Download Bacterial pathogenesis and infection

ARVO 2015 Annual Meeting Abstracts
413 Bacterial pathogenesis and infection
Wednesday, May 06, 2015 8:30 AM–10:15 AM
Exhibit Hall Poster Session
Program #/Board # Range: 4043–4078/A0220–A0255
Organizing Section: Immunology/Microbiology
Contributing Section(s): Clinical/Epidemiologic Research, Cornea,
Physiology/Pharmacology, Retina
Program Number: 4043 Poster Board Number: A0220
Presentation Time: 8:30 AM–10:15 AM
Pseudomonas aeruginosa adaptation to the ocular surface:
transcriptional changes and virulence determinants
Matteo M. Metruccio1, Yvonne Wu1, David J. Evans1, 2, Suzanne M.
Fleiszig1, 3. 1School of Optometry, University of California Berkeley,
Berkeley, CA; 2College of Pharmacy, Touro University California,
Vallejo, CA; 3Graduate Groups in Vision Science, Microbiology, and
Infectious Disease & Immunity, University of California, Berkeley,
Berkeley, CA.
Purpose: Pseudomonas aeruginosa is a leading cause of contact
lens-related corneal infection. Using a lens-wearing rodent model,
we previously showed that bacteria could infect the cornea more
efficiently if first pre-exposed to the ocular surface. This is consistent
with the increased risk of infection with extended lens wear in
humans. Here, we studied bacterial adaptions to the ocular surface
that subsequently enable them to penetrate the corneal epithelium.
Methods: RNA-seq was used to compare the transcriptional profile
of P. aeruginosa strain PAO1 exposed to human tear fluid for 5 h at
37 °C to PBS controls. Tn-seq was used to identify bacterial genes
required for traversal of human corneal epithelial cell multilayers in
vitro. For the latter, a pooled transposon mutant library of PAO1 was
generated, and ~106 cfu of bacterial mutants incubated with Transwell
filter-grown human telomerase-immortalized corneal epithelial cells
for 4 h at 37 °C. Transposon insertion sites were deep sequenced for
the input and traversed populations. HiSeq 2000 (Illumina) was used
for sequencing and data analysis performed with Galaxy and IGB.
Results: RNA-sequencing showed many P. aeruginosa genes
were deregulated (up- or down-regulated) by exposure to tear fluid
(~180 genes ≥ 8 fold). They included; the phoP/Q two-component
regulatory system (down-regulated), oprH for antimicrobial
resistance (down-regulated), and algF for biofilm formation and
antiphagocytic activity (up-regulated). Two small non-coding RNAs,
rsmZ and phrS, associated with virulence factor regulation, response
to oxygen availability and quorum sensing were also down- and
up-regulated respectively. Tn-seq identified ~200 gene insertions
differentially represented in traversed populations as compared to
the input (cut-off ≥ 18 fold), showing roles in epithelial traversal.
Insertions mapped in or near 150 genes belonging to numerous
important virulence categories, including pcrV (type three secretion),
motC (motility and attachment) and mexF (multidrug efflux pump).
Conclusions: Use of an unbiased global genetic approach to study
P. aeruginosa interaction with ocular surface components in vitro
identified genes and genomic regions involved in bacterial adaptation
to the host environment and ocular pathogenicity.
Commercial Relationships: Matteo M. Metruccio, None; Yvonne
Wu, None; David J. Evans, None; Suzanne M. Fleiszig, Allergan
Inc. (C)
Support: NIH EY024060
Program Number: 4044 Poster Board Number: A0221
Presentation Time: 8:30 AM–10:15 AM
Molecular mechanisms of host-pathogen interactions in
pseudomonas aeruginosa microbial keratitis
Ahmad Elsahn1, 2, Maria del Mar Cendra-Gascon1, Pawez Hossain1,
2
, Myron Christodoulides1. 1Infection, Inflammation & Immunity,
University of Southampton, Southampton, United Kingdom;
2
University Hospitals Southampton, Southampton, United Kingdom.
Purpose: To examine the molecular mechanisms of interactions
between P. aeruginosa bacteria and primary human corneal fibroblasts
(hCF) in an invitro model of microbial keratitis
Methods: Human CF were extracted from clinical samples, cultured
to confluence in vitro and incubated with live PAO1 wild type
bacteria as well as mutant strains deficient in type IV pilus (∆pilA),
flagella (∆fliM) or double mutants (∆pilA∆fliM) for 3h. Bacterial
association was quantified and compared across the strains. Confluent
hCF were also pre-treated with SRC kinase inhibitors genstein (GST)
and PP2 and actin microfilament inhibitor cytochalasin D (CD),
and bacterial internalization was compared across pre-treated cells
at 3h using a gentamicin protection assay. Wild type (WT) PA14 as
well as mutant strains deficient in type III secretion system (T3SS)
needle apparatus (∆popB) and flagella (∆flgK) were incubated with
confluent hCF for 9h, and bacterial cytotoxicity was assessed by a
lactate dehydrogenase (LDH) assay.
Results: Mutant PAO1strains ∆pilA, ∆fliM and ∆pilA∆fliM adhered
significantly less than WT to hCF (P<0.05). Bacterial internalization
in hCF pre-treated with CD, GST and PP2 was significantly less
than untreated cells (P<0.05). Mutant PA14 strains ∆popB and ∆flgK
caused significantly less cytotoxicity to hCF than WT PA14 (P<0.05).
Conclusions: PAO1 bacteria use type IV pilus and flagella to adhere
to hCF. Bacterial internalization to hCF is dependent on the actin
microfilament and SRC kinase systems. Bacteria use flagella to
adhere to hCF and T3SS to induce cytotoxicity.
Commercial Relationships: Ahmad Elsahn, None; Maria del
Mar Cendra-Gascon, None; Pawez Hossain, None; Myron
Christodoulides, None
Program Number: 4045 Poster Board Number: A0222
Presentation Time: 8:30 AM–10:15 AM
Correlation of P. aeruginosa Type III Secretion Profiles Diversity
and Ocular Disease Syndromes
Jorge Maestre, Edith Perez, Eduardo C. Alfonso, Harry W. Flynn,
Darlene Miller. Ophthalmology, University of Miami, Miami, FL.
Purpose: Pseudomonas aeruginosa associated ocular infections
are among the most diverse and difficult to manage. Clinical
presentations and pathology range from purulent conjunctivitis and
ulcerative keratitis to invasive scleritis and persistent, destructive
biomaterial centered infections. Virulence, disease course and patient
outcomes are strain specific. We used a combination of molecular,
metabolic and in vitro resistance markers to determine and correlate
Pseudomonas aeruginosa Type lll effector protein (T3SS) profiles
with biochemical patterns, ocular disease presentations and in vitro
susceptibility.
Methods: Type III Profiles: A multiplex PCR was used to
characterize and compare the prevalence of exoS (invasive), exoT
(invasive), exoU (cytotoxic) and exoY (adenyl cyclase) (genotypes
for 155 Pseudomonas aeruginosa strains collected from ocular
sources (cornea-n=96, contact lens-n=20, conjunctiva-n=23, lacrimal
system-n=8, Intraocular fluids-n=8) from 2008 to 2014. Biochemicals
and vitro susceptibility profiles were determined using the Vitek 2
system.
Results: Exo Y and T effector proteins were detected in all 155
isolates, while exoS ( N=70) and exoU (N=63) were generally
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
mutually exclusive. Prevalence of the four identified genotypes
were exoS+U- (45.2%), exoS-U+ (40.6%), exoS+U+ (7%) and exoU-S(9.7%). ExoU (cytotoxic strain) was the predominant profile of
isolates recovered from cornea (46.9%) and contact lens cases (70%)
but was evident in all sources. ExoS genotypes were documents
in 50% or higher for isolates recovered from conjunctiva (56.5%),
intraocular fluids (50%) and lacrimal system (50%). Strains with
absence of T3SS effectors were seen most often in isolates associated
with conjunctivitis (34.8%).
No significant correlation of metabolic profiles (N=100) were
associated with these isolates. Strains with genotype exoS-U+ had
the higher MIC90 (1 ug/ml) for ciprofloxacin vs 0.5 ug/ml or less
for exoS+U- and exoU-S- . Ciprofloxacin resistance ranged from 1%
(cornea) to 6% (conjunctiva). MIC90 for moxifloxacin was 2 ug/
ml and ranged from 3% (cornea) to 8% (conjunctiva and lacrimal
system).
Conclusions: Ocular Pseudomonas aeruginosa strains are diverse
and associated with specific disease syndromes and antibiotic
profiles. Characterizing the T3SS profiles may serve as an important
adjunct in understanding the pathology and management of these
destructive and recalcitrant infections
Commercial Relationships: Jorge Maestre, None; Edith Perez,
None; Eduardo C. Alfonso, None; Harry W. Flynn, None; Darlene
Miller, None
Support: Core Grant Department Ophthalmology Bascon Palmer
Eye Institute
Program Number: 4046 Poster Board Number: A0223
Presentation Time: 8:30 AM–10:15 AM
Photodynamic therapy to treat Methicillin-Resistant
Staphylococcus Aureus (MRSA) keratitis: An in vitro study
Heather A. Durkee1, Francisco Halili1, Mukesh Taneja2, Darlene
Miller3, 4, Alejandro Arboleda1, Cornelis J. Rowaan1, Mariela C.
Aguilar1, Guillermo Amescua4, Harry W. Flynn4, Jean-Marie A.
Parel1, 5. 1Ophthalmic Biophysics Center, Bascom Palmer Eye
Institute, University of Miami Miller School of Medicine, Miami, FL;
2
LV Prasad Eye Institute, Hyderabad, India; 3Ocular Microbiology
Laboratory, Bascom Palmer Eye Institute, University of Miami Miller
School of Medicine, Miami, FL; 4Anne Bates Leach Eye Hospital,
Bascom Palmer Eye Institute, University of Miami Miller School
of Medicine, Miami, FL; 5Brien Holden Vision Institute, UNSW,
Sydney, NSW, Australia.
Purpose: To assess the in vitro efficacy of rose bengal (RB) and
riboflavin (Ribo) mediated photodynamic therapy (PDT) for the
inhibition of a methicillin-resistant Staphylococcus aureus (MRSA)
strain.
Methods: A MRSA type II strain was isolated from the corneal
scraping of a patient with confirmed bacterial keratitis. Twentyfour hours prior to experimentation, a parent culture was plated on
nutrient agar. Next, a culture of MRSA was transferred into tryptic
soy broth and adjusted to a concentration of 1.5x108 colony forming
units per mL (cfu/mL). The MRSA suspension was diluted to a
concentration of 1.5x107cfu/mL with the appropriate solution and
1mL aliquots were inoculated in triplicate onto nutrient agar plates.
The eight groups were: (1) Control (high purity water) (2) UV-A
irradiation (3) 0.1% Ribo (4) 0.1% Ribo + UV-A (5) 0.1% RB (6)
0.1% RB + 518nm light (7) 0.03% RB (8) 0.03% RB + 518nm light.
All experiments were performed in minimal lighting conditions (4
lux) except for irradiation test plates. The UV-A and green lights
are custom built LED sources. The UV-A light, activate Ribo,
has a central wavelength of 375nm and an irradiance of 2.91mW/
cm2 over a 13.8cm2 surface. The 518nm light, activate RB, has a
central wavelength of 518nm and an irradiance of 2.2mW/cm2 over
a 28.3cm2 surface. Plates were either exposed to UV-A (Ribo) or
518nm (RB) irradiation for 20 minutes. All plates were immediately
placed upside down, wrapped in foil, and placed in an incubator at
30°C. Plates were photographed every 24 hours for 6 days.
Results: Riboflavin without irradiation did not inhibit MSRA growth;
however in Ribo with irradiation it did inhibit MRSA growth. 0.1%
RB with and without irradiation inhibited the growth of the MRSA.
0.03% RB inhibited MRSA growth with irradiation but did not
inhibit MRSA growth in the non-irradiated group. Inhibition in all
photosensitizer groups occurred as soon as 24 hours after irradiation.
Conclusions: Rose Bengal strips of 1.0% concentration are clinically
used to detect epithelial defects. Our study demonstrates MRSA
can be inhibited with 0.1% RB without irradiation. The RB will be
activated even when the patient is exposed to ambient light levels
in daily activities. PDT could be an excellent adjunct treatment for
MRSA keratitis as it provides a different mechanism of inhibition.
