
Inc., Cary, NC). A P value less than .05 was
considered statistically significant.
Table 1. Compares the median clinical scores
between eyes of the gatifloxacin and the BSS
treatment groups. (Click here to Enlarge)
Table 2. Comparison between the number of 0.3
gatifloxacin and BSS treated eyes that were
culture positive. (Click here to Enlarge)
Materials and Methods Rabbit Model (Click here
to enlarge) All rabbits were treated in
accordance with the guidelines set forth by ARVO,
and the DLAR at MUSC. Forty-nine female New
Zealand white rabbits of similar age and weight
(7 - 9 pounds) were divided into 4 groups. Three
groups of three rabbits each determined the
number of staphylococcal colony forming units
(cfu) that would be necessary for the remaining
40 rabbits utilized in the prophylactic study.
All right eyes were designated as the
experimental eye. Staphylococcus aureus Isolate
(Click here to enlarge) A clinical isolate of S.
aureus was provided by the Microbiology
Department at the MUSC. This blood culture
isolate was recovered from a patient diagnosed
with line-associated bacteremia. Beta hemolytic
activity on trypticase soy agar containing 10
sheep blood, and its Gram positive, catalase
positive, coagulase positive profile identified
this isolate as S. aureus. Susceptibility testing
demonstrated the isolate to be susceptible to
levofloxacin, gatifloxacin, oxacillin,
trimethoprim-sulfa, cefazolin, erythromycin,
clindamycin, and vancomycin. The MIC of the
bacterial isolate to gatifloxacin was 0.5 µg/mL.
Preparation of Inoculum (Click here to
enlarge) An overnight suspension of S. aureus was
made in saline equivalent to a McFarland 1
turbidity standard (Remel, Lenexa, KS),
representing approximately 2 x 108 cfu/mL and the
suspension was diluted 110, 1100, and 11000 in
trypticase soy broth to final concentrations of 2
x 107, 2 x 106, and 2 x 105 cfu/mL, respectively.
These inocula were packed on ice, transported to
the animal facility, and resuspended with a
junior vortex mixer immediately prior to
injection into the anterior chamber of 3 rabbits
per inoculum (5 x 105, 5 x 104, and 5 x 103 cfu,
respectively, per rabbit eye) using a 30-Gauge
half-inch needle attached to a 0.5 mL syringe,
and delivering an inoculum volume of 0.025 mL.
These preliminary studies revealed the 2 x 107
cfu/mL dilution (i.e., 5 x 105 cfu per rabbit
eye) was necessary to reproducibly create a
clinical picture consistent with endophthalmitis,
under these conditions, and thus became the
inoculum for the prophylactic study (these
preparation steps were repeated as needed for the
prophylactic study - 40 rabbits).

• Conclusions (Click here to enlarge)
• When compared to the control animals,
  gatifloxacin treated eyes demonstrated
  significantly less (P lt.05)
• Inflammation
• Infection
• Culture positive endophthalmitis
• It is important to further investigate the
  clinical implications from the results of this
  study.

Results (Click here to enlarge) The inoculum
determination for the prophylactic study
demonstrated that 5 x 105 cfu injected into the
anterior chamber produced a clinical presentation
consistent with endophthalmitis. Additionally,
this was the lowest concentration of S. aureus
that reproducibly produced clinical signs of
endophthalmitis in all three infected eyes, and
thus was the inoculum chosen for the Prophylactic
Study.

• References (Click here to enlarge)
• Speaker MG, Menikoff JA. Postoperative
  endophthalmitis pathogenesis, prophylaxis, and
  management (review). Int Ophthalmol Clin
  19933351-70.
• Bronner S, Jehl F, Peter JD, et al. Moxifloxacin
  efficacy and vitreous penetration in a rabbit
  model of Staphylococcus aureus endophthalmitis
  and effect on gene expression of leucotoxins and
  virulence regulator factor. Antimicrob Agents
  Chemother 2003471621-29.
• Yalvac IS, Basci NE, Bozkurt A, Duman S.
  Penetration of topically applied ciprofloxacin
  and ofloxacin into the aqueous humor and
  vitreous. J Cataract Refract Surg 200329487-91.
• Ozturk F, Kortunay S, Kurt E, et al. The effect
  of long-term use and inflammation on the ocular
  penetration of topical Ofloxacin. Curr Eye Res
  199919461-64.
• Kowalski RP, Romanowski EG, Mah FS, et al.
  Topical prophylaxis with moxifloxacin prevents
  endophthalmitis in a rabbit model. Am J
  Ophthalmol 200413833-7.

