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Bacterial infections of the eye Robert Shanks, PhD Eric Romanowski, MS.

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Presentation on theme: "Bacterial infections of the eye Robert Shanks, PhD Eric Romanowski, MS."— Presentation transcript:

1 Bacterial infections of the eye Robert Shanks, PhD Eric Romanowski, MS

2 MAJOR EYE INFECTIONS -Conjunctivitis – inflammation of the conjunctiva – pink eye -Keratitis – inflammation of the cornea -Endophthalmitis – inflammation of the interior of the eye frequencyseverity Kerri Walsh US Olympic Volleyball Bob Costas NBC Sports

3 Conjunctivitis – 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

4 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 bacterial – In UK, each year 1 in 8 children have symptoms of acute conjunctivitis annually Hovding G. Acta Ophthalmol. 2008;86:5-17

5 Bacterial Conjunctivitis - Types – Acute: Rapid onset of injection; purulent discharge; without pain, discomfort, or photophobia (most bacteria) – 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

6 Data from Regis Kowalski

7 Acute Bacterial Conjunctivitis

8 Neisseria gonorrhoeae Conjunctivitis

9 Neisseria meningitidis Conjunctivitis


11 Keratitis – 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

12 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

13 Data from Regis Kowalski



16 Pseudomonas aeruginosa Keratitis

17 Can you differentiate between a bacterial and fungal corneal ulcer by observation????

18 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 ulcers23% (95% CI 17-30) 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 (Supports the importance of microbiological testing) Can you differentiate between a bacterial and fungal corneal ulcer by observation????

19 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


21 Endophthalmitis – Infectious: Bacteria; viruses; fungi – Risk Factors: Ocular (cataract) surgery; trauma; intravitreal injections; systemic infections (endogenous endophthalmitis)

22 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

23 Data from Regis Kowalski


25 “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

26 Tears 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.

27 35-45 µm Gipson

28 Polar layer Surfactant-like Molecules Phospholipids, etc. Non-polar layer

29 The microbe has to get through this barrier to access the ocular surface Host-pathogen interactions From the eye and pathogen A bacterium (approximate relative to scale of the film)

30 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 Tear film factors that prevent infection

31 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 inflammation. 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

32 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 epithelium 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 Aqueous Layer Defenses

33 Lysozyme 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)

34 Lysozyme Some mucosal pathogens modify the peptidoglycan residues surrounding the lysozyme cleavage site to avoid cell wall damage and from lysozyme (Davis and Weiser 2011) Reported for some eye pathogens – Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Listeria monocytogenes, and Neisseria gonorrhoeae

35 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)

36 Lactoferrin – key role in host-pathogen interactions

37 Some bacteria have adapted to low iron conditions and can use lactoferrin Pseudomonas aeruginosa protease IV can cleaves lactoferrin Skaar PLoS Pathogen 2010

38 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 Lipocalins

39 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 1998 Cleaves membrane lipids of certain bacteria Also made by PMNs and induced in by bacterial infections Does not work against most Gram-negative bacteria Secretory Phospholipase A2

40 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 (phosphatidylglycerol) -form pores in bacterial membrane Cross-talk with adaptive immunity (Hancock, Lancet, 1997) + +

41 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…

42 Gram positive bacterium and defensin hBD-3

43 Mucin layer Tear film – mucin layer – 5 mucin genes expressed in tear film – MUC-1,2,4,5AC,7 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 mechanisms

44 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 How do human cells recognize bacteria?

45 Gram-positiveGram-negative Most bacteria can be divided into two groups based on their cell wall structure PAMP

46 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 Homodimers/Heterodimers – Intracellular signaling NF-kB signal Inler and Hoffman, Trends Cell Biol, 2001

47 Bacteria PRR (PAMPs): TLR5 (flagellin), TLR4 (LPS), TLR2 (LTA/lipoproteins/ExoS) TLR9 (unmethylated CpG in microbial DNA) PMNs major source of tissue damage in Pseudomonas Bact. Keratits In mice. Linda Hazlett Recruitment of Inflammatory cells Innate immune response* to ocular bacteria chemokines cytokines

48 Non-TLR transmembrane PRRs (pattern recognition receptors) AgonistReceptor S. aureus Lipoteichoic acid PAF/EGFR S. aureus Protein A TNFR1 S. pyogenesCD44 Pseudomonas aeruginosa CFTR Klebsiella pneumoniaCD46 (+ C3)

49 More PRRs

50 Inflammasomes – multiprotein complexes react to danger signals, e.g. PAMPS, and activate an immune response. Skeldon and Saleh Frontiers in Micro 2011

51 Pseudomonas aeruginosa biofilm 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 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

52 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 epithelium 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 Mucin layer

53 Bacteria PRR (PAMPs): TLR5 (flagellin), TLR4 (LPS), TLR2 (LTA/lipoproteins/ExoS) TLR9 (unmethylated CpG in microbial DNA) Recruitment of Inflammatory cells Innate immune response* to ocular bacteria chemokines cytokines Dendritic cells/MACs – IL-18 - - - INFg 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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