Results of 0.1% Ribo and 0.1% RB
Results of 0.03% Rose Bengal PDT at 72 hours
Commercial Relationships: Heather A. Durkee, None; Francisco
Halili, None; Mukesh Taneja, None; Darlene Miller, None;
Alejandro Arboleda, None; Cornelis J. Rowaan, None; Mariela C.
Aguilar, None; Guillermo Amescua, None; Harry W. Flynn, None;
Jean-Marie A. Parel, None
Support: Florida Lions Eye Bank, Drs KR Olsen and ME
Hildebrandt, NIH P30EY1481 (Center Grant), Research to Prevent
Blindness, Henri and Flore Lesieur Foundation (JMP). Technical
support was provided by: Karam Alawa, Victor Hernandez, and Nidhi
Relhan Batra.
Program Number: 4047 Poster Board Number: A0224
Presentation Time: 8:30 AM–10:15 AM
Interaction of Polymorphonuclear neutrophils (PMNs) with
Pseudomonas aeruginosa in biofilms in corneal infections in the
mouse
Padmanabhan Saraswathi1, Roger Beuerman1, 2. 1SERI, Singapore
Eye Research Inst (SERI), Singapore, Singapore; 2Department of
Ophthalmology, Yong Loo Lin School of Medicine, NUS, Singapore,
Singapore.
Purpose: Pseudomonas aeruginosa, is a common ocular pathogen
found in cornea infections, often associated with contact lens
wear. The survival of this bacteria as a “Biofilm” within a
extrapolysaccharide substances sheltered from innate immune
defence and antimicrobials. In this study, the interaction of
polymorphonuclear neutrophils (PMNs) with Pseudomonas in
planktonic and biofilm models was examined using a mouse model of
experimental keratitis infection
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Methods: A suspension (108 CFU/ml) of Pseudomonas aeruginosa
(ATTC 9027) was used to infect the mouse cornea (C57BL/6).
The infection was monitored by slit lamp. Infected eyes were
enucleated at post infection (PI) day1 to describe the planktonic
status and PI day3 and 5 for potential biofilm status. The response
of polymorphonuclear neutrophils (PMNs) was characterised
using histological, ultra-structural imaging and quantifying
myeloperoxidase levels
Results: Histology showed the expected large numbers of PMNs in
the stroma at PI day1, with diverse morphologies. At later stages of
infection they were seen on the corneal surface, but more spherical
shape. Scanning Electron Microscopy (SEM) demonstrated the
release of Neutrophil Extracellular Traps (NETs) as long fibrils and
entangling many bacteria which were seen as free cells at the early
stage of infection. However, at PI day3 and 5 the PMNs were seen
to change their morphology and lacking pseudopodia appeared
immobile, surrounding the clusters of Pseudomonas within a biofilm
while other PMNs were on the corneal surface. Transmission Electron
Microscopy (TEM) further confirmed the active phagocytosis at PI
day1 and immotile PMNs settled beneath the layer of the biofilm at
later stages of infection. Enhanced myeloperoxidase activity from 4.1
units at day1 to 16.9 units at day5 PI indicated the increased PMN
activity in the course of the infection
Conclusions: The murine neutrophils were seen to be highly
activated during an infection and produce NETs as an early defence
when the bacteria were planktonic. However, the PMNs were
concomitantly sluggish and not successful in phagocytosis when the
bacteria developed into biofilms at later times of infection. The early
role of PMN NETs as basic host defence may perhaps be considered
for novel drug targets to prevent biofilm formation and to augment
bacteria killing
Commercial Relationships: Padmanabhan Saraswathi, None;
Roger Beuerman, None
Program Number: 4048 Poster Board Number: A0225
Presentation Time: 8:30 AM–10:15 AM
Interactions of tear-film neutrophils with clinical bacteria
Maud Gorbet1, 2, Mark D. Willcox2. 1Systems Design Engineering,
University of Waterloo, Waterloo, ON, Canada; 2School of Optometry
and Vision Science, University of New South Wales, Sydney, NSW,
Australia.
Purpose: During sleep, in the closed-eye environment, a shift in tear
film composition leads to the recruitment of leukocytes to the ocular
surface. Neutrophils (also known as polymorphonuclear leukocytes;
PMN) are considered our first line of defense and play an essential
role in preventing infection. Following chemical stimulus, a lack of
upregulation of cell activation markers has previously been observed
on tear film neutrophils (TF-PMN). This pilot study was conducted
to investigate the response of TF-PMN to clinical bacteria and assess
whether overnight lens wear effected on their response to bacteria.
Methods: Acuvue Oasys lens wearers and non-lens wearers
were recruited to collect their cells upon awakening using an eye
wash “at-home collection kit”. Collected cells were counted and
resuspended in tubes containing either ATS-2% NHS (artificial tear
solution supplemented with 2% of normal human serum) or PBS10% Heat Inactivated (HI) FBS. Collected cells were incubated
with Pseudomona aeruginosa 6294 (an invasive clinical strain) and
P. aeruginosa 6206 (a cytotoxic clinical strain) at bacteria to PMN
ratios of 10:1 and 1:10. Following incubation, samples were serially
diluted in PBS, plated on agar and incubated overnight at 35oC. The
next day, bacteria colonies were counted. To further characterize the
response to bacteria, TF-PMN were analysed by flow cytometry for
receptor upregulation and oxidative burst.
Results: With a bacteria:TF-PMN ratio of 10:1 in ATS-2% NHS,
no reduction in bacteria growth was observed for 6206 and 6294.
Fewer 6294 CFU were counted when interactions with TF-PMN
occurred in PBS-HIFBS. Furthermore, regardless of incubation
medium, bacteria:TF-PMN interactions at a ratio of 1:10 resulted in
a significant increase in the number of 6206 cells (p<0.05). For both
6206 and 6294, interactions at the 1:10 ratio with TF-PMN collected
following overnight lens wear led to a significant increase in CFU
when compared to the 10:1 ratio (p<0.04). Flow cytometry results
confirmed the differential TF-PMN response to 6206 and 6294.
Conclusions: Our results suggest that at low bacteria to PMN ratio,
a condition that may closely mimic the closed-eye environment,
TF-PMN are unable to phagocytose clinical strains of Pseudomona
aeruginosa and may be releasing factors that promote bacterial
survival/growth. This may have an important impact during overnight
lens wear and may contribute to the higher risks of microbial
keratitis.
Commercial Relationships: Maud Gorbet, None; Mark D.
Willcox, None
Support: American Optometric Foundation VISTAKON® Research
Grant
Program Number: 4049 Poster Board Number: A0226
Presentation Time: 8:30 AM–10:15 AM
Pseudomonas aeruginosa Ocular Strains Resistant to Ceftazidime
in Northeast of Mexico
Pedro M. González, Alejandro F. Ibarra-Lozano, Jorge E. Valdez,
Jaime Torres. Ophthalmology, Instituto Tecnológico de Monterrey,
San Pedro Garza García, Mexico.
Purpose: The purpose of this case series is to report Pseudomonas
aeruginosa resistance to ceftazidime in keratitis isolates in northeast
of Mexico. Concern exists around the growing antibiotic resistance
in different ocular pathogens worldwide. Ceftazidime is known to be
one of the antimicrobial agents used with a high sensitivity profile in
bacterial keratitis caused by Pseudomonas aeruginosa.
Methods: Patient records from January 2010 to November of 2014
were revised. Twenty eight patients were found with the diagnosis of
infectious keratitis. Eight cases were culture positive for Pseudomona
aeruginosa. variables studied included: gender, age, time of
presentation to consultation, visual acuity, comorbidities, antibiotic
sensitivity/resistance pattern and complications encountered.
Results: Eight culture positive P. aeruginosa cases were found
between 2010 to the end of 2014. There were 2 females and 6 males,
the age range was from 19 to 67 years old. Ceftazidime resistance
was reported in seven cases. Susceptibility was reported for
ciprofloxacin, meropenem, amikacin and gentamicin. Ciprofloxaxin
and meropenem were the ones with the lowest minimal inhibitory
concentration. Final visual acuity had a strong relation with the
initially reported. Two cases were associated to traumatism and one
to soft contact lenses use. Two cases had corneal perforation.
Conclusions: This study reviews a series of cases of P. aeruginosa
keratitis in northeast of Mexico; the microbiologic profile showed
87.5% resistance to ceftazidime. Sensitivity to meropenem,
ciprofloxacin, amikacin and gentamicin was of 100%. This data
should influence the management of this entity in the geographic
zone examined.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Support: NIH Grant AI079192
Commercial Relationships: Pedro M. González, None; Alejandro
F. Ibarra-Lozano, None; Jorge E. Valdez, None; Jaime Torres,
None
Program Number: 4050 Poster Board Number: A0227
Presentation Time: 8:30 AM–10:15 AM
Corneal epithelial cells suppress vacuolar escape of P. aeruginosa
Abby Kroken, David J. Evans, Suzanne M. Fleiszig. School of
Optometry, University of California, Berkeley, Berkeley, CA.
Purpose: Previously we reported that invasive strains of P.
aeruginosa invade corneal epithelial cells wherein they replicate
and induce formation of plasma membrane blebs to which they
traffic. These events depend on the bacterial Type Three Secretion
System (T3SS), specifically the T3SS toxin ExoS. Other investigators
have studied ExoS using HeLa cells (not normally targeted by P.
aeruginosa) and PA103 (does not natively express ExoS), and have
not noted these phenomena. Here, we tested the hypothesis that the
impact of ExoS depends on bacterial strain and cell type.
Methods: Epithelial cell lines (HeLa or corneal) were infected with
P. aeruginosa strain PAO1 (natively expresses ExoS) or PA103
(engineered to express ExoS). Intracellular bacteria were quantified
using a gentamicin protection assay. A T3SS-driven GFP reporter was
used to monitor T3SS expression in individual bacteria. Images were
captured using time lapse wide-field or confocal microscopy.
Results: In both cell types, PAO1 and PA103 expressing ExoS
induced membrane blebbing, but only PAO1 invaded, replicated
intracellularly, or trafficked to blebs. In contrast to corneal cells,
HeLa cells even supported intracellular replication of PAO1
mutants lacking all known T3SS toxins (including ExoS). That
replication remained dependent on the T3SS, which was expressed
intracellularly, and occurred in the cytoplasm, without blebs, and in
cells possessing acidified vacuoles - all differentiating it from ExoSdependent replication in corneal cells. T3SS machinery mutants
remained in vacuoles in HeLa cells, showing that vacuolar escape is
mediated by a T3SS component or unknown effector, but not ExoS.
This contrasts with ExoS-dependent vacuolar escape in corneal cells.
Conclusions: ExoS has been mostly studied using PA103 and HeLa
cells. Our data show that neither accurately model how natively
encoded ExoS influences P. aeruginosa interactions with corneal
epithelial cells. This explains why its role in intracellular survival was
overlooked by investigators using those tools. Our data show that;
1) corneal epithelial cells have innate defenses against internalized
bacteria that are lacking in HeLa cells and which ExoS can overcome,
and 2) unknown T3SS factors allow intracellular replication in cells
lacking those innate defenses. Identifying mechanisms involved
could lead to novel strategies to combat infection.
Commercial Relationships: Abby Kroken, None; David J. Evans,
None; Suzanne M. Fleiszig, Allergan (C)
Program Number: 4051 Poster Board Number: A0228
Presentation Time: 8:30 AM–10:15 AM
Pseudomonas aeruginosa induces autophagy in human corneal
epithelial cells
VIDYARANI MOHANKUMAR1, LakshmiPriya Jeganathan1, Lalitha
Prajna1, Chidambaranathan Gowri Priya2. 1Ocular Microbiology,
Aravind Medical Research Foundation, Madurai, India; 2Immunology
and Stem Cell Biology, Aravind Medical Research Foundation,
Madurai, India.
Purpose: Keratitis caused by Pseudomonas aeruginosa is a
serious ocular infection which may lead to corneal perforation if
the intracellular bacteria are not cleared completely. Autophagy, a
normal catabolic process, has been shown to play a major role in
the clearance of intracellular pathogens. We propose that autophagy
induced by P. aeruginosa in human corneal epithelial cells (HCET),
may have a role in the clearance of intracellular bacteria.
Methods: HCET cells transfected with LC3-GFP plasmid were
infected with three different ocular isolates of P. aeruginosa and
autophagy was monitored after 1h using a Leica TCS SP8 confocal
microscope. EBSS treated (amino acid starvation) HCET cells were
used as positive control for autophagy. Another set of HCET cells
were infected with P. aeruginosa to study the mRNA expression of
autophagy related protein, beclin1 by real time PCR. To study the
intracellular survival and replication of the bacteria, HCET cells were
infected with P. aeruginosa in the presence of EBSS or 3 –methyl
adenine (3mM in EBSS), an inhibitor of autophagosome formation.