Figure 1 (Click here to enlarge)
Acknowledgement (Click here to enlarge) The
authors thank Dr. Luanna R. Bartholomew, Ph.D.,
Storm Eye Institute and Dr. Lisa L. Steed, Ph.D.,
Pathology and Laboratory Medicine, MUSC, for
their research assistance. Supported in part by
NIH/NEI EY-014793 an unrestricted educational
grant to Magill Research Center MUSC-SEI from
Allergan Laboratories, Irvine, CA, USA and an
unrestricted grant to MUSC-SEI from Research to
Prevent Blindness, New York, NY, USA.
Figure 1. Photographs of rabbit eyes taken 24
hours after anterior chamber inoculation with
Staphylococcus aureus (5 x 105 cfu). (A, B) Eyes
treated with 0.3 gatifloxacin. (C, D) Eyes
treated with balanced salt solution.
2
PURPOSE

• Infectious postoperative endophthalmitis is a
  rare but catastrophic complication of intraocular
  surgery.1 The incidence of postoperative
  endophthalmitis after cataract surgery is
  approximately 0.07. Early diagnosis is extremely
  important, as delay of treatment can
  substantially alter the visual prognosis. Despite
  advancements in the medical treatment of
  postoperative endophthalmitis, even with potent
  intravitreal injection of antibiotics, the
  prognosis of endophthalmitis remains poor.
  Therefore, in recent years there has been an
  increase in studies on the use of newly developed
  broad-spectrum antibiotics by various means of
  administration including the frequent and
  extended administration of antimicrobial eye
  drops.
• Although intravitreal injection of antibiotics is
  the primary route of antimicrobial management of
  endophthalmitis, topical prophylactic antibiotic
  treatment may also play a role. In fact, the
  ocular penetration of topically administered
  antibiotics has been improved with the newer
  fourth generation fluoroquinolones.2 As a result,
  there have been several studies evaluating the
  aqueous humor concentration and vitreous humor
  concentration after topical antibiotic
  administration.3,4 The purpose of this study was
  to investigate the prophylactic potential of the
  topically-applied fourth generation
  fluoroquinolone, (0.3) gatifloxacin, for the
  prevention of endophthalmitis.

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3
Materials and Methods Rabbit Model

• All rabbits were treated in accordance with the
  guidelines set forth by ARVO, and the DLAR at
  MUSC. Forty-nine female New Zealand white rabbits
  of similar age and weight (7 - 9 pounds) were
  divided into four groups. Three groups of three
  rabbits each determined the number of
  staphylococcal colony forming units (cfu) that
  would be necessary for the remaining 40 rabbits
  utilized in the prophylactic study.
• All right eyes were designated as the
  experimental eye.

Back to Poster
Previous Slide / Next Slide
4
Materials and MethodsStaphylococcus aureus
Isolate

• A clinical isolate of Staphylococcus aureus was
  provided by the Microbiology Department at the
  MUSC. This blood culture isolate was recovered
  from a patient diagnosed with line-associated
  bacteremia. Beta hemolytic activity on trypticase
  soy agar containing 10 sheep blood, and its Gram
  positive, catalase positive, coagulase positive
  profile identified this isolate as S. aureus.
  Susceptibility testing demonstrated the isolate
  to be susceptible to levofloxacin, gatifloxacin,
  oxacillin, trimethoprim-sulfa, cefazolin,
  erythromycin, clindamycin, and vancomycin. The
  minimum inhibitory concentration of the bacterial
  isolate to gatifloxacin was 0.5 µg/mL.

Back to Poster
Previous Slide / Next Slide
5
Materials and MethodsPreparation of Inoculum

• An overnight suspension of S. aureus was made in
  saline equivalent to a McFarland 1 turbidity
  standard (Remel, Lenexa, KS), representing
  approximately 2 x 108 cfu/mL and the suspension
  was diluted 110, 1100, and 11000 in trypticase
  soy broth to final concentrations of 2 x 107, 2 x
  106, and 2 x 105 cfu/mL, respectively. These
  inocula were packed on ice, transported to the
  animal facility, and resuspended with a junior
  vortex mixer immediately prior to injection into
  the anterior chamber of 3 rabbits per inoculum (5
  x 105, 5 x 104, and 5 x 103 cfu, respectively,
  per rabbit eye) using a 30-Gauge half-inch needle
  attached to a 0.5 mL syringe, and delivering an
  inoculum volume of 0.025 mL. These preliminary
  studies revealed the 2 x 107 cfu/mL dilution
  (i.e., 5 x 105 cfu per rabbit eye) was necessary
  to reproducibly create a clinical picture
  consistent with endophthalmitis, under these
  conditions, and thus became the inoculum for the
  prophylactic study (these preparation steps were
  repeated as needed for the prophylactic study -
  40 rabbits).

Back to Poster
Previous Slide / Next Slide
6
Materials and MethodsRabbit Examination

• Twenty-four hours after the anterior chamber
  infection, rabbit eyes were examined and graded
  by a masked examiner following the criteria
  described by Kowalski and coauthors.5 The
  presence of clinical signs of endophthalmitis
  (i.e., iritis, hypopyon, conjunctivitis, limbal
  injection, blepharitis, anterior chamber cells,
  anterior chamber flare, corneal infiltrates, red
  reflex, and fibrin) were evaluated and scored
  using a scale based on increasing severity (0,
  0.5, 1, 2, and 3), and the eye was photographed.
• At the end of the examination, all rabbits were
  anesthetized, and under sterile conditions the
  aqueous and vitreous material was aspirated using
  a 27-Gauge needle on a tuberculin syringe, to
  determine the number of viable bacteria present
  using standard colony count dilution methods. The
  animals were then euthanized as recommended by
  the Panel on Euthanasia of the American
  Veterinary Medical Association.

