Slide - Smith Lab

Bacterial infections of
the eye
Robert Shanks, PhD
Eric Romanowski, MS
Conjunctivitis – inflammation of the conjunctiva – pink eye
Keratitis – inflammation of the cornea
Endophthalmitis – inflammation of the interior of the eye
Kerri Walsh
US Olympic Volleyball
frequency severity
Bob Costas
NBC Sports
– Infectious: Bacteria; viral; chlamydia
– Non-infectious: Allergic; dry eyes; local trauma/FB;
iritis; glaucoma
– Risk factors: Allergies; contact lens use; being born;
pollution; smoking, diabetes…
– Generally self limiting
Conjunctivitis - Incidence
– Worldwide it is estimated that there are 5 million
cases of neonatal conjunctivitis per year
– In developed world it is estimated that annually
1-4% of all GP consultations are for red eyes, mostly
– In UK, each year 1 in 8 children have symptoms of
acute conjunctivitis annually
Hovding G. Acta Ophthalmol. 2008;86:5-17
Bacterial Conjunctivitis - Types
– Acute: Rapid onset of injection; purulent discharge;
without pain, discomfort, or photophobia (most
– Hyperacute: Rapid onset of injection; eyelid edema;
severe purulent discharge; chemosis; discomfort
and/or pain; (Neisseria gonorrhoeae)
– Chronic: Red eye with discharge lasting longer than
a few weeks (Chlamydia)
Tarabishy AB and Jeng BH. Cleveland Clinic Journal of Medicine 2008;75:507-512
Distribution of Bacteria Isolated from Conjunctivitis and Blepharitis (19932010) (n=1320)
70% Gram-positive : 30% Gram-negative
S. aureus
St. pneumoniae
Coagulase Negative
(n=183) 13.9%
(n=231) 17.5%
(n=442) 33.5%
Other Gram
(n=74) 5.6%
(n=17) 1.3%
(n=20) 1.5%
Other Gram
(n=134) 10.1%
(n=219) 16.6%
Data from Regis Kowalski
Acute Bacterial Conjunctivitis
Neisseria gonorrhoeae Conjunctivitis
Neisseria meningitidis Conjunctivitis
– Infectious: Bacteria; viruses; fungi; chlamydia
– Non-infectious: Exposure; immune; dry eyes; local
trauma/FB; neurotrophic; toxic (drugs)
– Risk Factors: Contact lens wear; trauma; smoking;
epithelial defects; cosmetic contact lens wear;
blepharitis; dry eye
Keratitis - Incidence
– Estimated 930,000 doctor’s office or outpatient clinic
visits per year in USA, 76.5% of cases result in
antimicrobial prescriptions
– Estimated 58,000 ER visits for keratitis or CL
disorders annually in USA
– Estimated $175 M in direct healthcare costs annually
– Estimated 250,000 hours of clinician time annually
Collier et al. MMWR Wkly. 2014;63:1027-1030
Distribution of Bacteria from Keratitis (N=1395)
Gram Positive Bacteria - 55%
Gram Negative Bacteria - 45%
coagulase negative Staphylococcus
9.2% (129)
Staphylococcus aureus
27.3% (380)
Streptococcus pneumoniae
5.7% (80)
Streptococcus viridans
6.7% (94)
Other Gram Positive
Bacteria - 6.2% (87)
Other Gram Negative
Bacteria - 11.5% (161)
Pseudomonas aeruginosa
16.1% (224)
Serratia marcescens
10.4% (145)
Haemophilus spp - 2.7% (38)
Moraxella species
4.1% (57)
Data from Regis Kowalski
Pseudomonas aeruginosa Keratitis
Can you differentiate between a bacterial and fungal corneal ulcer by observation????
Can you differentiate between a bacterial and fungal corneal ulcer by observation????