After 3h, the extracellular bacteria were killed with gentamicin
and the cells were incubated for another three hours to allow for
intracellular bacterial replication. Diluted cell lysates were plated on
to MacConkey agar, and the colonies were counted after an overnight
incubation.
Results: The HCET cells upon infection with three different P.
aeruginosa isolates showed an increased LC3 punctation, which is
a classical marker for autophagosome formation. The total number
of LC3 positive cells and the relative number of LC3-puncta per cell
varied upon infection with the different isolates which may be due to
the difference in the intracellular bacterial load. Correspondingly, the
mRNA expression of beclin1 was higher in P. aeruginosa infected
cells compared to uninfected controls.The ocular isolates were able
to efficiently invade and replicate inside the epithelial cells and the
bacterial load was relatively higher when the cells were pretreated
with 3-methyl adenine.
Conclusions: Altogether, the results suggest that P. aeruginosa
induces autophagy in human corneal epithelial cells, which inturn may limit the intracellular bacterial load. Since the ocular P.
aeruginosa isolates can efficiently invade and replicate inside the
epithelial cells, autophagy may represent a host defensive mechanism
to curtail infection in P. aeruginosa keratitis.
Commercial Relationships: VIDYARANI MOHANKUMAR,
None; LakshmiPriya Jeganathan, None; Lalitha Prajna, None;
Chidambaranathan Gowri Priya, None
Program Number: 4052 Poster Board Number: A0229
Presentation Time: 8:30 AM–10:15 AM
Role of IL-24 in Pseudomonas Aeruginosa Keratitis in a C57BL/6
Mouse Model
Bing Xu, Nan Gao, Fushin X. Yu. Anatomy & Cell Biology and
Ophthalmology, Wayne State University School of Medicine, Detroit,
MI.
Purpose: The cytokines in interleukin (IL)-10 family are known
by their anti-infection and anti-inflammatory activities. However,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
the biology of IL-24, a member of IL-10 family cytokines, is
largely unknown, particularly in the ocular tissue. In this study, we
investigated the role of IL-24 in a mouse Pseudomonas Aeruginosa
(PA) keratitis model.
Methods: Epithelium-injured B6 mouse corneas were pretreated
with or without flagellin for 24h, followed by PA (ATCC 19660)
inoculation. Samples were collected at various time points post
infection and subjected to PCR and immunofluorescence. Mice were
subconjunctivally injected with either IL-24 siRNA to knock down
IL-24 expression or mouse IL-24 recombinant protein before the
inoculation of PA to explore the biological functions of IL-24.
Results: PA infection induced IL-24 transcription on corneal
epithelial cells; flagellin pretreatment not only alleviated the
infection, but also reduced the mRNA expression of IL-24, but not
IL-19 and IL-20, the other two members of IL-10 family that share
common receptors. STAT3, a signal mediator in IL-24 pathway,
was phosphorated in response to PA infection, and p-STAT3 was
mainly expressed in the infiltrated cells, indicating that immune cells
were recruited and activated by the infection. IL-24 downregulation
alleviated the severity of PA keratitis, and the application of IL-24
recombinant protein exacerbated PA infection on mouse cornea.
Conclusions: These data demonstrate that IL-24 exhibits a
detrimental effect on the host defense against PA infection, suggesting
that IL-24 pathway may be a new intervention site for treating
infectious keratitis.
Commercial Relationships: Bing Xu, None; Nan Gao, None;
Fushin X. Yu, None
Support: NIH/NEI EY017960
Program Number: 4053 Poster Board Number: A0230
Presentation Time: 8:30 AM–10:15 AM
Thrombomodulin protects against bacterial keratitis and is not
angiogenic
Linda D. Hazlett, Sharon A. McClellan, Cui Li. Anatomy & Cell
Biology, Wayne State Univ Sch of Med, Detroit, MI.
Purpose: Thrombomodulin (TMB), a cell surface glycoprotein
composed of 5 domains, is expressed in a variety of cells. The
N-terminal lectin-like domain (TMD1) interacts with Lewis Y
antigen to inhibit angiogenesis and is responsible for TMB’s antiinflammatory properties; it also binds high mobility group box-1
(HMGB1), preventing its deleterious effects, while domains 2 and
3 (TMD23) induce neovascularization, with response regression
within 24 days. Despite data regarding activities of specific domains
of TMB, nothing has been reported regarding testing its protective
effects in experimental microbial keratitis, and whether it induces
corneal vascularity which is the purpose of this study.
Methods: C57BL/6 mice were injected (subconjunctivally and i.p.)
with recombinant (r)TMB and infected with P. aeruginosa. PBS
controls were similarly treated. Clinical score, photography with a slit
lamp, real time RT-PCR, MPO assay, and ELISA were used to assess
the disease response.
Results: Data show that treatment of C57BL/6 mice with rTMB
reduced clinical disease scores, corneal opacity and the neutrophil
infiltrate. mRNA levels for IL-1β, MIP-2, TLR4, and RAGE were
decreased, while anti-inflammatory molecules, including SIGIRR
and ST2 levels were increased when compared with controls. ELISA
confirmed the PCR data showing significant reduction of both IL-1β
and MIP-2 protein level (3 and 5 days post infection) after rTMB
treatment. VEGF, VEGF-R1 and VEGF-R2 were no different, or
reduced after TMB (5 days p.i.).
Conclusions: TMB is protective in keratitis, reducing disease
severity, and with no angiogenic effect .
Commercial Relationships: Linda D. Hazlett, None; Sharon A.
McClellan, None; Cui Li, None
Support: NIH Grants EY016058 and P30EY04068
Program Number: 4054 Poster Board Number: A0231
Presentation Time: 8:30 AM–10:15 AM
Conjunctival chemosis as a specific feature of Pseudomonas
aeruginosa corneal ulcers
Kaleena B. Michael. Tennents institute of ophthalmology, Glasgow,
United Kingdom.
Purpose: Corneal ulcer is a common ocular problem often
complicated by delayed treatment from late diagnosis. We looked
for presence of conjunctival chemosis in 44 consecutive cases of
confirmed infective corneal ulcers.
Methods: Retrospective examination of early ocular photographs of
44 consecutive cases of infective corneal ulcers.
Results: Conjunctival chemosis was observed in 13 out of 44 cases.
12 of these were culture positive for pseudomonas aeruginosa, and 1
for colliform bacilli. Statistical analysis with Fishers Exact test was
significant p>0.000001, Odds Ratio 112 (95% CI: 10.6 - 1190).
Conclusions: Our findings suggest a strong association between
conjunctival chemosis in pseudomonas aeruginosa corneal ulcers.
This ocular feature could potentially help predict the presence of of
pseudomonas aeruginosa in new corneal ulcers. This would enable
individuals to receive the treatment of choice at an earlier stage,
before microbiological confirmation.
Commercial Relationships: Kaleena B. Michael, None
Program Number: 4055 Poster Board Number: A0232
Presentation Time: 8:30 AM–10:15 AM
Microbiota and P. aeruginosa Genotype adhesion difference
on Worn Cosmetic Contact Lenses (CL) with Comparison of
Disinfectant Sensitivity
Elizabeth Shen1, Fung Rong Hu2. 1Ophthalmology, Taipei Tzu
Chi Hospital, Taipei, Taiwan; 2Ophthalmology, National Taiwan
University Hospital, Taipei, Taiwan.
Purpose: To analyze attached microbes found on worn cosmetic
CL materials and compare difference in adhesion of two Type III
secretion genotypes of P. aeruginosa. Disinfection ability of four
disinfectants is compared between two genotypes.
Methods: Healthy volunteers with myopia < -6 D and astigmatism
< -1.0 D with signed informed consent were recruited in this
prospective randomized study. Subjects wore nelfilcon (Alcon
Freshlook Colorblends gray), hilafilcon (Bausch & Lomb Naturelle
black),or etafilcon (Johnson & Johnson Acuvue 2 Define Accent style
black) daily disposable CL for at least 8 hours/day for 2 weeks. The
lenses are collected aseptically. Half of the lens is used to identify
attached microbes. The other half is incubated for 2 hrs with108cfu/
ml of cytotoxic PA103 or invasive PAK strains. Viable cell culturing
was done to determine number of bacteria attached. P. aeruginosa
incubated lenses were soaked with various disinfectants (Renu,
Aosept, Puremoist, and Replenish) at 25%, 50%, 75%, and 100% of
the suggested disinfection time. The number of bacteria remaining on
CL is counted and compared.
Results: The most commonly identified microbes attached to nonsymptomatic volunteers were coagulase negative Staphylococcus,
P. aeruginosa, and Streptococci. Adhesion of the cytotoxic strain
PA103 was found to be greatest for hilfilcon lenses (34.8x104cfu/
ml) > etafilcon (33.1 x104cfu/ml) > nelfilcon (15.5x104cfu/ml)
(P<0.05; ANOVA). Similar trend is noted for adhesion of the invasive
strain PAK: hilfilcon (30.9x104 cfu/ml)>etafilcon(37.5x104 cfu/ml)
>nelfilcon(10.1x104 cfu/ml) (P<0.05; ANOVA). At 100% suggested
time, all tested disinfectants were able to kill all bacteria. However,
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
at less than 75% of suggested disinfection time, significant number of
viable cytotoxic P. aeruginosa remained (P=0.03, t-test).
Conclusions: With increasing popularity of cosmetic CL, clinicians
are should be aware that different cosmetic materials attract different
microbial adhesions. P. aeruginosa, a commonly identified pathogen
in CL-associated microbial keratitis, is frequently found attached
to CL of asymptomatic individuals. Cytotoxic strains are more
resistant to MPS solution especially Renu. Proper lens care with
strict adherence to suggested disinfection time is strongly advised to
prevent sight threatening infections related to cosmetic lens wear.
Commercial Relationships: Elizabeth Shen, None; Fung Rong
Hu, None
Support: NSC-102-2628-B-002-051-MY3
Program Number: 4056 Poster Board Number: A0233
Presentation Time: 8:30 AM–10:15 AM
Effect of the interaction Fusarium solani-Staphylococcus aureus
over limbocorneal fibroblasts
Antonio Bautista-Hernandez1, 2, Beatriz Buentello-Volante3, Victor
M. Bautista1, José Luis Gómez Olivares2. 1Microbiology and
Ocular Proteomics, Inst of Ophthalmology “Conde de Valenciana”,
Mexico City, Mexico; 2Department of Health Sciences, Autonomous
Metropolitan University., Mexico City, Mexico; 3Genetics, Inst of
Ophthalmology “Conde de Valenciana”, Mexico City, Mexico.
Purpose: To study the effect of the interaction Fusarium solaniStaphylococcus aureus over limbocorneal fibroblasts.
Methods: The limborcorneal fibroblasts were obtained from
limbocorneal tissue and grown in media DMEM-F12 supplemented
with serum fetal bovine 10%, were evaluated expression of vimentin
and cytokeratin. F. solani and S. aureus were isolated from human
corneal ulcers. The microorganisms were identified by classical
microbiology. The biofilm formation was evaluated in media
DMEM-F12 by the Christensen method and fluorescence microscopy.
Fibroblasts were exposed to S. aureus and/or F. solani to evaluate the
expression of CD34 and production of INFγ.
Results: According to classical microbiology studies, bacterial strain
corresponded to S. aureus and fungal strain to F. solani. Fluorescence
microscopy showed a formed biofilm by the interactions between
S. aureus and F. solani, that generated a extracellular matrix. The
limbocorneal fibroblasts had characteristic morphology, vimentin
positive and cytokeratin negative. The expression of CD34 decreased
and increased content of INFγ with stimuli of S. aureus, F. solani and
S. aureus-F. solani.
Conclusions: Microorganism S. aureus and F. solani showed ability
to form biofilm. The limbocorneal fibroblasts were vimentin positive
and cytokeratin negative. Expression of CD34 was decreased and
increased INFγ production with stimuli of S. aureus, F. solani and S.
aureus-F. solani.
Commercial Relationships: Antonio Bautista-Hernandez, None;
Beatriz Buentello-Volante, None; Victor M. Bautista, None; José
Luis Gómez Olivares, None
Support: This work was supported by “Conde de Valenciana”
Foundation
Program Number: 4057 Poster Board Number: A0234
Presentation Time: 8:30 AM–10:15 AM
Involvement of endoplasmic reticulum (ER) stress in the
pathogenesis of bacterial endophthalmitis
Ajay Kumar1, Pawan Kumar Singh1, Ashok Kumar1, 2.