Back to Poster
Previous Slide / Next Slide
7
Materials and MethodsProphylactic Study Protocol

• The 40 rabbits were randomly placed into two
  groups. Group A (20 rabbits) received one drop of
  0.3 gatifloxacin (commercially available Zymar
  Allergan, Inc., Irvine, CA), and Group B (20
  rabbits) received one drop of balanced salt
  solution (commercially available BSS Alcon
  Laboratories, Inc., Fort Worth, TX) in their
  right eye every 15 min (i.e., 0, 15, 30, and 45
  minutes post-infection) respectively. Immediately
  after the final pre-treatment drop was placed,
  the anterior chamber of each rabbit was injected
  with an inoculum of 0.025 mL containing 5 x 105
  cfu of S. aureus. Following the staphylococcal
  injection, the treatment regimen continued Group
  A (1 drop of 0.3 gatifloxacin) or Group B (1
  drop of BSS), each received immediately
  post-infection, and at 6, 12, 18, and 24 hours.
• Twenty-four hours after the staphylococcal
  injection, rabbit examination was performed as
  previously mentioned. The animals were then
  euthanized.

Back to Poster
Previous Slide / Next Slide
8
Materials and MethodsStatistical Analysis

• Statistical analysis was performed using the
  Wilcoxon Rank-sum test, Fisher and Chi square
  with SAS System 9.1 for Windows (SAS Institute
  Inc., Cary, NC).
• A P value less than .05 was considered
  statistically significant.

Back to Poster
Previous Slide / Next Slide
9
Results

• The inoculum determination for the prophylactic
  study demonstrated that 5 x 105 cfu injected into
  the anterior chamber produced a clinical
  presentation consistent with endophthalmitis.
  Additionally, this was the lowest concentration
  of S. aureus that reproducibly produced clinical
  signs of endophthalmitis in all three infected
  eyes, and thus was the inoculum chosen for the
  Prophylactic Study.

Back to Poster
Previous Slide / Next Slide
10
Figure 1

• Photographs of rabbit eyes taken 24 hours after
  anterior chamber inoculation with Staphylococcus
  aureus (5 x 105 cfu). (A, B) Slit-lamp
  photographs of representative eyes treated with
  0.3 gatifloxacin. (C, D) Slit-lamp photographs
  of representative eyes treated with balanced salt
  solution.

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11
Table 1
Table 1. Compares the median clinical scores
between eyes of the gatifloxacin and the BSS
treatment groups.
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12
Table 2
Table 2. Comparison between the number of 0.3
gatifloxacin and BSS treated eyes that were
culture positive.
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13
Conclusions

• When compared to the control animals,
  gatifloxacin treated eyes demonstrated
  significantly less (P lt.05)
• Inflammation
• Infection
• Culture positive endophthalmitis
• It is important to further investigate the
  clinical implications from the results of this
  study.

Back to Poster
Previous Slide / Next Slide
14
References

• Speaker MG, Menikoff JA. Postoperative
  endophthalmitis pathogenesis, prophylaxis, and
  management (review). Int Ophthalmol Clin
  19933351-70.
• Bronner S, Jehl F, Peter JD, et al. Moxifloxacin
  efficacy and vitreous penetration in a rabbit
  model of Staphylococcus aureus endophthalmitis
  and effect on gene expression of leucotoxins and
  virulence regulator factor. Antimicrob Agents
  Chemother 2003471621-1629.
• Yalvac IS, Basci NE, Bozkurt A, Duman S.
  Penetration of topically applied ciprofloxacin
  and ofloxacin into the aqueous humor and
  vitreous. J Cataract Refract Surg
  200329487-491.
• Ozturk F, Kortunay S, Kurt E, et al. The effect
  of long-term use and inflammation on the ocular
  penetration of topical Ofloxacin. Curr Eye Res
  199919461-464.
• Kowalski RP, Romanowski EG, Mah FS, et al.
  Topical prophylaxis with moxifloxacin prevents
  endophthalmitis in a rabbit model. Am J
  Ophthalmol 200413833-37.

Back to Poster
Previous Slide / Next Slide
15
Acknowledgement

• The authors thank Dr. Luanna R. Bartholomew,
  Ph.D., Storm Eye Institute and Dr. Lisa L. Steed,
  Ph.D., Pathology and Laboratory Medicine, MUSC,
  for their research assistance.
• Supported in part by NIH/NEI EY-014793 an
  unrestricted educational grant to Magill Research
  Center MUSC-SEI from Allergan Laboratories,
  Irvine, CA, USA and an unrestricted grant to
  MUSC-SEI from Research to Prevent Blindness, New
  York, NY, USA.

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