The Clinical Differentiation of Bacterial and Fungal Keratitis: A Photographic Survey
Cyril Dalmon, et al (Proctor and Aravind)
IOVS, April 2012, Vol. 53, No. 4, 1787-91
15 fellowship trained corneal specialists
Presented with ~80 photos of fungal or bacterial corneal ulcers and asked to:
Predict the etiology
Bacteria vs. Fungal etiology
66% (95% CI 63-68%)
Gram stain accuracy of bacterial ulcers 46% (95% CI 40-53)
Genus and species of bacterial ulcers
23% (95% CI 17-30)
(Supports the importance of microbiological testing)
The Clinical Differentiation of
Bacterial and Fungal
Keratitis: A Photographic
Cyril Dalmon, et al
(Proctor and Aravind)
IOVS, April 2012, Vol. 53, No. 4,
– Infectious: Bacteria; viruses; fungi
– Risk Factors: Ocular (cataract) surgery; trauma;
intravitreal injections; systemic infections
(endogenous endophthalmitis)
Endophthalmitis - Incidence
– Incidence of endophthalmitis after cataract surgery
varies, ranging from 0.01% to 0.367%
– WHO estimated 20M cataract surgeries in 2010,
expected to rise to 32M in 2020
– Estimated 2,000 – 73,400 cases in 2010
– Estimated 3,200 – 117,440 cases in 2020
Data from Regis Kowalski
• “The ocular surface is in constant contact with
microorganisms but rarely becomes colonized or
infected with these agents because of these ocular
defenses, especially the tear film.” Davidson and
Kuonen Vet Ophthalmol 2004
• Tear film
• eyelid mechanical action of blinking – a
nonspecific sheer stress that limits the contact
time between a potential infecting microbe and
the corneal surface
In humans, the tear film coating the eye, known as the precorneal film, has three
distinct layers, from the most outer surface:
1- The lipid layer (0.11 µm thick), produced by the Meibomian glands, it coats the
aqueous layer, providing a hydrophobic barrier that reduces the evaporation of
tears , and prevents tears spilling onto the cheek.
2- The aqueous layer, (7.0 µm thick), which is secreted by the lacrimal gland which
Promotes spreading of the tear film, control of infectious agents, and promotes
osmotic regulation.
3- The Mucous layer , (7-30 µm thick) produced by the conjunctival goblet cells
made up mainly of mucin , coating the cornea with a hydrophilic layer which
allows for even distribution of the tear film.
Few commensal organisms – although DNA of many bacteria and bacteriophages
can be found using PCR amplification. Staphylococcus epidermidis,
Propionibacterium acnes, and other skin microbes.
Polar layer
Phospholipids, etc.
A bacterium (approximate relative to scale of the film)
The microbe has
to get through
this barrier to
access the ocular
From the eye and
Tear film factors that prevent infection
Surfactant Protein-D
• SP-D and similar collectins bind to microorganisms (bacteria, viruses,
protozoa, etc.)
• associated with inhibition of microbial growth, complement activation,
stimulation of macrophage cytokine production, and the enhancement of
microbial phagocytosis by immune cells
• SP-D can interact with gram-negative bacteria via the core region of the
bacterial lipopolysaccharide (LPS)
• Has a small effect on invasion of P. aeruginosa into rabbit corneal
epithelial cells – Fleiszig 2005 I&I
• SP-D found at 2-5µg/ml in human tears
Surfactant Protein-D
• Brauler et al, 2007, showed that SP-A and SP-D were found in
human tears and were induced by HSV-1 and Staphylococcus aureus
Microbial response to lipid layer
Many bacteria secrete lipases and esterases that could disrupt or alter the tear film, but their
role in ocular pathogenesis is unknown. Staphylococcus aureus makes several lipases. Bugs
like Pseudomonas and Serratia can use lipids as an energy source.
Do lipases from eye-lid commensal bacteria effect the tear film?
This may be the case in some cases of of blepharitis as lipase cleavage products can induce
Aqueous Layer Defenses
• Lysozyme – causes bacteriolysis through hydrolysis of peptidoglycan and
has chitinase activity against fungi
• Lactoferrin (0.6-2.0 mg/ml) sequesters iron preventing bacterial growth.