1
Ophthalmology, Wayne State University School of Medicine,
Detroit, MI; 2Anatomy and Cell Biology, Wayne State University
School of Medicine, Detroit, MI.
Purpose: Endoplasmic Reticulum (ER), through the unfolded protein
response (UPR), regulates various cellular functions, including
inflammation. Here, we have investigated the role of UPR signaling,
specifically IRE1α in regulating intraocular inflammation in an
experimental model of Staphylococcus aureus (SA) endophthalmitis
Methods: Endophthalmitis was induced by intravitreal injection of S.
aureus in WT (C57BL/6), TLR2-/-, and MyD88-/- mice. After 24 hours
of infection, retinal tissue was collected; RNA was extracted, and
RT-PCR was performed to assess ER stress marker genes (XBP-1s,
IRE1α, PD1, CHOP, WSF, BiP, and ERDj4) using specific primers.
To determine the role of IRE1α, inhibition studies were performed
using IRE1α specific inhibitor, 4μ8C, given prior to S. aureus
challenge. Secretion of inflammatory mediators and the activation of
TLR-downstream signaling pathways (JNK1/2, MAPK, and NF-kB)
were assessed using ELISAs and western blot analyses respectively.
Mechanistic studies were performed using cultured BV2 microglial
cells.
Results: S. aureus did not induce the classical UPR response as
evidenced by upregulation of ER stress markers (PD1, CHOP,
WSF, BiP, and ERDj4) in the infected retina. However, S. aureus
caused the splicing of XBP1 in both the mouse retina and the BV2
microglia. Concomitant with increased XBP-1s levels, the level of
IRE1α was also increased in infected cells/tissue. The IRE1 inhibitor
(4μ8C) reduced the levels of both XBP-1s and IRE1α in WT mice
and microglia, but not in TLR2-/- and MyD88-/- mice. Similarly, the
expression and secretion of inflammatory cytokines were attenuated
in 4μ8C-treated WT mice and microglia cells, while no reduction
was observed in TLR2-/- and MyD88-/- mice. Furthermore, S. aureusinduced the activation of ERK1/2, p38 MAPK, and NF-kB signaling
was down regulated by the IRE1α inhibition.
Conclusions: Taken together, our study, for the first time, provides
evidence of IRE1α-mediated UPR stress signaling in generating
retinal innate responses in bacterial endophthalmitis. Furthermore,
these findings suggest a potential cross-talk between TLR and UPRsignaling in orchestrating inflammatory responses in the retina.
Commercial Relationships: Ajay Kumar, None; Pawan Kumar
Singh, None; Ashok Kumar, None
Support: NIH EY019888
Program Number: 4058 Poster Board Number: A0235
Presentation Time: 8:30 AM–10:15 AM
Bacterial Impediment of Corneal Cell Migration
Kimberly Brothers, Nicholas A. Stella, Kristin M. Hunt, Regis
P. Kowalski, Jes Klarlund, Robert M. Shanks. Ophthalmology,
University of Pittsburgh, Pittsburgh, PA.
Purpose: The loss of corneal epithelium in patients with microbial
keratitis is well known but the underlying mechanisms are poorly
understood. This study set out to determine the impact of bacterial
secreted factors on ocular wound healing.
Methods: Bacterial secretomes from ocular keratitis isolates were
prepared from overnight cultures and filtered to remove bacteria.
Using a plate based cell migration assay, secretomes were added
to stratified human corneal limbal epithelial (HCLE) cells and
incubated. Calcein AM viability stained cell layers were imaged
by confocal microscopy. Porcine corneal organ culture was used
to assess epithelial wound healing phenotypes ex vivo. Transposon
mutagenesis of the Serratia marcescens genome was conducted
to identify bacterial genes responsible for corneal cell migration
inhibition. LPS was purified by the hot phenol method. The waaG
lipopolysaccharide (LPS) gene was cloned using yeast homologous
recombination into vector pMQ131.
Results: Corneal cells treated with bacterial secretomes from
4/5 Pseudomonas aeruginosa, 26/27 S. marcescens, and 2/14
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Staphylococcus aureus isolates showed dose dependent inhibition
of HCLE migration. No inhibition was seen with secretomes
from 4 other ocular pathogens. Using an ex vivo porcine corneal
wound model, we have recapitulated S. marcescens inhibition of
wound healing. A transposon mutation in the S. marcescens LPS
biosynthetic locus that prevented bacterial inhibition of epithelial
cell migration was identified and complemented with the wild-type
gene on a plasmid. Supporting the importance of LPS in the cell
migration phenotype, depletion of LPS with polymyxin B agarose
inactivated the inhibitory ability of the bacterial secretomes. Purified
S. marcescens LPS, but not E. coli LPS was able to inhibit corneal
cell migration.
Conclusions: Together these data support that multiple ocular
pathogens secrete factors able to inhibit corneal epithelial wound
healing. Genetic and biochemical data using S. marcescens as a
model organism indicate that S. marcescens LPS is sufficient to
prevent corneal epithelial cell migration and wound healing. This
study presents a novel host-pathogen interaction with implications
for corneal ulcers and other medical problems where bacteria impact
wound healing, such as chronic wounds and provides evidence that
LPS may be a key factor in the inhibitory mechanism.
Commercial Relationships: Kimberly Brothers, None; Nicholas
A. Stella, None; Kristin M. Hunt, None; Regis P. Kowalski, None;
Jes Klarlund, None; Robert M. Shanks, None
Support: NIH 2T32 EY017271-06A1 NIH EY08098 NIH AI085570
Program Number: 4059 Poster Board Number: A0236
Presentation Time: 8:30 AM–10:15 AM
Toxicity and Virulence of Viridans Group Streptococci Isolated
from Endophthalmitis
Mary E. Marquart, Hannah R. Rice. Microbiology, Univ of
Mississippi Med Ctr, Jackson, MS.
Purpose: Endophthalmitis due to viridans group streptococci (VGS)
has been shown to result in poor visual prognosis despite antibiotic
therapy. We hypothesized that VGS possess toxins that are involved
in ocular pathogenesis. The purpose of this study was to identify the
virulence characteristics of endophthalmitis strains of VGS.
Methods: Concentrated extracellular milieu from 20 endophthalmitis
strains of VGS, and concentrated culture media (controls), were
subjected to 3 assays: lysis of sheep erythrocytes, retinal pigmented
epithelial (RPE) cell toxicity, and Western blot for detection of
pneumolysin (the major toxin of S. pneumoniae). Each strain was
assigned a profile based on assay results. Four strains with different
profiles were chosen for intravitreous injection of 100 colonyforming units (CFU) of each strain into the left eyes of each of 3
rabbits, followed by clinical examination and bacterial recovery from
the vitreous. The concentrated extracellular milieu from each of the 4
strains was also tested for protease activity by gelatin zymography.
Results: Eight of 20 strains (40%) lysed sheep erythrocytes and 15
(75%) were cytotoxic at a level of ≥2 on a scale from 0 (no activity)
to 4 (full activity). Pneumolysin was detected in 6 (30%). Two of
the 4 strains tested in vivo caused anterior chamber inflammation in
addition to vitreous haze, with severity increasing with time up to 72
hours after infection. The most severe eyes, which were infected with
strain E664, had clinical scores with a mean of 25.33 ± 2.08 (scale
of 0 to 32) and bacterial recovery with a mean of 6.95 ± 0.53 log10
CFU/mL at 72 hours. Interestingly, E664 was not hemolytic nor did it
produce pneumolysin, but was highly toxic to RPE cells. The secondmost virulent strain in vivo, 144065, was negative for all of the in
vitro assays. The least virulent strain in vivo, E618, was positive for
all of the in vitro assays. Zymography of the 4 strains tested in vivo
showed protease activity in E664 and 144065, but not in the other 2
strains.
Conclusions: VGS from endophthalmitis are diverse in toxin activity,
and toxin activity cannot necessarily predict virulence in the rabbit
eye. Strains with protease activity induce more damage in the rabbit
eye than those without detectable protease. Determination of whether
protease activity contributes to pathogenesis will aid in the first steps
of characterizing the virulence of VGS in the eye.
Commercial Relationships: Mary E. Marquart, None; Hannah R.
Rice, None
Program Number: 4060 Poster Board Number: A0237
Presentation Time: 8:30 AM–10:15 AM
Measuring Severe Inflammatory Trachoma (TI) when prevalence
is low provides indirect data on infection with C. trachomatis in
endemic communities
Andrea I. Zambrano2, Beatriz E. Munoz1, Laura Dize2, Harran
Mkocha4, Charlotte Gaydos3, Thomas Quinn2, 3, Sheila K. West1.
1
Ophthalmology, Wilmer Eye Inst Johns Hopkins Univ, New York,
NY; 2Infectious Disease, International Chlamydia Laboratory, Johns
Hopkins School of Medicine, Baltimore, MD; 3National Institute of
Allergy and Infectious Disease, Bethesda, MD; 4Kongwa Trachoma
Project, Kongwa, United Republic of Tanzania.
Purpose: Inflammatory trachoma (TI) is not measured when
assessing the impact of trachoma programs because it is felt to
indicate non-trachoma disease; only follicular trachoma (TF) is
measured. We tested the supposition that TI was not associated
with infection when disease prevalence is low, and does not add to
understanding the effect of programs.
Methods: In each of 52 communities in Kongwa Tanzania,
100 children ages 1-9 years were randomly selected for survey
at baseline, 6, and 12 months. In 17 communities, Mass Drug
Administration (MDA) was stopped at baseline because infection
rates were 1% or less; 35 had MDA. Both eyelids were graded for
evidence of TF and TI and a swab for detection of infection was
taken. The swabs were tested using Aptima (GenProbe/Hologic)
for presence of C. trachomatis. Overall prevalence rate of active
trachoma in communities with and without MDA at each time point
were compared, and the proportion of active trachoma cases that
were TI alone was estimated. Proportion of active trachoma cases
that were TI alone were compared among the treated and not treated
communities. All comparisons were analyzed using Fisher’s exact
test.
Results: Overall prevalence of active trachoma at baseline was 6%
(318 cases); 15% were TI alone. The prevalence of infection in TF
cases was 36% and 37% in TI alone. At 6 months, the prevalence of
active trachoma was 5.5% in communities where MDA was stopped;
18% of the cases were TI alone, for whom the rate of infection
was 41.2%. In treated communities, prevalence of active trachoma
was 3.9% and 10% of cases were TI alone for whom the rate of
infection was 30.8%. At 12 months, prevalence of active trachoma
in communities with MDA stopped was 5.8% and 17% of cases
were TI alone; 59% had infection with C. trachomatis. In treated
communities, prevalence rate was 5.1% with 22% of cases being TI
alone for whom the rate of infection was 37.5%. Infection in the TI
cases in communities where MDA was stopped was significantly
greater than in treated communities (p=0.02).
Conclusions: Despite low prevalence, the clinical sign of TI was
better correlated with infection than TF, and particularly so when the
survey was a year or more after MDA. TI should be measured as part
of impact surveys and particularly at surveys several years after the
last MDA.
Commercial Relationships: Andrea I. Zambrano, None; Beatriz
E. Munoz, None; Laura Dize, None; Harran Mkocha, None;
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Charlotte Gaydos, None; Thomas Quinn, None; Sheila K. West,
None
Support: National Eye Institute EY022584
Program Number: 4061 Poster Board Number: A0238
Presentation Time: 8:30 AM–10:15 AM
Evaluation of effectiveness of real-time PCR for bacterial
keratitis diagnosis
Daisuke Shimizu, Dai Miyazaki, Keiko Yakura, Tomoko Haruki,
Yoshitsugu Inoue. Ophthalmology and Visual Science, Tottori
University Faculty of Medicine, Yonago city, Japan.
Purpose: To determine the effectiveness of measurement of bacterial
DNA amount by real-time PCR for the diagnosis of bacterial
keratitis, we characterized the sensitivity and specificity profile and
determined cut-off value for diagnosis by using Receiver Operating
Characteristic (ROC) analysis.
Methods: Consecutive case series (241 eyes of 241 cases), suspected
of infectious keratitis and measured for the amount of bacterial
DNA in corneal tissue samples, were retrospectively analyzed. The
measurement of bacterial DNA amount (16S rDNA by real-time
PCR), smear examination by Gram and
Fungiflora staining, and culture testing were evaluated for diagnostic
efficacy by ROC analysis.
Results: One hundred and nine eyes were diagnosed as definitive
bacterial keratitis. Eighty four eyes were diagnosed as keratitis
of other causes. In the definitive bacterial keratitis eyes, average
bacterial DNA copy number was 1.6x106, culture positive rate was
54%, and the smear positive rate was 45%. In the non bacterial
keratitis eyes, they were 6.1x103, 0.0%, and 7.0%, respectively. Area
under the curve (AUC) was calculated to show diagnostic efficacy
based on ROC analysis of these testing. The AUC for definitive
bacterial keratitis was 0.73, 0.65, and 0.60 for smear testing,
bacterial DNA copy measurement, and culture testing, respectively.
To determine the most useful combination of each testing, AUC of
combined outcome was calculated using propensity scoring analysis.
Combination of smear testing and bacterial DNA amount indicated
highly efficacious diagnostic value (AUC: 0.80), and cut off value of
bacterial DNA copy for diagnosis of bacterial keratitis was 1.0X103.
Conclusions: Combined testing of bacterial DNA quantification and
smear testing was highly efficacious to definitively diagnose bacterial
keratitis at initial visit.
Commercial Relationships: Daisuke Shimizu, None; Dai
Miyazaki, None; Keiko Yakura, None; Tomoko Haruki, None;
Yoshitsugu Inoue, Alcon Japan Inc. (F)
Program Number: 4062 Poster Board Number: A0239
Presentation Time: 8:30 AM–10:15 AM
Evaluation of real-time PCR for the diagnosis of intra-ocular
tuberculosis
Soumyava Basu, Manas R. Barik, Praveen K. Balne, Soveeta S.
Rath, Mamatha Reddy, Savitri Sharma. LV Prasad Eye Institute,
Bhubaneswar, India.
Purpose: Definitive diagnosis of intraocular tuberculosis has
remained challenging despite recent advances in molecular
diagnostic techniques. Here we report the development of a realtime polymerase chain reaction (PCR) assay for detection of
Mycobacterium tuberculosis complex in aqueous and vitreous
samples from eyes with intraocular tuberculosis.
Methods: Aqueous or vitreous humor samples were collected
from patients with clinically suspected ocular tuberculosis (based
on previously published diagnostic criteria; Gupta et al, Surv
Ophthalmol’ 2007) and non-uveitis eyes undergoing vitrectomy or
cataract surgeries (controls). mpb64 gene of M. tuberculosis genome
and human RPPH1 (RNase P RNA component H1) were amplified
from the extracted DNA and detected real-time by customized
FAM-labeled probes. The ratio of copy numbers of mpb64 and
RPPH1, obtained from each test and control sample was used to
generate Receiver Operating Characteristic (ROC) curves. The
optimum cut-off value of real-time PCR was identified from the
experimental data that had the highest Youden index (Youden index =
sensitivity+specificity-1).
Results: M. tuberculosis complex genome was detected in 33 of 47
test samples (70.2%) and 2 of 18 healthy controls (11.2%) based
on optimum cutoff value of copy number ratios (0.025) obtained
from ROC curve having highest Youden index number, 0.727. At
this cutoff value the sensitivity was 81.0% and specificity 91.7%.
The copy number ratios varied widely in different clinical samples,
with the highest median value seen in intermediate uveitis sub-group
(0.387±0.664). The numbers were not sufficient to compare aqueous
and vitreous samples.
Conclusions: We could develop a highly specific and sensitive
PCR assay for detection of M. tuberculosis complex in aqueous
and vitreous samples. There was wide variation in copy numbers in
different disease sub-types that need to be analyzed in larger studies.
Commercial Relationships: Soumyava Basu, None; Manas R.
Barik, None; Praveen K. Balne, None; Soveeta S. Rath, None;
Mamatha Reddy, None; Savitri Sharma, None
Program Number: 4063 Poster Board Number: A0240
Presentation Time: 8:30 AM–10:15 AM
Models of microbial keratitis in ex vivo rabbit and human
corneas
Abigail Pinnock1, Nagaveni Shivshetty2, Sanhita Roy2, Stephen
Rimmer1, Ian Douglas1, Sheila MacNeil1, Prashant Garg2. 1University
of Sheffield, Sheffield, United Kingdom; 2LV Prasad Eye Institute,
Hyderabad, India.
Purpose: To study microbial keratitis, cultured cells and in
vivo animal models are commonly used but, these are either not
representative of the in vivo situation or involve the use of many
animals. Recently, there is increased use of ex vivo corneal models
to replace or reduce animal use but there is no direct comparison of
bacterial and fungal keratitis in these models. Accordingly, we aimed
to establish reproducible models of bacterial and fungal infections in
rabbit and human ex vivo corneal models to aid studies of keratitis.
Methods: Wild brown rabbit and donated human corneas were
maintained in corneal organ culture as previously described
(Deshpande et al., Biomater. 2013). Corneas were wounded
with a scalpel, and exposed to, or injected intrastromally with,
108Staphylococcus aureus, Pseudomonas aeruginosa or Candida
albicans for 24 or 48h at 37°C. Corneas were lysed, the resulting
suspension serially diluted and spotted onto agar plates for colony
enumeration. Corneal tissue was histologically processed and
sections were Gram-stained. Corneas not exposed to microbes were
used as controls.
Results: After 24h, using the scalpel wounding method, S.aureus,
P.aeruginosa and C.albicans were recovered at 6.4±1.5x105,
5.5±0.8x106 and 2.2±0.8.1x104 CFU/rabbit cornea respectively
and 3.8±0.8x106, 4.4±0.6x108 and 1.9±0.3x105 CFU/human cornea
respectively. No difference in the CFU/cornea after 48h was observed
compared with 24h. The injection method yielded a 10-fold increase
(p<0.05) in detectable organisms for P.aeruginosa but no difference
for S.aureus or C.albicans, compared with the scalpel method.
Histology of the scalpel-wounded and injection models indicated
extensive infiltration of P.aeruginosa, throughout the entire cornea,
with less infiltration observed for S.aureus and C.albicans.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Conclusions: Bacterial and fungal infections were initiated in ex
vivo corneal models after 24 and 48h, with both scalpel wounding
and injection methods suitable for inducing infection. Differences
between the CFU/cornea for rabbit and human corneas may be due to
the expression of different antimicrobial peptides or surface receptors
influencing colonisation of the tissue. These simple and reproducible
ex vivo models will be useful as an alternative to monolayer cells and
in vivo models for investigating microbial keratitis.
Commercial Relationships: Abigail Pinnock, None; Nagaveni
Shivshetty, None; Sanhita Roy, None; Stephen Rimmer, None; Ian
Douglas, None; Sheila MacNeil, None; Prashant Garg, None
Support: Wellcome-DBT grant 0998800/B/12/Z
Program Number: 4064 Poster Board Number: A0241
Presentation Time: 8:30 AM–10:15 AM
The use of pediatric blood culture bottles in the diagnosis of acute
postoperative endophthalmitis
Tatiana Tanaka1, Joao N. Almeida2, Thais S. Di Gioia2, Flavia
Rossi2, Juliana M. Kato3, Bruno F. Ferreira1, Aline D. Ruppert1,
Yoshitaka Nakashima1, Sergio L. Pimentel1, Joyce H. Yamamoto1.
1
Ophthalmology, University of São Paulo, São Paulo, Brazil;
2
Microbiology, University of São Paulo, São Paulo, Brazil; 3Faculty
of Medicine, University of São Paulo, São Paulo, Brazil.
Purpose: Sample culture is an essential laboratory procedure
necessary to confirm the microbiological etiology and to prompt and
appropriate treatment of endophthalmitis. Techniques for culturing
vitreous samples vary. The traditional method for culturing the
undiluted vitreous uses solid media plates and tioglicolate. Direct
inoculation of blood culture bottles may be alternative.1 Pediatric
blood culture bottles may be suitable for samples of small amount.2
The present study evaluated the culture yield in the diagnosis of
endophthalmitis using for inoculation conventional methodology
(CM) or pediatric blood culture bottle (PBCB).
Methods: This retrospective study included cases of clinically
suspected acute postoperative endophthalmitis treated between
January 2010 and December 2013 at Department of Ophthalmology,
University of São Paulo, SP, Brazil. Undiluted vitreous were
cultivated in CM from January 2010 to December 2011 and in PBCB
from January 2012 to December 2013. The isolated agents and
culture yield were analysed for each methodology. The study was
approved by the Institutional Ethics Committee.
Results: Fourty two cases were included during this 4-year period.
These cases were associated with phacoemulsification (n=20, 47.6%),
trabeculectomy (n=9, 21.4%), extracapsular cataract extration (n=6,
14.3%), pars plana vitrectomy (n=4, 9.5%), phacoemulsification/
trabeculectomy (n=2, 4,8%) and intravitreal bevacizumab injection
(n=1, 2.4%). The most prevalent agents were Staphylococcus
epidermidis (n=5, 23.8%), Streptococcus viridans (n=3, 14.3%),
coagulase-negative Staphylococus (n=2, 9.5%) and Enterococcus
faecalis (n=2, 9.5%). The culture yield of the 23 eyes cultured in CM
was 36.4 % and of the 20 eyes cultured in PBCB was 65% (Table).
Conclusions: In spite of this non-comparative and retrospective
study, pediatric blood culture bottle yielded substantially high
positivity and seems a good alternative to CM if access to
microbiological facilities is suboptimal. PBCB had some advantages
over conventional methodology: easy inoculation, reduce the risk
of contamination with transport and possibility of storage at room
temperature.
1
Joondeph BC et a. A new culture method for infectious
endophthalmitis. Arch Ophthlamol 1989;107:1334-7; 2 Heggers JP et
al. The efficacy of pediatric bllod culture sets in the determinations of
burn bacteremia. J Burn Care Rehabil 1990; 11:419-22
Table
Commercial Relationships: Tatiana Tanaka, None; Joao N.
Almeida, None; Thais S. Di Gioia, None; Flavia Rossi, None;
Juliana M. Kato, None; Bruno F. Ferreira, None; Aline D.
Ruppert, None; Yoshitaka Nakashima, None; Sergio L. Pimentel,
None; Joyce H. Yamamoto, None
Program Number: 4065 Poster Board Number: A0242
Presentation Time: 8:30 AM–10:15 AM
Gene expression analysis of severe bacterial, fungal and
acanthamoeba keratitis: role of immune and inflammatory
biomarkers of disease
Jaya D. Chidambaram1, 2, Namperumalsamy Venkatesh Prajna2,
3
, Palepu Srikanthi2, Manisha Shah3, Lalitha Prajna3, Elakkiya
Shanmugam3, Julien Bauer4, Martin Holland1, Matthew J. Burton1,
5 1
. Clinical Research Dept, London School of Hygiene & Tropical
Medicine, London, United Kingdom; 2Cornea Department,
Aravind Eye Hospital, Madurai, India; 3Aravind Medical Research
Foundation, Madurai, India; 4Pathology Dept, University of
Cambridge, Cambridge, United Kingdom; 5London School of
Hygiene & Tropical Medicine, International Centre for Eye Health,
London, United Kingdom.
Purpose: Microbial keratitis (MK) is a leading cause of blindness
worldwide. Excessive host inflammatory responses are thought to
be the cause of tissue damage in MK. In this study, we investigated
the expression of 45 genes involved in immune and inflammatory
pathways, and extracellular matrix (ECM) modulation in corneal
cells taken from late stage human bacterial (BK), fungal (FK) and
acanthamoeba keratitis (AK), as compared to normal cadaver corneal
tissue.
Methods: Corneal swab samples from 239 patients presenting to
Aravind Eye Hospital Cornea Clinic in India from Feb 2012 to
Feb 2013 with MK (>=3mm diameter stromal infiltrate). Final
outcome was recorded at 21 days post-enrolment (i.e. “nonhealing”=perforation or descemetocoele or intervention, e.g.
corneal glue, transplant or intrastromal antifungal). Cadaver
corneal tissue (n=13) was collected from Aravind Eye Bank. Total
RNA was extracted from swabs/tissue (Qiagen, Netherlands), and
RTqPCR performed with custom Taqman Low Density Arrays (Life
Technologies, USA). Genes were normalized to HPRT1. Pairwise
comparisons between BK, FK or AK versus Controls, BK versus FK
and FK healed versus FK non-healed were performed and statistical
significance of differential expression assessed with Wilcoxon rank
sum test in Stata 12.1 (Bonferroni adjusted p-values).
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Results: 218 patients were microbiologically positive for fungus
(n=185, mainly Fusarium sp. or Aspergillus flavus), bacteria (n=20,
mainly Streptococcus pneumoniae) and Acanthamoeba sp. (n=13).