• Lactoferrin also binds IgA, IgG and complement protein and thereby
modulates the immune system
• Immunoglobulins – IgA (0.1-0.8 mg/ml) – predominantly sIgA
• sIgA coats microorganisms preventing bacterial adherence to corneal
• IgG – concentration increases during inflammation. IgG participates in
phagocytosis and complement-mediated bacterial lysis.
• Secretory phospholipase A2 – cleaves bacterial phospholipids
• Complement – activates the immune system, aids in killing bacteria
Muramidase, N-acetylmuramide glycanohydrolase
Cationic protein
Mol wt. 14.3 kD
~1.5 mg/ml in tears
Activity: cleaves -1,4 linkage between NAM-NAG in
bacterial cell wall peptidoglycan.
• Cleavage products are pro-inflammatory
• Due to its charge, has some non-enzymatic microbicidal
activity against bacteria and fungi (Laible and Germaine, 1985;
Tobji et al, 1988)
• Some mucosal pathogens modify the peptidoglycan
residues surrounding the lysozyme cleavage site to avoid
cell wall damage and from lysozyme (Davis and Weiser
• Reported for some eye pathogens – Staphylococcus
aureus, Streptococcus pneumoniae, Enterococcus
faecalis, Listeria monocytogenes, and Neisseria
Lactoferrin (lactotransferrin)
• LF iron chelating glycoprotein
• 3 mg/ml in tears; 10-20 mg/L saliva; 1 g/L in milk
• LF single polypeptide; MW 80 kD; 2 homologous
domains that each binds one Fe+2 ion
• Activity: blocks growth by limiting iron
– (nutritional immunity)
Lactoferrin – key role in host-pathogen interactions
Skaar PLoS Pathogen 2010
Some bacteria have adapted to low iron conditions and can use lactoferrin
Pseudomonas aeruginosa protease IV can cleaves lactoferrin
• Tear lipocalins (1.5 mg/ml in human tears)
bind hydrophobic compounds –
(phospholipids, fatty acids)–
• Binds to siderophores – a broad spectrum
of bacterial and fungal – and inhibited
growth under iron limiting conditions
• Human tear lipocalin exhibits antimicrobial
activity by scavenging microbial
siderophores. Glukinger et al 2004, AAC
Secretory Phospholipase A2
• Secreted enzyme found at 30 µg/ml in human tears
• “the principle bactericide for staphylococci and other
gram-positive bacteria in human tears” Qu and Lehrer
• Cleaves membrane lipids of certain bacteria
• Also made by PMNs and induced in by bacterial
• Does not work against most Gram-negative bacteria
Human -Defensins
Produced by epithelial cells
• Cationic, peptides
- hBD1, constitutive
- hBD2, inducible
- hBD3, inducible
- hBD4, inducible ?
- antibacterial, antifungal, antiviral
• Mechanism of action
- anionic targets: LPS, LTA, phospholipids
- form pores in bacterial membrane
• Cross-talk with adaptive immunity
(Hancock, Lancet, 1997)
• Antimicrobial peptides continued:
• 29-45 amino acids in length – have 6 cysteine residues that
interact to form 3 disulfide bonds and a B-sheet structure
• Human beta-defensins (bBD) 1-4 are expressed mainly by
epithelial tissues, but also immune cells
• Made as larger precursors and stored in cytoplasmic granules
• Human neutrophil peptides 1-3 (alpha-defensins) are found in
human tears at 0.2-1µg/ml
• hBD-2 made by ocular epithelium – induced by LPS via TLR4
and LTA and lipoproteins through TLR2 – not detected in
normal tear film
• LL-37 another antimicrobial peptide was detected in ocular
cells and upregulated by IL-1B
• Response: Bacteria can alter their LPS to prevent antimicrobial
peptides from working…
Gram positive bacterium and
defensin hBD-3
Mucin layer
• Tear film – mucin layer – 5 mucin genes expressed in tear film –
• Serves as barrier against toxins, hydrolytic enzymes
• Traps various host defense factors, providing high
concentrations of these factors near surface
• ex. SIgA concentrated in mucin layer overlying epithelium
• Mucin and epithelial glycoproteins trap contaminants including
bacteria and aid in their removal through tear clearance
How do human cells recognize bacteria?