Differential expression (FC >2 or <2, p<0.0005) was found in 15, 9
and 3 genes in FK, BK and AK versus Controls, with no significant
difference between BK and FK expression. Upregulated genes
included ECM modifiers MMP9 (BK), MMP10 (FK, BK) and
COL5A1 (BK, FK, AK); cytokines IL8 (BK, FK), IL18 (FK), and
neutrophil antimicrobial effectors S100A9 and PI3 (all 3). In FK, PI3
was upregulated in non-healing ulcers (FC 3.6, p=0.002).
Conclusions: Several MMPs and immune effectors are significantly
upregulated in BK, FK and AK. PI3, expressed in healing epithelia
and known to be an immune modulator, correlates with poor
prognosis in other diseases (e.g. cutaneous graft-vs-host disease).
Further research is needed into PI3 as a possible prognostic
biomarker for FK.
Commercial Relationships: Jaya D. Chidambaram, None;
Namperumalsamy Venkatesh Prajna, None; Palepu Srikanthi,
None; Manisha Shah, None; Lalitha Prajna, None; Elakkiya
Shanmugam, None; Julien Bauer, None; Martin Holland, None;
Matthew J. Burton, None
Support: Wellcome Trust PhD Research Fellowship Grant no.
097437/Z/11/Z
Program Number: 4066 Poster Board Number: A0243
Presentation Time: 8:30 AM–10:15 AM
Microbiome of contact lens cases following corneal infiltrative
events
Ajay Kumar Vijay1, Jacqueline Tan1, Lily Ho1, Anahit Penesyan2,
Ian Paulsen2, Mark D. Willcox1. 1School of Optometry & Vision
Science, University of New South Wales, Sydney, NSW, Australia;
2
Department of Chemistry and Biomolecular Sciences, Macquarie
University, Sydney, NSW, Australia.
Purpose: Contact lens cases become contaminated with bacteria
during use and this can lead to corneal infiltration or infection. Many
bacterial species remain non-culturable with standard laboratory
techniques. We performed culturing and next-generation DNA
sequencing to identify both culturable and non-culturable organisms
that reside on contact lens cases of patients during corneal infiltrative
events.
Methods: Contact lens cases were collected from patients with
corneal infiltrative events and both wells were individually swabbed.
Swabs from the right wells were cultured to isolate and identify
viable microbes using standard culture techniques. Microbial DNA
was extracted from the swabs of the left wells, and 16S rRNA
amplicon sequencing performed using PCR primers 515/806
targeting the V4 variable region of 16S rRNA gene.
Results: Six lens cases were collected from five subjects with corneal
infiltrative events: microbial Keratitis (n=1), contact lens peripheral
ulcer (n=1) and infiltrative keratitis (n=3). All six lens cases were
culture positive for Gram-negative bacteria; no Gram-positive
bacteria, fungi or Acanthamoeba were grown. Several bacterial
strains were cultured from 5/6 of the lens case wells; S. marcescens
(n=5), A. xylosoxidans (n=2), S. maltophilia (n=1), A. faecalis
(n=1) and P. fluorescens (n=1). 16S rRNA sequencing of DNA from
lens cases identified multiple microbial species (median = 30, max
= 79, min = 21) including members of Proteobacteria (95.9%),
Bacteroidetes (2.2%), Actinobacteria (1.8%), Firmicutes (0.1%),
Cyanobacteria (0.01%), Candidate division OP11 and Deinococcus
thermos (combined 0.01%).
Conclusions: The results of this study confirm that a large number
of microbes remain non-culturable and DNA analysis of lens cases
can provide valuable information that may help understand the
pathogenesis of contact lens related microbial keratitis and corneal
infiltrative events.
Commercial Relationships: Ajay Kumar Vijay, Bausch+Lomb (F);
Jacqueline Tan, None; Lily Ho, None; Anahit Penesyan, None; Ian
Paulsen, None; Mark D. Willcox, Bausch+Lomb (F)
Program Number: 4067 Poster Board Number: A0244
Presentation Time: 8:30 AM–10:15 AM
Human Ocular Surface Microbiome Composition Revealed By
Next-Generation Sequencing
Thuy Doan1, Lakshmi Akileswaran1, Dallin Andersen1, Narae
Ko1, Angira Shrestha1, Cecilia S. Lee1, Aaron Lee2, Russell Van
Gelder1. 1Ophthalmology, University of Washington, Seattle, WA;
2
Ophthalmology, University of British Columbia, Vancouver, BC,
Canada.
Purpose: Human mucosal surfaces are thought to be colonized
by a diverse community of microorganisms that help shape the
immune system and when altered may lead to infections or cause
inflammation in the host. Conventional culture techniques have failed
to identify the composition and to characterize the diversity of these
communities because a majority of these microbes are unculturable.
In this study, we sought to characterize the ocular surface bacterial
community in healthy subjects by using 16S rRNA gene deep
sequencing on the Illumina platform.
Methods: Conjunctiva samples of the upper and lower fornices of
both eyes were collected using forensic DNA recovery swabs from
35 healthy volunteers. Along with appropriate negative control
samples, the conjunctiva samples underwent DNA extraction, library
preparation, and 16S rRNA gene deep sequencing on the Illumina
platform. Quantitative PCR for Torque Teno Virus (TTV) was also
performed. Data were analyzed in R.
Results: Sequencing resolution to the genus and species levels were
obtained for 140 conjunctiva samples from 35 healthy volunteers.
Propionibacterium acnes, Arthrospira fusiformis, Corynebacterium
tuberculostearicum, Enterobacter hormaechei, and Chryseobacterium
indologenes were the most abundant species across all samples.
Principal component analyses showed that the genera responsible
for the majority of the variance across all conjunctiva samples were
Corynebacterium, Propionibacterium, and Staphylococcus. TTV was
detected in 63% of the patients (17/27). Subgroup analyses revealed
that the TTV load was statistically higher in men compared to women
(0.012 TTV copy/epithelial cell ± 0.0028 TTV copy/epithelial cell
vs. 0.001 TTV copy/epithelial cell ± 0.0006 TTV copy/epithelial cell,
mean ± SEM, p = 0.0078).
Conclusions: The ocular surface microbiome bacterial composition
in healthy volunteers is diverse. The variability across samples
is largely determined by Corynebacterium, Propionibacterium,
and Staphylococcus. Low TTV levels are found in the majority
of the samples and TTV viral load is dependent on gender.
Future experiments using unbiased next-generation sequencing
to characterize the bacterial, fungal, and viral composition of the
ocular surface will further our understanding of ocular infectious and
inflammatory diseases.
Commercial Relationships: Thuy Doan, None; Lakshmi
Akileswaran, None; Dallin Andersen, None; Narae Ko, None;
Angira Shrestha, None; Cecilia S. Lee, None; Aaron Lee, None;
Russell Van Gelder, None
Support: EY022038, P30EY001730, Unrestricted Research
Departmental Grant From Research To Prevent Blindness
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 4068 Poster Board Number: A0245
Presentation Time: 8:30 AM–10:15 AM
Mucosal microbiome in Sjögren Syndrome
Stephen C. Pflugfelder1, Cintia S. De Paiva1, Dan B. Jones2, Quianta
Moore1, Shani Corbiere5, Joseph Petrosino3, Diane Smith3, Michael
E. Stern4, 1, Nadim Ajami3. 1Ophthal-Ocular Surf Ctr, Baylor College
of Medicine, Houston, TX; 2Ophthalmology, Baylor College of
Medicine, Houston, TX; 3Alkek Center for Metagenomics and
Microbiome Research, Baylor College of Medicine, Houston, TX;
4
Biological Sciences, Allergan, Irvine, CA; 5Sjogren Syndrome
Support Group, Houston, TX.
Purpose: To compare the ocular, oral and fecal microbiome in
patients with Sjögren syndrome (SS) with control subjects
Methods: Conjunctival, tongue and fecal samples were obtained
from 10 patients with primary SS meeting revised ACR criteria and
controls (normal eyes and fecal samples, and tongue samples from
patients with rosacea). Severity of oral and ocular surface disease was
graded. Conjunctival goblet cell density was counted in impression
cytology. 16s ribosomal DNA gene sequencing was performed
by 454 (conjunctival) and MiSeq sequencing and sequences were
mapped to microbial databases. Relative abundance of phyla and
genera and alpha and beta diversity of observed OTUs between
groups were compared.
Results: A low abundance ocular surface microbiome consisting
of core phyla Actinobacteria,, Bacteroidetes, Proteobacteria and
Firmicutes was identified. There were no differences in alpha or beta
diversity between normal and dry eyes; however, there was greater
abundance of Firmicutes in the SS group. Compared to control, alpha
diversity was greater in the tongue and reduced in the stool in the
SS group. Between group differences in relative abundance were
observed with greater Streptococcus and Hemophilus and reduced
Neisseria and Fusobacterium genera in the SS tongue and greater
abundance of Blautia, Escherichia/Shigella, and Streptococcus and
reduced Akkermansia, Subdoligranulum, Faecalibacterium and
Prevotella in the SS stool. Distinct clustering of OTUs was seen in SS
stool and subjects with most severe clinical severity scores had the
least diversity of observed OTUs in the stool.
Conclusions: Minimal differences in abundance and diversity were
found in the SS ocular microbiome. In contrast, SS stool showed
a less diverse microbiome that contained a greater number of
inflammatogenic and lower number of homeostatic flora.
Commercial Relationships: Stephen C. Pflugfelder, None; Cintia
S. De Paiva, None; Dan B. Jones, None; Quianta Moore, None;
Shani Corbiere, None; Joseph Petrosino, None; Diane Smith,
None; Michael E. Stern, None; Nadim Ajami, None
Support: Sjögren Syndrome Metagenomics Research Fund, NIH
Grant EY11915 (SCP), Research to Prevent Blindness, Oshman
Foundation, William Stamps Farish Fund, Hamill Foundation
Program Number: 4069 Poster Board Number: A0246
Presentation Time: 8:30 AM–10:15 AM
Metaproteomic analysis in infected ocular surface
Diana Gabriela Ponce-Angulo1, 2, Antonio Bautista-Hernandez1, 3,
Gerardo Aparicio-Ozores2, Victor M. Bautista1. 11. Research Unit/
Microbiology and Ocular Proteomics, Institute of Ophthalmology
“Fundación de Asistencia Privada Conde de Valenciana”, Mexico
City, Mexico; 22. Laboratory of Medical Bacteriology, Department of
Microbiology, National School of Biological Sciences- IPN, Mexico
City, Mexico; 33. Doctoral Program in Biological Sciences and
health, Autonomous Metropolitan University (UAM), Mexico city,
Mexico.
Purpose: To realize a comparative metaproteomic analysis of the
ocular surface in patients with ocular infection.
Methods: The samples were obtained from patients with healthy
and infected ocular surface. The human tears samples were taken
following the internal protocols in both groups. Steril saline solution
was applied over the ocular surface and collected by capillar
tubes. Microorganisms were identified by automated microbiology
methods. Chocolate agar, blood agar, Sabouraud agar were seeded
with samples from both groups, samples also were added to Brain
Heart Infusion. The bacterial identification was determined by Vitek
2 Compact System. The fungi were identificated by morphologycal
caracteristics. The tears samples of each patient were analysed by 2D
electrophoresis and differential protein expression between groups
was performed using Dymensión 2 Software.
Results: Microbiological analysis showed the presence of
Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea,
Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella
adiacens and Candida guillermondi in healthy group; S. capitis, S.
epidermidis and C. albicans were isolated and identified in infected
group, three patients not shown microbiology growth. Proteomic
analysis showed
In the results of healthy group, there were differential growt of
Staphylococus aureus, S. epidermidis, S. lentus, Kokuraria rosea,
Streptococcus thoraltensis, Rothia mucilaginosa, Granulicatella
adiacens and Candida guillermondi. In the infection group, the
findings in the isolated microorganism were S. capitis, S. epidermidis
and C. albicans and in three of all them had not microbiology grown.
At the end of the analysis of differential expression in 2D gels
without infection group compared with the control group the
following results: in the first sample were obtained 5 differentially
expressed spots, in the second sample 4 spots, en the third sample
2 spots, in the fourth sample 3 spots, in the fifth sample 2 spots and
finally in the sixth sample 2 spots. The metaproteomic analysis shows
the relevant expression of diferential proteins. The diferential proteins
will be identificated by mass spectrometry.
Conclusions: The bacterial species were obtained in both groups
were Staphylococcus epidermidis and Staphylococcus capitis.
The analysis have difencial protein expression in helthy group in
compararising with infection group.
Commercial Relationships: Diana Gabriela Ponce-Angulo, None;
Antonio Bautista-Hernandez, None; Gerardo Aparicio-Ozores,
None; Victor M. Bautista, None
Support: This work was supported by “Conde de Valenciana”
Foundation
Program Number: 4070 Poster Board Number: A0247
Presentation Time: 8:30 AM–10:15 AM
Microbiologic spectrum of post-injection endophthalmitis by
indication for intravitreal anti-VEGF therapy
Charles Calvo1, Nadim Rayess1, Ehsan Rahimy1, Chirag Shah2,
Jeremy D. Wolfe3, Eric Chen4, Francis DeCroos5, Sunir J. Garg1,
Jason Hsu1. 1Retina Service, Wills Eye Hospital, Philadelphia,
PA; 2Ophthalmic Consultants of Boston, Boston, MA; 3Associated
Retinal Consultants at William Beaumont Hospital, Royal Oak, MI;
4
Retina Consultants of Houston, Houston, TX; 5Southeastern Retina
Associates, Chattanooga, TN.
Purpose: To describe the microbiologic spectrum of endophthalmitis
following intravitreal anti-VEGF injections for treatment of
neovascular age-related macular degeneration (AMD), diabetic eye
disease, and retinal vein occlusion (RVO).
Methods: A multicenter, retrospective, consecutive case series.
Results: Between January 1, 2011 and September 30, 2013, a total
of 503,890 intravitreal anti-VEGF injections were performed at the
five participating clinical sites. Presumed infectious endophthalmitis
occurred in 159 of 416,133 injections performed for neovascular
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
AMD (1/2617), 16 of 40,982 for diabetic eye disease (1/2561), and
8 of 46,775 for RVO (1/5846). For patients with neovascular AMD,
61 of the 159 cases (38%) of endophthalmitis were culture positive,
corresponding to a culture positive rate of 1/6822. The cultured
organisms included 13 cases of coagulase-negative Staphylococcus,
12 of S. epidermidis, 5 of S. pneumonia, 5 of S. mitis, 5 of
methicillin-sensitive S. aureus, 4 of Staphylococcus lugdunesis, 4
of Enterococcus fecalis, 3 of S. viridans, 3 of non-differentiated
gram-positive cocci, and 1 case of each of the following organisms:
Staphylococcus auricularis, Staphylococcus homininis, Streptococcus
sanguis, alpha-hemolytic Streptococcus, Candida parapsicolosis,
Propionibacterium, and Lactobacillus. For patients treated for
diabetic eye disease, 8 of the 16 cases (50%) were culture positive,
providing a culture positive rate of 1/5123. Four eyes grew
coagulase-negative Staphylococcus, 2 had S. epidermidis, and there
was 1 case of S. pneumonia and 1 case of H. influenzae. For patients
with RVO, 4 of the 8 cases (50%) were culture positive, resulting in
an overall culture positive rate of 1/11,694. There was 1 case of S.
pneumoniae, methicillin-sensitive S. aureus, S. mitis, and coagulasenegative Staphylococcus.
Conclusions: Gram-positive coagulase-negative Staphylococcus
was the most common bacteria isolated in cases of post-injection
endophthalmitis. A greater proportion of coagulase-negative
Staphylococcus was found in endophthalmitis following injections
for diabetic eye disease (75%) compared to AMD (51%) and RVO
(25%). Oral flora pathogens accounted for 22% of the cases.
Commercial Relationships: Charles Calvo, None; Nadim Rayess,
None; Ehsan Rahimy, None; Chirag Shah, None; Jeremy D.
Wolfe, None; Eric Chen, None; Francis DeCroos, None; Sunir J.
Garg, None; Jason Hsu, None
Program Number: 4071 Poster Board Number: A0248
Presentation Time: 8:30 AM–10:15 AM
Microbiologic Analysis of Dacryocystitis at LAC + USC Medical
Center
Mica Bergman1, 2, Jesse berry1, 2. 1USC Eye Institute, Los Angeles,
CA; 2Ophthalmology, LAC + USC Medical Center, Los Angeles, CA.
Purpose: To investigate the microbiologic profile of dacryocystitis at
a major county hospital in Southern California.
Methods: IRB approved retrospective review of
dacryocystorhinostomy (DCR) operations performed from 1/1/2000
– 11/3/2014. Patients with a diagnosis of dacryocystitis in whom at
least one culture was performed during the course of their care were
included in this study.
Results: Sixteen cases of dacryocystitis were identified in fifteen
patients. Of these, eleven underwent culture one time, four underwent
culture two times, and one underwent culture three times, yielding
a total of 22 cases. Of the 22 cases, 19 cases had a positive yield,
12 cases were monomicrobial, 6 cases were bimicrobial, and 1 case
was trimicrobial. In lacrimal sacs undergoing culture two or more
times, the same organism was found in multiple specimens two out
of five times. In total, there were 14 isolates of gram-positive cocci,
10 isolates of gram-negative bacilli, 1 isolate of gram-negative
diplococci, and 2 fungal isolates. The most commonly identified
bacteria was klebsiella pneumoniae (4 isolates), followed by
staphylococcus aureus and streptococcus viridans (3 isolates each).
Bacteria thought to be nonpathogenic (eg staphylococcus epidermidis
and diphtheroids) were excluded.
Conclusions: This series demonstrates that there is a wide range
of pathogens implicated in dacryocystitis in our county hospital in
Southern California. The most commonly isolated bacteria were
klebsiella pneumoniae, staphylococcus aureus, and streptococcus
viridans. One third of patients had polymicrobial culture results.
Given that gram-negative and gram-positive pathogens were found
in similar numbers, it is advisable to treat initially with a broadspectrum antibiotic.
Commercial Relationships: Mica Bergman, None; Jesse berry,
None
Support: An Unrestricted grant from Research to Prevent Blindness,
New York, NY 10022
Program Number: 4072 Poster Board Number: A0249
Presentation Time: 8:30 AM–10:15 AM
15 Years of Microbial Keratitis at an Urban University Practice
in Saint Louis: The Isolates
Hugo Y. Hsu1, 2, Sean Edelstein3. 1Doheny Eye Institute, Los Angeles,
CA; 2Ophthalmology, David Geffen School of Medicine at UCLA,
Los Angeles, CA; 3Ophthalmology, Saint Louis University School of
Medicine, Saint Louis, MO.
Purpose:
The spectrum of micro-organisms causing infectious keratitis varies
between geographic areas and through time. We wish to identify
the isolated micro-organisms from infectious keratitis cases and
to observe any trends in the spectrum of causative organisms
over a 15-year period at Saint Louis University’s Department of
Ophthalmology.
Methods:
We searched the database of the microbiology department and the
diagnosis database of the ophthalmology department at Saint Louis
University to identify cases of microbial keratitis from 1999-2013.
Records of culture-positive cases were reviewed retrospectively.
Non-contaminant isolates were tabulated into three 5-year periods
(1999-2003; 2004-2008; and 2009-2013) and compared.
Results:
229 non-contaminant isolates were identified: 45 from 1999-2003; 84
from 2004-2008; and 100 from 2009-2013. Overall, Gram-positive
organisms were the most commonly isolated (47%) followed by
Gram-negative organisms (34%). Fungi represented 18% of the
overall isolates. Separated into the three 5-year periods, Gram+
organisms represented 53%, 44%, and 46% of the total; Gramrepresented 38%, 27%, and 33% of the total; and fungal organisms
represented 9%, 20%, and 20% of the total. Pseudomonas was the
most commonly isolated organism overall (21%) as well as in each
of the 3 time periods. Streptococcus species were the next most
common isolates overall (15%) followed by coagulase-negative
staphylococcus and Staphylococcus aureus (14% each). The
percentage of Staphylococcus aureus isolates that were oxacillinresistant (ORSA) increased in each of the three 5-year periods from
22% to 44% to 69%.
Conclusions:
The number of non-contaminant isolates increased in each of the
three 5-year periods. Pseudomonas was the most common isolate
found in infectious keratitis at Saint Louis University over the
15-years period. The two biggest changes we observed were in the
number and percentage of fungal isolates as well as the increase
in the proportion of ORSA isolates over time. Fungal organisms
accounted for 9% of isolates at the start of the 15-year period but then
doubled to 20% during the mid 2000’s and continued through the
end of the 15-year period in 2013. The percentage of ORSA isolates
tripled over the 15 years period.
Commercial Relationships: Hugo Y. Hsu, None; Sean Edelstein,
None
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Program Number: 4073 Poster Board Number: A0250
Presentation Time: 8:30 AM–10:15 AM
Clinical and Microbiological Characteristics of Infectious
Keratitis in Mexico
Alejandro F. Ibarra-Lozano, Pedro M. Gonzalez, Jaime Torres, Jorge
E. Valdez. Ophthalmology Residence, Instituto Tecnológico y de
Estudios Superiores de Monterrey, San Pedro Garza Garcia, Mexico.
Purpose: Geographical differences play a major role to the clinical
and microbiological characteristics of infectious keratitis. The
purpose of this observational retrospective study is to present these
aspects of the infectious keratitis found in Mexico to be able to
establish empiric treatment guidelines.
Methods: Patient records from January 2010 to November of 2014
were revised. Cases in which corneal scrape cultures were made
were selected. Variables studied included were: gender, age, time of
presentation to medical attention, visual acuity, microbiologic agent
encountered, comorbidities, and complications.
Results: Twenty-eight cases of microbial keratitis with corneal scrape
culture were found. Male:Female ratio was 18:10 respectively and
age at presentation varied from 2 to 85 years. The most commonly
encountered microbiologic agent was Pseudomona aeruginosa, found
in 8 patients; 5 cases were positive to fungal agents, 2 for Fusarium
spp and 2 for Candida spp; S. pneumonie was found in two patients,
Acantamoeba spp in one patient, and Corynebacerium matruchotti
in another patient. Other isolated microorganisms included M.
catharralis, H. influenzae, A. xyloxidans, Aspergillus, and S.
marcescens. Twenty one percent of the cultures were negative.
Ocular trauma was associated to 32.1% of the cases, including
vegetal and metallic objects. Diabetes Mellitus was found as a
comorbidity in 32.1% of the cases and one case was HIV positive.
Only 3 contact lens users were identified in the total of cases. Time
from the beginning of symptoms to time of medical attention ranged
from 1 to 60 days. Patients who waited more time to get medical
attention were those with fungal and parasitic infections. Gramnegative bacteria dominated the microbiologic pattern being the
encountered organism in 36.3% of the positive cultures. Four patients
had corneal perforation, two of them associated to Pseudomona
aeruginosa.
Conclusions: This study revised the epidemiology of microbial
keratitis in northern Mexico. The microbiologic pattern encountered
varies importantly from that in other geographic zones, especially
in the unusually high Gram-negative predominance. There was a
tight relation between microbial keratitis with trauma and Diabetes
Mellitus. This study will aid the generation of new treatment
guidelines in the geographic zone studied.
Commercial Relationships: Alejandro F. Ibarra-Lozano, None;
Pedro M. Gonzalez, None; Jaime Torres, None; Jorge E. Valdez,
None
Program Number: 4074 Poster Board Number: A0251
Presentation Time: 8:30 AM–10:15 AM
Gram-negative Isolates from Patients with Endophthalmitis:
Incidence Rates and Antibiotic Susceptibilities
Benjamin D. Wilson, Darlene Miller, Harry W. Flynn. Bascom Palmer
Eye Institution, Miami, FL.
Purpose: To report incidence rates and antibiotic susceptibilities
among gram-negative isolates from patients with endophthalmitis.
Methods: Microbiology reports were reviewed to identify the
incidence rates of gram negative pathogens (Enterobacteriaceae vs
NonEnterobacteriaceae) recovered from endophthalmitis patients
during a 25 year period (1990-2014). A combination of disk diffusion,
Vitek 2 and Etests were used to susceptibility trends for amikacin,
ceftazidime, and ciprofloxacin. Etests were used to evaluate
moxifloxacin. Identifications were confirmed using conventional
methods and Vitek 2.
Results: In period I (1990-1999, period I) Gram negative pathogens
represent 11.4% (n=125/1095) of the cases. Organism spectrum
included: Enterobacteriaceae (32%, n=40/125). Top pathogens were
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Proteus mirabilis (n=12/30%), Serratia marcescens (n=8/20%), and
Klebsiella pneumoniae (n=7/17.5%). NonEnterobacteriaceae were the
predominant group -68% (n= 85/125). Top pathogens: Pseudomonas
aeruginosa (41/48%), Haemophilus influenzae (n=15/17.6%) and
Moraxella species (n=8/9.4%).