Pattern recognition receptors (PRRs)
• Transmembrane and cytosolic
• Recognize and discriminate a diverse
array of microbial patterns
• Recognize PAMPs (pathogen associated
molecular patterns)
• Activate intracellular signalling cascades
• Regulate gene expression
Most bacteria can be divided into two groups based on their cell wall structure
Innate Immunity
• Recognition of bacteria
– PAMPs (pathogen associated molecular patterns)
• Lipopolysaccharide, peptidoglycan, lipoteichoic acid, flagella,
mannans, bacterial DNA, glucans
– Toll-like Receptors (TLRs)
TLR-1: bacterial lipoproteins
TLR-2: peptidoglycan/LTA/lipoprots/B-glucan
TLR-4:LPS, bac HSP60, ExoS
TLR-5: bacterial flagella
TLR-9: pathogen DNA
TLR-13: bact. Ribosomal sequence
– Intracellular signaling
• NF-kB signal
Inler and Hoffman, Trends Cell Biol, 2001
Innate immune response* to ocular bacteria
PRR (PAMPs): TLR5 (flagellin), TLR4 (LPS), TLR2 (LTA/lipoproteins/ExoS)
TLR9 (unmethylated CpG in microbial DNA)
Recruitment of
Inflammatory cells
PMNs major source of tissue damage in Pseudomonas Bact. Keratits
In mice. Linda Hazlett
Non-TLR transmembrane PRRs
(pattern recognition receptors)
S. aureus Lipoteichoic acid
S. aureus Protein A
S. pyogenes
Klebsiella pneumonia
CD46 (+ C3)
More PRRs
Inflammasomes – multiprotein complexes react to
danger signals, e.g. PAMPS, and activate an immune response.
Skeldon and Saleh
Frontiers in Micro 2011
Pathogenic bacteria make molecules that
Enable infection known as virulence factors
P. aeruginosa virulence factors:
Pili, type I, type IV – attachment factors
MDR efflux pumps – antibiotic tolerance
Biofilm formation – antibiotic/immune system tolerance
Pseudomonas aeruginosa biofilm
Type III secretion system – syringe like secretion system
- ExoS – GTPase/ADP-ribosyltransferase – cytoskelleton rearrangements and cell death
invasive PA
- ExoU-intracellular phospholipase – rapid cell death – cytotoxic PA
Proteases – LasB, type IV protease, Alk prot – degrade immune system components,
cause tissue damage
LPS – endotoxin induces inflammation
Mucin layer
• Prevents bacterial adherence – a study by Fleiszig showed
that removal of corneal mucous increased adherence of Pa
to rabbit corneas by 3-10 fold – and this could be rescued
using ocular mucus from porcine cells
• Others showed that mucin aggregated PA could not invade
or cause cytotoxicity
• Bind pathogens before they reach ocular surface
• Competitively block microbial receptors found on the
• MUC1 extends above the cell membrane preventing the
approach of pathogens
• MUC1 had high levels of sialic acid residues that are
negatively charged and may repel some pathogens
• Sialic acid residues may bind to bacterial adhesins and
prevent bacteria from binding epithelial cells
• MUC1 induced by bacteria and activated lymphocytes
Innate immune response* to ocular bacteria
PRR (PAMPs): TLR5 (flagellin), TLR4 (LPS), TLR2 (LTA/lipoproteins/ExoS)
TLR9 (unmethylated CpG in microbial DNA)
Dendritic cells/MACs – IL-18 - - - INFg
Recruitment of
Inflammatory cells
Th1 CD4+ T cells maximize the killing efficacy of macrophages and proliferation of
Cytotoxic CD8+ T cells – in mice this is associated with corneal perforation

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