In period II (2000-2014), gram negative pathogen frequency
was 11.6% (n=121/1039), 75/62% were nonEnterobacteriaceae.
Top pathogens were: Pseudomonas aeruginosa (n=37/49.3%),
Haemophilus influenzae (15/20%), Moraxella osloensis (8/10.7%).
For Enterobacteriaceae, the rate was 38% (n= 46). Serratia
marcescens (n=16/34.8%), Enterobacter cloacae (9/19.6%), and
Klebsiella pneumoniae (5/10.9%).
Antimicrobial susceptibilities for Enterobacteriaceae from period
I were: amikacin-80%; ceftazidime-83% and ciprofloxacin-89%.
Non-Enterobacteriaceae: amikacin-100%; ceftazidime-90%; and
ciprofloxacin-98%. Susceptibilities for Enterobacteriaceae from
period II: amikacin, 95%; ceftazidime, 97%; and ciprofloxacin- 89%.
Non-Enterobacteriaceae: amikacin, 84%; ceftazidime, 91%; and
ciprofloxacin-98%.
Moxifloxacin susceptibility for the Enterobacteriaceae (N=12) was
83% versus 41% for the Non-Enterobacteriaceae (N=17).
The highest resistance and/or nonsusceptible trends for all pathogens
were observed for moxifloxacin-41% (n=12/29), followed by
amikacin (10%), ceftazidime (6%) and ciprofloxacin (4%).
Conclusions: Gram negative isolates from patients with
endophthalmitis have remained stable over the 25 year period.
Emerging resistance trends to commonly used antibiotics amikacin,
ceftazidime, ciprofloxacin and moxifloxacin have been identified.
Commercial Relationships: Benjamin D. Wilson, None; Darlene
Miller, BPEI (E); Harry W. Flynn, BPEI (E)
Support: RPB Unrestricted Award, NIH Core Grant P30EY014801
Program Number: 4075 Poster Board Number: A0252
Presentation Time: 8:30 AM–10:15 AM
Detection of virulence factors with multiplex PCR Technique of
Conjunctival Coagulase Negative-Staphylococcus (CNS) from
patients undergoing cataract surgery
Veronica E. Castillo1, Yolanda Lopez2, Margarita Samudio2, Norma
Farina2, Sonia Abente2, Nilsa Gonzalez2, Florentina Laspina2,
Agustin Carron1, Diogenes Cibils1, Herminia Mino de Kaspar3.
1
Ophthalmology, Hospital de Clínicas, National University of
Asunción, Asuncion, Paraguay; 2Research Institute for Health
Sciences, National University of Asunción, Paraguay, Asuncion,
Paraguay; 3Ophthalmology, Ludwig-Maximilians-University,
Munich, Germany.
Purpose: We performed a prospective study to identify by multiplex
PCR (Polymerase Chain Reaction), genes encoding virulence factors
(ica, Atle and mecA) in CNS isolates from the ocular microbiota
of patients undergoing cataract surgery and to investigate possible
changes in the Coagulase Negative-Staphylococcus (CNS) profile
due to antibiotic prophylaxis.
Methods: After approval of the Institutional Review Board of our
institution had been obtained, patients undergoing cataract surgery
were recruited for this study at the Department of Ophthalmology,
National University of Asuncion, Paraguay. Patients (n= 162)
received in the eye to be operated moxifloxacin 0.5% eye drops, four
times the day before surgery and a last drop one hour before surgery
(T1). The other eye remained as control (T0). Conjunctival swabs
were taken from both eyes one hour after the last drop. Presence of
genes encoding biofilm formation (ica and atlE) and gene encoding
methicillin resistance (mecA) were detected by a multiplex PCR in
CNS isolates from both timepoints.
Results: Of the 162 patients, 87 eyes were positive for CNS in T0,
yielding 96 CNS isolates and in T1, 70 eyes were positive, yielding
77 isolates CNS. We used for this study 43 CNS isolates from T0 and
45 from T1. Of the total CNS isolates, 81,8% (72/88) had at least one
virulence factor gene (37/43 from T0 and 35/45 from T1 (p=0.314),
simultaneous detection of ica and Atle genes was more frequent in
T0 (n=25, 58.0%) than T1 (n=21, 46.7%), but the difference was not
significant (p=0.28).
Conclusions: A high frequency of genes encoding virulence factors
was observed in the coagulase-negative Staphylococcus isolates. The
use of moxifloxacin did not modify significantly the CNS virulence
factor profiles.
Commercial Relationships: Veronica E. Castillo, None; Yolanda
Lopez, None; Margarita Samudio, None; Norma Farina, None;
Sonia Abente, None; Nilsa Gonzalez, None; Florentina Laspina,
None; Agustin Carron, None; Diogenes Cibils, None; Herminia
Mino de Kaspar, None
Program Number: 4076 Poster Board Number: A0253
Presentation Time: 8:30 AM–10:15 AM
Comparison of two different evaluation methods for biofilm
formation of S.epidermidis isolated from ocular surface
Berna Akova Budak1, Sertac Argun Kivanc1, Meral Yildiz1, Merih
Kivanc2, Gulay Gullulu3. 1Uludag University, Bursa, Turkey;
2
Anadolu University, Eskiehir, Turkey; 3Armedica Eye Center, Izmit,
Turkey.
Purpose: To compare of two different evaluation methods for
biofilm formation of Staphylococcus epidermidis isolated from ocular
surface.
Methods: S.epidermidis strains that were isolated from ocular surface
previously were studied. Microtiter plate method (MPA) and Congo
red agar (CRA) method were used for measuring biofilm formation.
Ica A, ica D, bap genes positivity and multi-antibiotic resistance were
determined. Accurracy of measuring biofilm production capacity of
MPA and CRA were compared.
Results: 8 of the strains produced strong biofilm according to MPA
method, and 4 of these strains also produced strong biofilm according
to CRA method. 8 strains produced strong biofilm according to CRA
method. 11 strains were biofilm negative with MPA however 7 (64
%) of these were positive with CRA. 15 strains were resistant to 3
or more antibiotics, 7 (46%) and 6 (40 %) of these strains produced
strong biofilm according to MPA and CRA methods respectively. 8
strains were resistant to 4 or more antibiotics, 6 (75 %) and 3 (37 %)
of these strains produced strong biofilm according to MPA and CRA
methods respectively.
Conclusions: Strains that produced strong biofilm with MPA had
more multi-antibiotic resistance than with CRA.These two methods
may not be consistent with each other.
Commercial Relationships: Berna Akova Budak, None; Sertac
Argun Kivanc, None; Meral Yildiz, None; Merih Kivanc, None;
Gulay Gullulu, None
Program Number: 4077 Poster Board Number: A0254
Presentation Time: 8:30 AM–10:15 AM
Microbiology and Biofilm Growth on Clinically Infected Silicone
Implants within the Lacrimal System: A Thirty Year Review
Lilangi S. Ediriwickrema1, David Samimi2, 1, Brett Bielory2, Darlene
Miller2, Thomas V. Johnson2. 1Ophthalmology, USC Eye Institute,
Los Angeles, CA; 2Bascom Palmer Eye Institute, University of
Miami Miller School of Medicine, Miami, FL.
Purpose: To investigate the pathogens and biofilms responsible for
clinically significant infection of silicone stents implanted within the
lacrimal system.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].
ARVO 2015 Annual Meeting Abstracts
Methods: Retrospective review of culture results for silicone
lacrimal stents removed early for clinically significant infection over
a thirty year period. As a control, routinely removed, clinically noninfected stents were prospectively sent for culture over a six month
period. Four clinically infected stents and six clinically non-infected
stents showing mucus within the lumen at removal were sent for
scanning electron microscopy to grade the presence of organisms,
matrix deposits, organisms within a matrix, and a significant biofilm
by a masked electron micrographer.
Results: Nineteen stents were included in the study; 100% of
clinically non-infected stents (n=9) and of those removed for
infection (n=10) were culture positive. None of the non-infected
stents were culture positive for mycobacteria compared with 90% of
infected specimens (p < 0.001). Of non-infected stents, 89% grew
gram-positive organisms compared with 50% of infected stents
(p=0.07). Sixty-seven percent of non-infected stents had gramnegative organisms versus 50% of infected stents (p=0.46). Electron
microscopy of the four infected stents revealed organisms consistent
with mycobacteria (size, shape) encased within a matrix. Of the six
non-infected stents examined, organisms were identified within the
lumens that were consistent with culture results but were without
clear biofilm formation. A masked electron micrographer was able
to identify and grade the presence of organisms, matrix deposits,
organisms within a matrix, and a significant biofilm all with statistical
significance.
Conclusions: In our study population, atypical mycobacteria
comprise the primary pathogen responsible for clinically significant
infection of silicone stents in the lacrimal system. Robust biofilm
production by this organism likely plays a role in pathogenesis.
Development of biofilm resistant implant material, targeted liposomal
antibiotic delivery, and physical or chemical disruption strategies are
potential methods towards reducing rates of infection.
Commercial Relationships: Lilangi S. Ediriwickrema, None;
David Samimi, None; Brett Bielory, None; Darlene Miller, None;
Thomas V. Johnson, None
Support: Research to Prevent Blindness, New York, NY 10022.
Results: After 6 hours exposure, MPS-1, MPS-2 and MPS-6 showed
>2.4 log kill against combined inoculum of all ISO bacteria and
clinical isolates and did not exhibit reduced efficacy or regrowth
over 14 days. MPS-3 showed <1 log kill against combined inoculum
of all ISO bacteria and clinical isolates after 6 hours exposure, and
exhibited regrowth over 14 days. MPS-4 and MPS-5 exhibited <1
log kill against combined inoculum of all ISO bacteria and Serratia
marcescens and all ISO bacteria and Achromobacter xylosoxidans
clinical isolates after 6 hours exposure and showed reduced efficacy
over 7 days storage. MPS-4 and MPS-5 exhibited <1.5 log kill
against combined inoculum of all ISO bacteria and Achromobacter
sp. clinical isolate after 6 hours exposure and showed reduced
efficacy after 24 hours. Low numbers of ISO bacteria S. aureus and
S. marcescens were observed for MPS-3, MPS-4 and MPS-5 after
6 hours exposure when combined inoculum of these bacteria and
Achromobacter sp. was used.
Conclusions: MPS-1, MPS-2 and MPS-6 showed high ability
to reduce microbial load after 14 days storage, while MPS-4 and
MPS-5 exhibited reduced disinfection efficacy. MPS-3 exhibited low
disinfection efficacy at 6 hours and regrowth over 14 days.
Commercial Relationships: Marina Milenkovic, Abbott Meducal
Optics (E); James Cook, Abbott Medical Optics (E)
Program Number: 4078 Poster Board Number: A0255
Presentation Time: 8:30 AM–10:15 AM
Antimicrobial Efficacy of Multipurpose Disinfecting Solutions
against Combined Inoculum of ISO Bacteria and Clinical Isolates
Marina Milenkovic1, James Cook2. 1Corneal R&D Microbiology,
Abbott Medical Optics, Santa Ana, CA; 2Corneal R&D, Abbott
Medical Optics, Santa Ana, CA.
Purpose: To compare antimicrobial efficacy of multipurpose
disinfecting solutions (MPS) against combined inoculum of ISO
bacteria and Gram-negative clinical isolates during 14 days storage.
Methods: The multipurpose disinfecting solutions studied were MPS-1: polyquaternium (PQ1)+alexidine dihydrochloride (ALX),
MPS-2: polyhexamethylene biguanide (PHMB)+poloxamer, MPS-3:
PQ1+myristamidopropyl dimethylamine (ALDOX)+nonanoylEDTA, MPS-4: PQ1+ALDOX+polyoxyethylene-polyoxybutylene
(EOBO-41), MPS-5: PQ1+ALDOX and MPS-6: PHMB+PQ1. Test
solutions were inoculated with combined inoculum of ISO bacteria
and Gram-negative clinical isolates in the test tube according
to ISO 14729. Test solution efficacy was evaluated at minimum
recommended manufacturer disinfection time of 6 hours, 24 hours,
7 days and 14 days. ISO bacteria tested were: Staphylococcus
aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027 and
Serratia marcescens ATCC 13880. Clinical isolates tested were:
Achromobacter sp., Serratia marcescens and Achromobacter
xylosoxidans.
©2015, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at [email protected].